Flow path device, nucleic acid purification method, and sample pretreatment system

Abstract

In order to remove impurities from a sample that includes nucleic acids and perform purification and treatment for purifying and recovering the nucleic acids in a highly efficient manner, the present disclosure (see fig. 9A and 9B) proposes a flow path device comprising: a purification membrane capable of recovering nucleic acids; a membrane accommodation section that is a space formed in a substrate, is connected to the flow path, and accommodates the purification membrane; and an annular resin ring press fitted into the membrane accommodation section, wherein the membrane accommodation section has a base surface formed such that the purification membrane can be installed between the upper surface and the lower surface of the substrate, the membrane accommodation section is divided with the base surface serving as a boundary into a membrane accommodation section upper part with a cylindrical shape and a membrane accommodation section lower part having a smaller diameter than the membrane accommodation section upper part, the flow path includes a first flow path that is connected with at least one valve mechanism, formed on the upper surface of the substrate, and connected to a side wall of the membrane accommodation section upper part, and a second flow path that is connected with at least one valve mechanism, formed on the lower surface of the substrate, and connected with the membrane accommodation section lower part, the resin ring has an outer diameter equal to or larger than the inner diameter of the membrane accommodation section upper part, the purification membrane is fixed between the base surface and the resin ring, the outer diameter of the press-fitted resin ring is smaller than the outer diameter of the resin ring when not accommodated in the membrane accommodation section, the inner diameter of the membrane accommodation section upper part is substantially the same as the inner diameter of the membrane accommodation section upper part when the resin ring is not accommodated in the membrane accommodation section, and the inner diameter of the membrane accommodation section upper part and the outer diameter of the press-fitted resin ring are substantially the same at least at a position where the side wall of the membrane accommodation section upper part and the press-fitted resin ring are closest to each other.

Description

Flow channel device, nucleic acid purification method, and sample pretreatment system

[0001] The present disclosure relates to a flow path device, a nucleic acid purification method, and a sample pretreatment system.

[0002] Rapid technological advances, such as improved analytical accuracy and generalization, have led to the widespread use of genetic analysis technology in a variety of fields, including in vitro diagnostics, forensic medicine, and environmental measurement. While genetic testing methods and analytical targets vary widely, a typical analytical process consists of the steps of collecting a specimen, extracting a sample to extract genes from the specimen, purifying the sample to remove inhibitors, amplifying the target gene through a gene amplification reaction, detecting the amplified product, and analyzing the detected signal. Generally, collected specimens are transported to specialized facilities equipped with benchtop genetic analyzers and specialized equipment, and subsequent processing is performed manually by skilled, specialized technicians (laboratory approach).

[0003] In recent years, there has been an increasing demand for sample-to-answer genetic analysis systems that use a gene pretreatment / detection device and a dedicated disposable cartridge to enable fully automated operations from sample introduction to measurement and data analysis, with the aim of simplifying and speeding up operations and reducing costs, etc. For example, Patent Document 1 proposes a fully automated DNA typing system that includes a sample introduction port, a nucleic acid extraction unit, a purification unit, a PCR unit, and a detection unit within the cartridge.

[0004] Methods for purifying nucleic acids include organic phase solidification and solid-phase extraction using porous membranes or magnetic beads. Solid-phase extraction is particularly widely used due to the availability of many commercially available kits and its excellent purification efficiency. Solid-phase extraction involves recovering nucleic acids by binding them to a solid carrier. The carrier may be a porous membrane made of silica or glass fiber, or silica-coated magnetic beads. Generally, manual purification often involves the use of a spin column method, as shown in Patent Document 2, in which a solution is placed on a porous membrane installed in a spin column and transported by centrifugation. This method can be performed using standard laboratory equipment and is relatively easy to operate.

[0005] On the other hand, when applied to automated systems, the magnetic bead method is often adopted, which allows solution exchange without centrifugation by accumulating magnetic beads on a magnet. For example, Non-Patent Document 1 discloses a method for automating gene extraction and purification steps using the magnetic bead method. Non-Patent Document 2 also discloses a nucleic acid purification method using a flow channel chip, in addition to the magnetic bead method. In particular, the Boom method is widely used as a method that enables highly efficient and sample-robust nucleic acid purification with simple processing. The Boom method is a DNA purification technique that utilizes the action of silica to tightly bind to DNA in the presence of chaotropic salts. The Boom method is applied to spin columns, numerous magnetic bead method lysis and purification kits, and nucleic acid purification on flow channel chips.

[0006] Japanese Patent No. 5815572, Japanese Patent Application Laid-Open No. 2014-030397, Japanese Patent Publication No. 2002-512688, Japanese Patent Application Laid-Open No. 2005-257647, Japanese Patent Application Laid-Open No. 2016-63475, Japanese Patent Application Laid-Open No. 2007-111653, Japanese Patent Application Laid-Open No. 2019-144164, Japanese Patent Application Laid-Open No. 2010-257691, Japanese Patent Application Laid-Open No. 2002-39984, International Publication No. 2022 / 030605

[0007] “Microfluidic Devices for Nucleic Acid (NA) Isolation, Isothermal NA Amplification, and Real-Time Detection,” Methods Mol. Biol. 1256, 15-40(2015). “Magnetic particles for integrated nucleic acid purification, amplification and detection without pipetting,” Trends Analyt. Chem. 127:115912(2020).”Purification of Nucleic Acids in Microfluidic Devices,” Anal. Chem. 80, 6472-6479(2008)

[0008] The magnetic bead method requires a magnet for collecting beads, a magnet drive circuit, and a mechanism for transporting beads with high precision to perform multi-stage analysis flows. This poses the problem of complex configurations of the device and flow channel chip, resulting in high costs. For example, as shown in Non-Patent Document 1, when providing micropillars or monolith structures as binding carriers in the flow channel, special processing is required for the flow channel chip, which increases the manufacturing cost of the flow channel chip.

[0009] The aforementioned Patent Documents 1, 3, and 4 disclose examples of nucleic acid purification within a channel chip containing a membrane-like carrier. However, none of these documents even considers the application of these techniques to highly sensitive analysis at a level that allows for sensitive testing of even small amounts of sample (e.g., a level that can be used to test forensic samples (such as artifacts left at crime scenes)). This is supported by the fact that there are no publicly known examples that address the purification efficiency of these techniques, even though it is generally known that the amount and purity of nucleic acids recovered from a sample have a significant impact on the accuracy and sensitivity of the analysis.

[0010] One of the challenges of performing highly efficient nucleic acid purification using a membrane-like carrier on a channel chip is that the purification efficiency is lower than that of laboratory methods and channel chip purification methods using carriers other than membranes. This is a new challenge that has only recently become apparent through the studies of the present inventors. Furthermore, the main cause of this challenge was also revealed through the studies of the present inventors. The background and details of how this challenge was discovered will be explained in the embodiments and the accompanying drawings, as described below.

[0011] One possible solution to the above problem is to seal and fix the purification membrane to prevent leakage of the solution. Not limited to nucleic acid purification applications, a method of holding a thin film object in a predetermined storage unit so that it is in contact with a liquid or gas without leakage includes a membrane fixation method using a gasket such as an O-ring or a C-ring, as exemplified in Patent Documents 4 to 8.

[0012] Patent Document 4 discloses a structure in which a reinforcing plate, a gas-permeable membrane, and an O-ring (rubber gasket) are sequentially housed in an outer container, and an inner container is fitted from above the O-ring to press the O-ring toward the membrane. When the configuration of Patent Document 4 is applied to high-sensitivity analytical applications such as those described above (e.g., forensic sample testing), solution is drawn into the gap between the inner container and the outer container, or between the reinforcing plate and the outer container, resulting in liquid leakage, and the solution absorbed in the gap becomes impossible to recover. This is because Patent Document 4 is intended to allow gas components to pass through, but does not take into account the passage of liquid toward the membrane. At the same time, incorporating the configuration of Patent Document 4 into a channel chip substrate requires the assembly of at least four substrate layers: an outer container containing the membrane and ring, an inner container against which the ring is pressed, a lower lid for providing the channel, and an upper lid against which the lower lid is pressed, resulting in a large chip size and high cost.

[0013] Patent Document 5 describes a configuration in which a C-ring is fitted into a groove in an inner fitting member and then fitted into an outer container, continuously delivering a solution to a separation membrane without leaking the solution outside the container. This is also a common method of using a gasket ring. When the configuration of Patent Document 5 is applied to high-sensitivity analytical applications such as those described above (e.g., forensic sample testing), while it prevents liquid leakage outside the container, liquid penetrates the gap between the outer container and the ring, making the solution absorbed in the gap impossible to recover. This is because Patent Document 5 is intended for continuous delivery of liquid and does not take into account the possibility of flowing a trace amount of solution and recovering it later. Furthermore, as with Patent Document 4, applying the configuration of Patent Document 5 to a substrate-like chip device would result in complex chip components, which would again result in a large size and high cost.

[0014] Patent Document 6 discloses a configuration in which a filter is stored between two chip substrates each having a flow channel and a filter storage section, and the filter is fixed by bonding the chip substrates. The configuration of Patent Document 6 requires a structure in which a flow channel is provided within two chip substrates. Therefore, it cannot be applied to a flow channel chip formed by laminating or bonding a thin, soft material such as a film onto a single chip substrate. Therefore, an on-chip diaphragm valve, as exemplified in Patent Document 7, can be considered. However, in this case, an additional bonding layer is required to provide the valve, complicating the layer structure of the chip. At the same time, precise control of the bonding conditions and positioning accuracy is required. This places significant restrictions on the flow channel shape and chip material that can be formed, and increases manufacturing costs.

[0015] Patent Documents 8 and 9 disclose a configuration in which a gas-permeable membrane, a sealing ring, and a fixing member are sequentially stored, and the ring is deformed under pressure to prevent leakage of the electrolyte contained inside the device. This allows the device to be filled with a trace amount of solution without leakage. However, since this configuration is not intended for liquid delivery, a flow path cannot be provided. Furthermore, even if a flow path were provided to apply this technology to high-sensitivity analytical applications such as those described above (e.g., forensic sample testing), it is clear that unrecoverable residual liquid would still be produced.

[0016] Patent Document 10 describes a configuration in which a nucleic acid extraction membrane is placed in a membrane containment vessel constructed on a flow channel chip, and a rubber O-ring with a notch is pressed against the membrane to prevent blockage of the flow channel, thereby allowing a constant amount of liquid to pass through the membrane. The inventors' investigations revealed that when the configuration of Patent Document 10 is applied to high-sensitivity analysis applications such as those described above (e.g., forensic sample testing), the purification efficiency is low and the amount of recovered liquid is significantly reduced. Furthermore, the inventors' investigations revealed that the sealing and fixing strength of the membrane in the configuration of Patent Document 10 is insufficient for high-sensitivity analysis applications, and that the phenomenon of reduced liquid recovery is due to residual liquid remaining due to the notch. Because the O-ring in Patent Document 8 elastically deforms, the notch is more susceptible to deformation than the surrounding area, resulting in uneven deformation. In this case, if the notch is small, the flow channel becomes narrower, increasing flow channel resistance and impairing liquid delivery controllability. On the other hand, if the notch is large, the notch will deform more significantly, further narrowing the flow channel. At the same time, liquid is likely to remain in the notch, and because the cross-sectional shape of the ring is O-shaped, liquid is likely to penetrate into the gap between the ring and the membrane housing, resulting in an increase in the amount of solution that cannot be recovered. The configuration example exemplified in Patent Document 10 is intended to transport at least several hundred μL of liquid, whereas in the above-mentioned high-sensitivity analysis application, it is necessary to pass a trace amount of liquid, on the order of several tens of μL, through the membrane before recovery. Therefore, even with the configuration exemplified in Patent Document 10, the above-mentioned high-sensitivity analysis application is not anticipated, and it can be said that the expected effect cannot be achieved.

[0017] As described above, while there are many conventional membrane sealing and fixing methods, they are all limited to those intended for gas flow, continuous liquid delivery, or liquid collection, and are not intended for the collection of trace amounts of liquid. In other words, the technical significance of all known membrane fixing methods is to prevent gas or liquid from leaking from a channel or container and entering areas where the gas or liquid should not enter. In contrast, for high-sensitivity analytical applications, it is necessary for the flowing trace amount of liquid to pass through the membrane without loss and be able to be recovered with high yield in the subsequent stage.

[0018] Another method for holding a thin-film object in a predetermined storage section so that it is in contact with the liquid or gas without leakage is a membrane fixation method using a hard ring, as exemplified by Patent Document 2. Patent Document 2 describes a configuration in which a circular porous member, a membrane, and a hollow resin member are sequentially press-fitted into a resin spin column to fix the membrane. This is a typical spin column configuration, allowing trace amounts of liquid to pass through the membrane without loss and recover with high yield. However, the inventors' investigations revealed that when attempting to apply the configuration of Patent Document 2 to high-sensitivity analysis applications, the press-fit ring or flow path chip would be damaged or deformed, making it unsuitable. It was also found that this is because, unlike spin columns, the flow path chip has a high rigidity, and therefore the strain generated when the ring is pressed into the flow path chip cannot be absorbed by both the ring and the flow path chip.

[0019] Another issue when applying the configuration of Patent Document 2 to high-sensitivity analysis applications is that solution remains inside the circular porous support member that serves as the membrane support base, resulting in a reduced yield. It was found that this occurs because the solution transport pressure is lower in applications without centrifugation, compared to solution transport by centrifugation, which is a prerequisite for implementing Patent Document 2. Another issue when applying the configuration of Patent Document 2 to high-sensitivity analysis applications is that even after taking measures to address the above issues and employing the Boom method, a standard technology for highly efficient nucleic acid purification, the purification efficiency is still low or the downstream PCR reaction may be inhibited. This was found to be due to sample clogging and inhibitor residues caused by insufficient liquid transport pressure. When performing a series of purification flows using a chip with a firmly fixed purification membrane so that the sample solution passes through the purification membrane without loss, a high liquid transport pressure (generally >200 kPa) is required. On the other hand, with typical sample-to-answer type channel chips, it is often difficult to obtain sufficient liquid transport pressure due to considerations of channel pressure loss and the pressure resistance of the valves within the channel. To achieve a liquid delivery pressure equivalent to that achieved by centrifugation using a combination of known technologies, it is necessary to improve the chip's pressure resistance and increase the output of the liquid delivery pressure source. This inevitably leads to higher costs and larger systems for the chip and system, making it impossible to realize a fully automated, highly sensitive genetic analysis system using a simple, low-cost device and flow channel chip.

[0020] In view of this situation, the present disclosure proposes a technology that enables the removal of impurities from samples containing nucleic acids and the purification and recovery of nucleic acids to be carried out highly efficiently using a simple, low-cost device and flow path chip.

[0021] In order to solve the above-mentioned problems, the present disclosure provides a flow path device in which a flow path is formed on a substrate, the device comprising: a purification membrane capable of recovering nucleic acids; a membrane containment section which is a space formed in the substrate and connected to the flow path and which contains the purification membrane; and an annular resin ring press-fitted into the membrane containment section, the membrane containment section having a base surface formed between the upper and lower surfaces of the substrate so that the purification membrane can be placed thereon; the membrane containment section is divided by the base surface into a cylindrical upper membrane containment section and a lower membrane containment section having a smaller diameter than the upper membrane containment section; the flow path is connected to at least one valve mechanism; and a second flow path formed on the underside of the substrate and connected to the lower part of the membrane storage section, the resin ring having an outer diameter equal to or larger than the inner diameter of the upper part of the membrane storage section, the purification membrane being fixed between the base surface and the resin ring, the outer diameter of the pressed-in resin ring being smaller than the outer diameter of the resin ring when not stored in the membrane storage section, and the inner diameter of the upper part of the membrane storage section being approximately the same as the inner diameter of the upper part of the membrane storage section when the resin ring is not stored in the membrane storage section, and the inner diameter of the upper part of the membrane storage section and the outer diameter of the pressed-in resin ring are the same at least at the position where the side wall of the upper part of the membrane storage section and the pressed-in resin ring are closest to each other.

[0022] Further features related to the present disclosure will become apparent from the description of this specification and the accompanying drawings. Also, aspects of the present disclosure are achieved and realized by the elements and combinations of various elements and the aspects of the following detailed description and the appended claims. The description of this specification is merely exemplary and does not limit the scope or application of the claims of the present disclosure in any way.

[0023] According to the technology of the present disclosure, the removal of impurities from a sample containing nucleic acid and the purification and recovery of nucleic acid can be performed highly efficiently using a simple, low-cost device and channel chip.

[0024] 1 is a diagram showing the processing steps of a nucleic acid purification method using a standard Boom method. It is a diagram showing the processing steps shown in FIG. 1 and the composition of the sample solution used in the study. It is a diagram showing the reagents and solution transport method used in the purification processing steps shown in FIG. 1. It is a conceptual diagram showing an example configuration of a sample pretreatment system 301 that performs the processing steps of FIG. 1 using a flow path device. It is a diagram showing the configuration of the A-A cross section in FIG. 3A in a state where the flow path device is installed. It is a top view of a flow path device (example 1) 101 that applies a known membrane installation method. It is a diagram showing the A-A cross section configuration in FIG. 4A. It is a top view of a flow path device (example 2) 101 that applies a known membrane installation method. It is a diagram showing the A-A cross section configuration in FIG. 5A. It is a top view of a flow path device (example 3) 101 that applies a known membrane installation method. It is a diagram showing the A-A cross section configuration in FIG. 6A. It is a diagram showing the results of quantitative evaluation of nucleic acid purification efficiency and recovered liquid volume when using a spin column and a flow path device with a known configuration. 9A , 9B, 9C, 9D, 9E, 9F, 9G, 9H, 9H, 9I, 9J ... 12A is a diagram showing the results of a quantitative evaluation of the recovered eluate volume in a nucleic acid purification experiment using the sample pretreatment system shown in FIG. 3A and the flow channel device 101 of FIG. 9. FIG. 12B is a diagram showing the results of a quantitative evaluation of the DNA purification efficiency in a nucleic acid purification experiment using the sample pretreatment system shown in FIG. 3A and the flow channel device 101 of FIG. 9. FIG. 12C is a diagram showing the top view configuration of the flow channel device 101 to which a spin column configuration is applied. FIG. 12B is a diagram showing the A-A cross-sectional configuration in FIG. 12A.18 is a diagram (enlarged photograph) showing an example of a resin ring 204 that has failed to be press-fitted and deformed on the membrane containment unit 104. FIG. 19 is a diagram (photograph) showing an example of a resin ring 204 that has failed to be press-fitted and deformed on the membrane containment unit 104. FIG. 20 is a table summarizing actual examples of the outer diameter dimensions of the resin ring 204 and the success or failure of press-fitting and fixation. FIG. 21 is a diagram showing an example of the internal configuration of a liquid delivery mechanism 302. FIG. 22 is a diagram showing an example of the internal configuration of a liquid delivery mechanism 302 that includes a multi-way valve mechanism (V0) that can be opened to the atmosphere. FIG. 23 is a diagram showing an example of a configuration for achieving the same function as a multi-way valve mechanism 3006 by combining multiple on-off valve mechanisms 3005. FIG. 24 is a diagram showing an example of the internal configuration of a reagent holding unit 303. FIG. 25 is a flowchart for explaining a nucleic acid purification process in the nucleic acid purification system shown in FIG. 16. FIG. 26 is a diagram showing another example of the internal configuration (variant) of the reagent holding unit 303 in the nucleic acid purification system. FIG. 27 is a flowchart for explaining a nucleic acid purification process in the nucleic acid purification system shown in FIG. 21A . FIG. 21B is a diagram showing an example of the system configuration of a sample pretreatment system 301 in which a reagent tank-integrated flow path device (nucleic acid purification device) 101 is installed. FIG. 21C is a diagram showing an example of the top view configuration around the reagent tank 3105. FIG. 21D is a diagram showing an example of the cross-sectional configuration along A-A in FIG. 21A. FIG. 21E is a flowchart (example) for explaining the nucleic acid purification process performed by the nucleic acid purification system shown in FIG. 20. FIG. 21F is a diagram showing an example of the overall configuration of a sample-to-answer type DNA analysis system composed of a flow path device 101 and a sample pretreatment system 301, which can automatically perform nucleic acid extraction, nucleic acid purification, nucleic acid amplification and detection from a biological sample. FIG. 21G is a diagram showing an example of the configuration of a nucleic acid extraction unit 1201 in the sample-to-answer type DNA analysis system. FIG. 21H is a flowchart for explaining the nucleic acid extraction process in the sample-to-answer type DNA analysis system. FIG. 21I is a diagram showing an example of the configuration around the nucleic acid purification unit 1101 in the sample-to-answer type DNA analysis system. FIG. 21IH is a diagram showing an example of the configuration around the nucleic acid amplification unit 1301. 27A. FIG. 27B is a diagram showing an example of a cross-sectional configuration taken along line A-A in FIG. 27A. FIG. 27C is a flowchart for explaining a nucleic acid amplification process in a sample-to-answer type DNA analysis system. FIG. 27D is a diagram showing an example of a configuration of the periphery of the membrane containing section 104 in each modified example, viewed from above. FIG. 27E is a diagram showing an example of a cross-sectional configuration taken along line A-A in FIG. 29A in modified example 1.29A in Modification 2. FIG. 29A in Modification 3. FIG. 29A in Modification 4. FIG. 29A in Modification 5. FIG. 29A in Modification 6. FIG. 29A in Modification 7. FIG. 29A in Modification 8. FIG. 29A in Modification 9. FIG. 30A in Modification 10. FIG. 31A in Modification 11. FIG. 32A in Modification 12. FIG. 33A in Modification 13. FIG. 34A in Modification 14. FIG. 35A in Modification 15. FIG. 36A in Modification 16. FIG. 37A in Modification 17. FIG. 38A in Modification 18. FIG. 39A in Modification 2. FIG. 40A in Modification 2. FIG. 41A in Modification 2. FIG. 42A in Modification 2. FIG. 43A in Modification 2. FIG. 44A in Modification 2. FIG. 45A in Modification 2. FIG. 46A in Modification 2. FIG. 47A in Modification 2. FIG. 48A in Modification 2. FIG. 49A in Modification 3. FIG. 50A in Modification 4.

[0025] An embodiment of the present disclosure proposes a technology that enables highly efficient nucleic acid purification by allowing minute amounts of reaction solution to pass through a purification membrane without leakage in a channel device (channel chip), enabling highly efficient recovery. This specification first describes the issues that were first identified in the study of the present disclosure, followed by a description of the features of a sample pretreatment system (sample pretreatment device) according to an embodiment. This specification then moves on to a description of modified examples of this embodiment. In the drawings of this specification, functionally identical elements may be designated by the same number. While the accompanying drawings show specific embodiments and implementation examples in accordance with the principles of the present disclosure, these are intended to facilitate understanding of the present disclosure and are not intended to limit the interpretation of the present disclosure. Furthermore, while the present embodiment is described in sufficient detail to enable those skilled in the art to implement the present disclosure, it should be understood that other implementations and forms are possible, and that configurations and structures can be modified and various elements can be substituted without departing from the scope and spirit of the technical concept of the present disclosure. Therefore, the following description should not be interpreted as being limited to these examples.

[0026] (1) Further Consideration of Issues in Nucleic Acid Purification (Detailed Study) <Nucleic Acid Purification Process> Figure 1 shows the process steps of a standard nucleic acid purification method. First, a sample solution containing sample DNA is passed through a purification membrane to bind the DNA to the purification membrane (Step I). When using the Boom method for nucleic acid purification, a chaotropic agent such as guadium salt is added to the sample solution obtained by lysing a biological sample. Next, a washing solution is passed through the purification membrane to wash away impurities adhering to the membrane (Step II). Typically, a solution containing ethanol is used as the washing solution, and washing may be performed multiple times. Furthermore, the purification membrane is dried to remove the washing solution components (Step III). Finally, the target DNA is recovered by passing a DNA eluate through the purification membrane to which the DNA has bound and from which the impurities and washing solution components have been removed according to the above flow (Step IV).

[0027] Figure 2A shows the composition of the sample solution used in the process steps shown in Figure 1 and in this study. Figure 2B shows the reagents and solution transport method used in the purification process steps shown in Figure 1. Here, a human genome sample (2800 M) was used as the sample, and the QIAamp DNA Investigator Kit (Qiagen), a standard kit for laboratory-based lysis and purification, was used as the dissolution and purification reagent. In the case of the spin column method used in laboratories, the transport of each solution is achieved by centrifugation.

[0028] <Sample Pretreatment System for Nucleic Acid Purification> FIG. 3A is a conceptual diagram showing an example configuration of a sample pretreatment system 301 that performs the processing steps of FIG. 1 using a flow path device. The sample pretreatment system 301 includes a liquid delivery mechanism 302, a reagent holding unit 303, a PEEK (polyether ether ketone) chip holding unit 304, a PEEK cover 305 having a fitting 306, and piping 307. The liquid delivery mechanism 302 is composed of a liquid delivery power source and a pressure control mechanism that can apply a solution delivery pressure at any timing, and is connected to the reagent holding unit 303 via piping 307. The reagent holding unit 303 includes an area for holding a solution and an area for recovering the solution after delivery, each of which is connected to the flow path device 101 via piping 307. The reagent holding unit 303 can deliver and recover any solution in response to pressurization or depressurization controlled by the liquid delivery mechanism 302. Specific examples of the internal system configuration of the liquid delivery mechanism 302 and the reagent holding unit 303 will be described later.

[0029] 3B is a diagram showing the configuration of the cross section A-A in FIG. 3A in a state in which the flow channel device is installed. The flow channel device 101 is installed so as to be able to be held at a predetermined position between the chip holder 304 and the cover 305. When the flow channel device 101 is installed, the first vent port 107 and the second vent port 108 provided in the flow channel device 101 are connected to the fitting 306 provided in the cover 305.

[0030] The liquid delivery power source of the liquid delivery mechanism 302 can be one that can apply a pressure sufficient to deliver the solution but that does not exceed the chip's withstand pressure. For example, a diaphragm pump or syringe pump that can control the applied pressure in the range of 0.1 kPa to 250 kPa can be used. In this example, air pressure is used as the liquid delivery pressure source, but mechanical compression, centrifugal force, etc. can also be used.

[0031] The material of the chip holder 304 and the cover 305 does not have to be PEEK, and is not particularly limited as long as it is a material that is rigid enough to hold the flow path device 101. In addition to PEEK, resin materials such as polycarbonate (PC) and acrylic (PMMA), metal materials such as aluminum and stainless steel, etc. can be selected as the material. In consideration of the possibility of leakage of solutions such as surfactants and salts contained in the sample solution and ethanol contained in the cleaning solution, it is preferable that the material has chemical resistance to these solutions. <Flow path device for nucleic acid purification>

[0032] Next, the flow channel device 101 used here (in this study) will be described. Figures 4A, 5A, and 6A are top views of flow channel devices (Examples 1 to 3) 101 to which a known membrane installation method is applied. Figures 4B, 5B, and 6B are views showing the cross-sectional configurations taken along line A-A in Figures 4A, 5A, and 6A, respectively.

[0033] Each flow channel device 101 commonly includes a substrate 102 on which a flow channel structure is formed, polyolefin (PO) sealing tape 103 attached to the top and bottom surfaces of the substrate 102, and a purification membrane 201 installed in a membrane containment section 104. First, a 20 mm long, 50 mm wide, and 4 mm thick PC substrate 102 was machined to form a coaxial, cylindrical membrane containment section 104 with an upper section of 6 mm diameter and 1 mm depth and a lower section of 4 mm diameter and 2 mm depth, a first flow channel 105 and a second flow channel 106 with a width and depth of 1 mm, and a first vent 107 and a second vent 108 with a diameter of 1 mm. The first vent 107 is located at the end of the first flow channel 105, and the second vent 108 is located at the end of the second flow channel 106. These are machined as through-holes on the substrate 102, with an open top. The first flow channel 105 is formed on the top surface of the substrate 102 and connects to the sidewall of the upper membrane containment section 110. The second flow channel 106 is formed on the lower surface of the substrate 102 and is connected to the side wall of the membrane housing lower portion 111 .

[0034] 4A and 4B show the configuration of a flow channel device 101 without a membrane fixing structure, i.e., a configuration in which a purification membrane 201 is placed on a base surface 109. Before applying sealing tape 103 to the upper surface of substrate 102, a purification membrane 201 (silica membrane filter, GF / F, Whatman) with a diameter of 6 mm was placed so as to be pressed from the upper membrane storage section upper portion 110 toward base surface 109. After confirming that the outer periphery of purification membrane 201 had been installed without lifting, sealing tape 103 was applied to the upper surface of substrate 102 to form a flow channel device 101 without a membrane fixing structure.

[0035] 5A and 5B show the configuration of a flow path device 101 equipped with a membrane fixation structure using an O-ring with a notch. Before attaching sealing tape 103 to the top surface of substrate 102, a 6 mm diameter purification membrane 201 (silica membrane filter, GF / F, Whatman) was installed so as to be pressed from the membrane containment upper portion 110 toward the base surface 109. Furthermore, a notched rubber O-ring 202 (outer diameter 6 mm × inner diameter 4 mm) was inserted so that the notch coincided with the first flow path 105 so as not to block the first flow path 105. After confirming that the purification membrane 201 and O-ring 202 were installed without floating, sealing tape 103 was attached to the top surface of substrate 102, and a 10 mm square PMMA block was attached to the sealing tape 103 on the top of membrane containment portion 104 to crush the O-ring. This completed the formation of a flow path device 101 equipped with a membrane fixation structure using an O-ring.

[0036] 6A and 6B show the configuration of a flow channel device 101 equipped with a membrane fixing structure using an adhesive. Before applying sealing tape 103 to the upper surface of substrate 102, double-sided tape (#9969, 3M) was applied to base surface 109. Furthermore, a 6 mm diameter purification membrane 201 (silica membrane filter, GF / F, Whatman) was placed so as to be pressed against base surface 109 from the upper membrane storage section 110 side toward base surface 109, and adhered to base surface 109. After confirming that the outer periphery of purification membrane 201 was installed without lifting, sealing tape 103 was applied to the upper surface of substrate 102, thereby forming flow channel device 101 equipped with a membrane fixing structure using an adhesive.

[0037] <Experimental Results> Nucleic acid purification experiments were conducted using the sample pretreatment system shown in Figures 3A and 3B and the flow channel devices 101 shown in Figures 3A and 3B through 5A and 5B. Figures 7A and 7B show the results of quantitative evaluations of the recovered eluate volume and DNA purification efficiency from the nucleic acid purification experiments. As shown in Figure 7A, the final recovered eluate volume using the spin column method was an average of 17 μL, while the recovered eluate volume using the chip device was an average of 10 μL or less. In particular, in the case of the flow channel device 101 equipped with a membrane fixation structure using an O-ring with a notch, the recovered eluate volume was reduced to less than 1 / 10 of the input volume. Furthermore, Figure 8 is a diagram (photograph) showing the phenomenon of a pool of eluate 403 forming near the notch (apparently liquid being trapped on the notch) in a flow channel device 101 (one example) equipped with a membrane fixation structure using an O-ring after eluate recovery.

[0038] In Figure 7B, the dark bars (left) for each method represent the purification efficiency (DNA recovery efficiency) calculated by quantifying the amount of DNA contained in the eluate recovered in Figure 7A using real-time PCR. Also, the light bars (right) for each method represent the DNA leakage rate calculated by quantifying the amount of DNA contained in the sample solution (hereinafter referred to as waste liquid) that passed through the purification membrane. The DNA leakage rate was evaluated by loading the waste liquid into a new spin column, performing the purification process of this embodiment, and quantifying the DNA content using real-time PCR. As shown in Figure 7B, the purification efficiency of the spin column method averaged approximately 50%, and DNA leakage was 0% in all six trials. Meanwhile, the purification efficiency of the chip device was less than half that of the spin column method, indicating a high DNA leakage rate. Furthermore, in the flow channel device 101 equipped with an adhesive membrane fixation structure, there were cases where PCR inhibitors were suspected to be present in the recovered eluate.

[0039] The reason for the lower recovered liquid volume using the chip device compared to the spin column method may be due to the residual liquid on the ring observed in the flow channel device 101 equipped with an O-ring membrane fixation structure (which is simply the insertion of an elastic body, unlike the press-fitting technique described below in this disclosure). The difference in the liquid delivery method may also be responsible. Because the purification membrane is a porous material with a certain water retention capacity, the amount of eluate remaining within the membrane depends on the membrane's material, density, and delivery pressure. The purification membrane 201 used in this study was a silica membrane with similar density in both the spin column method and the chip device. Therefore, this is unlikely to contribute to the difference in recovered liquid volume observed in this study. Meanwhile, the delivery pressure differs between the spin column method and the chip device due to the different solution delivery methods. In the spin column method, the solution introduced into the column was passed through the purification membrane by centrifugal force and delivered to the collection tube. During this process, the solution was constantly subjected to a pressure of 20,000 g (equivalent to a maximum delivery pressure of approximately 100-150 kPa under the conditions of this study). On the other hand, the chip device used in this study uses air pressure liquid transfer, and the liquid transfer pressure for the eluate is low at a maximum of about 50 kPa. Therefore, it is thought that in the flow channel device method, the sample solution held within the membrane is difficult to discharge into the flow channel, resulting in a decrease in the amount of recovered liquid.

[0040] Regarding purification efficiency, in the case of the spin column method, the waste liquid contains almost no DNA. This suggests that the sample solution passes through the purification membrane without leakage and that the binding efficiency between the purification membrane and the DNA is extremely high. On the other hand, in the case of the flow channel device 101 without a membrane fixation structure, there is no mechanism to fix the purification membrane. This is thought to have caused the membrane to float up and seep out from the periphery. In the flow channel device 101 equipped with a membrane fixation structure using an O-ring, membrane floating was suppressed compared to the flow channel device 101 without a membrane fixation structure, which is thought to have reduced the DNA leakage rate and improved purification efficiency. However, the purification efficiency was lower compared to the spin column method, suggesting that the ability to suppress DNA leakage, i.e., membrane fixation, was insufficient.

[0041] Even in the flow channel device 101 equipped with a membrane fixation structure using an adhesive, a high DNA leakage rate and significantly low purification efficiency were observed. This is thought to be due to insufficient adhesion, resulting in unintended gaps, or clogging of the purification membrane by the adhesive. PCR inhibition did not occur in any flow channel device other than the one using the adhesive 203. This suggests that contamination by the adhesive 203 was the cause of PCR inhibition. The adhesive 203 used in this study has been confirmed to be chemically resistant to purification reagents. However, the adhesive 203 is not a product with guaranteed biocompatibility. Therefore, slight component elution, which is not considered a problem in other applications, may have inhibited PCR analysis. Furthermore, because the flow channel device 101 in this study was assembled manually, the adhesive condition could not be reproducibly controlled, and it is likely that the adhesive components protruded from the base or were unevenly attached. Contact with the solution may have made PCR inhibitors more likely to be mixed into the solution. To resolve this issue, it is necessary to more precisely control the device manufacturing process and use adhesive components that do not contain PCR inhibitors. However, considering the size of the membrane housing section 104 and the base surface 109 (the portion to be attached) of the flow channel device 101 in this study, it is expected that stable prototyping in a laboratory will be difficult. At the same time, since the purification reagent in this study contains a highly concentrated solution of ethanol, which is an organic solvent, the types of adhesives and double-sided tapes that can be selected are greatly limited, which is a serious concern.

[0042] (2) Embodiments of the Present Disclosure A sample pretreatment system 301 and a flow path device 101 according to this embodiment, which provide a solution to the problems discovered in the above discussion (examination), will be described.

[0043] <System and Flow Channel Device> The sample pretreatment system 301 is the same as the system shown in FIG. 3A. FIG. 9A is a top view of a flow channel device 101 according to this embodiment. FIG. 9B is a cross-sectional view taken along line A-A in FIG. 9A. The flow channel device 101 includes a membrane containment section 104 formed on a substrate 102, a first flow channel 105, a second flow channel 106, a first vent 107, a second vent 108, sealing tape (film sheet) 103 attached to the top and bottom surfaces of the substrate 102, and a purification membrane 201 installed in the membrane containment section 104. The membrane containment section 104 is composed of an upper membrane containment section 110 and a lower membrane containment section 111 having different inner diameters across the pedestal surface 109, such that a pedestal surface 109 on which the purification membrane 201 can be installed is formed midway between the top and bottom surfaces of the substrate 102. The inner diameter of the upper membrane containment section 110 is approximately the same as that of the purification membrane 201, and the inner diameter of the lower membrane containment section 111 is smaller than those of the purification membrane 201 and the upper membrane containment section 110. The first vent port 107 is located at the end point of the first flow path 105. The second vent port 108 is located at the end point of the second flow path 106. Both the first vent port 107 and the second vent port 108 are, for example, processed as through-holes on the substrate 102, and are open on their upper surfaces. The first flow path 105 is formed on the upper surface of the substrate 102 and connects to the side wall of the upper membrane containment section 110. The second flow path 106 is formed on the lower surface of the substrate 102 and connects to the side wall of the lower membrane containment section 111.

[0044] Specifically, the flow path device 101 according to this embodiment is formed by cutting a PC substrate 102 measuring 20 mm in length, 50 mm in width, and 4 mm in thickness, into which a coaxial cylindrical membrane storage section 104 having an upper portion with a diameter of 6 mm and a depth of 1 mm and a lower portion with a diameter of 4 mm and a depth of 2 mm, a first flow path 105 and a second flow path 106 having a width of 1 mm and a depth of 1 mm, and a first air vent 107 and a second air vent 108 having a diameter of 1 mm.

[0045] The materials for the substrate 102 and the sealing tape 103 are not particularly limited as long as they are materials commonly used in the relevant technical field. For example, materials with low DNA adsorption can be PC, polypropylene (PP), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate, and polyurethane. Furthermore, the amount of DNA adsorption can be reduced by modifying the surface to be negatively charged. Other materials include glass materials such as glass, quartz glass, fused silica, synthetic quartz, alumina, sapphire, ceramics, forsterite, and photosensitive glass; plastics such as polyester resin, polystyrene, polyethylene resin, dimethylpolysiloxane (PDMS), nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, and vinyl chloride resin; and any combination thereof. The sealing tape (film sheet) 103 made of the material described above is attached (adhered with an adhesive that does not inhibit PCR) to the top and bottom of the substrate 102 on which the first flow path 105, the second flow path 106, and the membrane storage section 104 are formed, and at least a portion of the openings of the flow paths, etc. is sealed (sealing will be performed except for at least the first air vent 107 and the second air vent 108), thereby forming the flow path device 101.

[0046] <Method of Fixing Purification Membrane> Figures 10A to 10C are diagrams illustrating a method of installing purification membrane 201. Before applying sealing tape 103 to the upper surface of substrate 102, purification membrane 201 with diameter P and thickness Q was installed on pedestal surface 109 from membrane storage section upper portion 110 with diameter R and depth D (Figure 10A). Furthermore, resin ring 204 with outer diameter a, inner diameter b, and height c (where a > R) was press-fitted to fix purification membrane 201 between pedestal surface 109 (Figures 10B and 10C). After confirming that the inner diameter of the membrane containment upper portion 110 and the outer diameter of the resin ring 204 are the same (R' = a': R' indicates the diameter of the membrane containment upper portion 110 after the resin ring 204 is pressed into the membrane containment upper portion 110; a' indicates the outer diameter of the resin ring 204 after the resin ring 204 is pressed into the membrane containment upper portion 110), and that the purification membrane 201 and the resin ring 204 have been installed without floating, sealing tape 103 is attached to the upper surface of the substrate 102 to form the flow path device 101. Note that the height of the resin ring 204 is preferably such that it does not block the first flow path 105 in the assembled state (FIG. 10C). More preferably, the upper surface of the resin ring 204 (the end surface not in contact with the purification membrane 201) is located lower than the bottom surface of the first flow path 105, at least around the connection between the membrane containment upper portion 110 and the first flow path 105 (thickness T of the chip - (depth of the first flow path 105) ≧ Q + c). In other words, it is desirable that the upper surface of the resin ring 204 is lower than the bottom surface of the first flow path 105. However, it is desirable that the side surface of the resin ring 204 be in contact with the side surface of the membrane containing section 104 over as large an area as possible. For this reason, it is advisable to determine the height of the resin ring 204 so that the upper surface of the resin ring 204 is at the same height as the bottom surface of the first flow path 105. This not only prevents the transported solution from remaining in the gap between the resin ring 204 and the membrane containing section 104, but also reduces flow path resistance, making it possible to recover trace amounts of solution without loss.

[0047] In the technology disclosed herein, "press-fit" refers to a fitting state equivalent to a push-in to press-fit state based on the standard of mechanical fitting accuracy. In other words, after press-fitting is complete, the membrane housing section 104 and the resin ring 204 cannot move relative to each other, and can only be removed by applying a strong force, or they cannot be disassembled without destroying the components. In the case of the membrane fixation structure using an O-ring as shown in Patent Document 10 or the above-mentioned study (Discussion of the Problems), the O-ring 202 undergoes elastic deformation (returns to its original shape when the external force is removed). Therefore, after being attached to the membrane housing upper section 110, the O-ring returns in a direction away from the membrane, as if repelling the external force that caused it to be attached. At the same time, frictional force acts between the outer periphery of the O-ring 202 and the side wall of the membrane housing upper section 110. Therefore, once housed, the O-ring 202 will not pop out, but the force pressing down on the purification membrane is weak. Therefore, this does not qualify as a "press-fit" state in the technology disclosed herein.

[0048] On the other hand, when the resin ring 204 is inserted (press-fitted) into the membrane storage upper portion 110, the resin ring 204 undergoes plastic deformation (the deformation remains even after the external force is removed, and the shape does not return to its original state) and is held in place by the frictional force between the resin ring 204 and the side wall of the membrane storage upper portion 110. As a result, a force pressing down on the purification membrane 201 is maintained in the membrane storage portion 104. At this time, by appropriately setting the dimensional range with the membrane storage upper portion 110, the resin ring 204 can be held in a fitted state that goes from being pressed in to being pressed in. This allows for a high membrane fixing force to be achieved. In the flow channel device 101, whether a membrane fixing structure is achieved by press-fitting the resin ring 204 can be confirmed by checking whether R' = a' at least at the position where the side wall of the membrane containment section upper part 110 and the resin ring 204 are closest in the assembled state, or more precisely, by closely observing the flow channel device 101 in the assembled state and checking whether |R' - a'| < 1 μm (below the resolution of an optical microscope) at least at the position where the side wall of the membrane containment section upper part 110 and the resin ring 204 are closest. Implementation of the technology of the present disclosure can also be confirmed by measuring the inner diameter of the membrane containment section upper part 110 and the outer diameter of the resin ring 204 in the unassembled state ( FIG. 10B ) and the assembled state ( FIG. 10C ) and checking that R / R' (≈ 1) > a' / a holds. That is, in both the unassembled and assembled states, the membrane housing upper portion 110 is highly rigid and therefore hardly deforms, and the resin ring 204, which has an outer diameter a larger than the inner diameter R of the membrane housing upper portion 110, is installed so as to contract, which is an important feature of the technology disclosed herein. A known example of a membrane fixation method by press-fitting using a resin ring is the spin column configuration exemplified in Patent Document 2. However, if the inner diameters of the spin column in the unassembled and assembled states are S and S', respectively, and the outer diameters of the ring are A and A', respectively, then S / S'<A' / A (≈1) holds, which is the opposite relationship to that in the technology disclosed herein. This is because, in the case of the spin column configuration, the spin column is a thin-walled container with low rigidity and can be deformed by press-fitting the ring. For example, in the case of the spin column included in the QIAamp DNA Investigator Kit (Qiagen), P=7 mm, S=approximately 6.4 to 6.7 mm, and A=approximately 7 to 7.2 mm, and S'=A'≈A holds.

[0049] Specifically, in this embodiment, in the flow path device 101, a purification membrane 201 (silica membrane filter, GF / F, Whatman) having a diameter of 6 mm and a thickness of 0.4 mm and a PE resin ring 204 having an outer diameter of 6.2 mm, an inner diameter of 4 mm, and a height of 0.5 mm were installed in the membrane storage section 104.

[0050] The purification membrane 201 may be any membrane capable of retaining particles of 100 μm or larger. Furthermore, a membrane thickness of 1 μm or larger is preferable. Furthermore, since the finer the mesh, the more efficiently DNA can be recovered, it is desirable that the purification membrane 201 be capable of retaining particles of 10 μm or larger, more preferably 1 μm or larger, and even more preferably 0.1 μm or larger. The type of membrane does not have to be a silica membrane; a solid substrate primarily composed of cellulose capable of adsorbing DNA, carboxylated particles, or a purification membrane made of an ion exchange resin may also be used. If the volume of the purification membrane is too small, the amount of biomolecules that can be adsorbed will be reduced. On the other hand, if the volume is too large, there is a risk of unintended molecular adsorption during purification or subsequent steps, or reduced solution transport efficiency. Therefore, the volume must be appropriately set, but there are no restrictions on the size.

[0051] The material of the resin ring 204 does not have to be PE, and is preferably a material that does not elastically deform, such as polyester resin, polystyrene, polyethylene resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, or vinyl chloride resin, and does not have to be made of resin. Specific physical properties include a Young's modulus of the material of 5 MPa to 1 GPa, an elastic modulus of 100 kPa or more, or an elastic strain limit of less than 100%.

[0052] The outer diameter a of the resin ring 204 may be any dimension that allows it to be fitted into the membrane housing upper portion 110 and press-fitted and fixed. Specifically, the inventors have found that the outer diameter a of the resin ring 204 is larger than the inner diameter R of the membrane housing upper portion 110 and is preferably designed to be in a range that satisfies 0≦a−R<400 μm or (a−R) / R≦0.2. Details of the outer diameter a of the resin ring 204 will be described later.

[0053] The inner diameter b of the resin ring 204 may be any diameter that allows the solution to pass through, but it is desirable that it be approximately the same as the inner diameter r of the lower membrane containment section. This allows the solution that comes into contact with the purification membrane 201 to pass efficiently into the lower membrane containment section 111, maximizing the amount of solution that can be recovered. It is also desirable that the difference between the outer diameter a and the inner diameter b of the resin ring 204 be as small as possible. Specifically, it is desirable that it be approximately 2.5 mm or less. This prevents the solution from being captured and remaining on the upper surface of the resin ring 204, allowing for more efficient solution recovery.

[0054] 11A and 11B show the results of quantitative evaluation of the recovered eluate volume and DNA purification efficiency in a nucleic acid purification experiment conducted using the sample pretreatment system shown in Fig. 3A and the flow path device 101 of Fig. 9. In this embodiment, as in the above "Further consideration of problems in nucleic acid purification," the operation was also carried out using the sample solution and treatment steps shown in Fig. 1.

[0055] As shown in Figure 11A, the chip device of the present disclosure recovered an average 13 μL of eluate. While this recovered eluate volume is slightly lower than that of the spin column method, a good recovery efficiency (over 50%) was achieved compared to conventional chip devices. In Figure 11B, the dark bar graphs (left side) for each method represent the purification efficiency (DNA recovery efficiency) calculated by quantifying the amount of DNA contained in the eluate recovered in Figure 11A using real-time PCR. The light bar graphs (right side) for each method in Figure 11B represent the DNA leakage rate calculated by quantifying the amount of DNA contained in the sample solution (hereinafter referred to as waste liquid) that passed through the purification membrane. As shown in Figure 11B, when using the chip device of this embodiment, the high DNA leakage rate, a problem with conventional chip devices, was resolved and purification efficiency equivalent to that of the spin column method was achieved. Note that the maximum solution transport pressure required in the purification process of this embodiment is 65 kPa, which is the same level as that of conventional chip devices. As described above, according to this embodiment, a chip device with a simple configuration and a small apparatus can be used to achieve strong membrane immobilization without sample leakage and stable delivery of a small amount of solution, thereby achieving highly efficient purification with performance equivalent to that of the spin column method. The above technical effects also apply to the embodiments and their derivatives described below.

[0056] (3) Consideration of the Issues with Flow Channel Devices Using a Spin Column Configuration As shown in Fig. 11B, highly efficient purification can be achieved even when using a spin column configuration. Here, we will examine and consider the issues with the flow channel device 101 using a spin column configuration, which is the only known example of achieving membrane fixation by press-fitting a resin ring, and further clarify the advantages of the flow channel device 101 according to the present disclosure (configuration without a porous holding member 205) shown in Fig. 9.

[0057] Figure 12A is a diagram showing the top view of a flow channel device 101 employing a spin column configuration. Figure 12B is a diagram showing the cross-sectional configuration taken along line A-A in Figure 12A. The flow channel device 101 used for this study includes, in the membrane housing section 104, a porous holding member 205 having a diameter of 6.7 mm and a thickness of 2 mm, a purification membrane 201 having a diameter of 7.0 mm and a thickness of 0.4 mm, and a PE resin ring 204 having an outer diameter a (seven types: a = 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1), an inner diameter of 5 mm, and a height of 0.5 mm. The flow channel device 101 employing a spin column configuration is formed by cutting a PC substrate 102 measuring 20 mm long, 50 mm wide, and 5 mm thick. The substrate 102 is then fitted with a tapered membrane housing section 104, which has a diameter of 7.1 mm at the top and a diameter of 5 mm at the bottom. The first and second flow channels 105 and 106 are 1 mm wide and 1 mm deep, and the first and second vents 107 and 108 are 1 mm in diameter. The first vent 107 is located at the end of the first flow channel 105. The second vent 108 is located at the end of the second flow channel 106. Both the first and second vents 107 and 108 are machined as through-holes on the substrate 102, with their upper surfaces open. The first flow channel 105 is formed on the upper surface of the substrate 102 and connects to the side wall of the membrane housing section 104. The second flow channel 106 is formed on the lower surface of the substrate 102 and connects to the side wall of the membrane housing section 104.

[0058] The porous retaining member 205 has the function of holding the purification membrane 201 in a fixed position in the membrane housing unit 104, and when pressed into the spin column and flow path device 101, it deforms so as to be crushed in the circumferential direction. First, the porous retaining member 205 was pressed into the membrane housing unit 104 so that it was horizontal in the planar direction (Procedure 1). The purification membrane 201 was placed on the fixed porous retaining member 205 (Procedure 2), and the resin ring 204 was pressed into the membrane housing unit 104 so that it was horizontal in the planar direction (Procedure 3). It was found that the following several problems actually existed in the purification membrane fixing procedures 1 to 3.

[0059] (i) The first issue in the purification membrane immobilization procedure when using a flow path device 101 with a spin column configuration having a porous retention member 205 is the difficulty in controlling the immobilization position of the porous retention member 205 in step 1. The porous retention member 205 is easily deformed by stress from the side wall of the membrane housing section 104 when pressed into place. Therefore, if pressure is continued from above after the desired immobilization position is reached, the porous retention member 205 will be placed on the connection between the membrane housing section 104 and the second flow path 106, increasing flow path resistance. Eventually, the porous retention member 205 will reach the bottom of the membrane housing section 104 and completely block the second flow path 106. If the sample solution 401 is introduced through the first flow path 105 in this state, the flow path resistance will prevent efficient recovery of the sample solution 401. Conversely, if pressure from above is stopped before the desired immobilization position is reached, the resin ring 204 will be placed on the connection between the membrane housing section 104 and the first flow path 105 when the purification membrane 201 and resin ring 204 are immobilized, increasing flow path resistance. If sample solution 401 is introduced from first channel 105 in this state, sample solution 401 may not come into efficient contact with purification membrane 201, or sample solution 401 may find its way around into unintended gaps.

[0060] When a flow channel is provided on a substrate 102 and a purification membrane 201 is installed, as in the flow channel device 101 (see FIG. 9 ) according to the present disclosure, it is generally preferable to provide the flow channel in the plane direction of the purification membrane 201, because the thinner the substrate 102, the simpler, smaller, and less expensive the flow channel device can be. On the other hand, in the case of the spin column method, there is no incentive to provide a flow channel in the plane direction of the purification membrane, so the disadvantage described in the first problem does not occur. In other words, in the case of the spin column method, the disadvantage of using a porous retaining member 205 does not exist.

[0061] (ii) A second problem in the purification membrane fixation procedure when using a flow channel device 101 with a spin column configuration having a porous holding member 205 is that, in step 3 above, the resin ring 204 cannot be press-fitted and fixed into the membrane housing section 104 using dimensional control equivalent to that used in the spin column method. As described in the configuration of the flow channel device 101 of this embodiment ( FIG. 9 ), the resin ring 204 is deformed by stress from the side wall of the membrane housing section 104 when it is press-fitted into the membrane housing section 104. At this time, because the resin ring 204 is not made of an elastic material such as rubber, if the resin ring 204 is too large for the membrane housing section 104, the resin ring 204 will either not fit into the membrane housing section 104 and will be crushed, or the substrate 102 around the membrane housing section 104 will crack and be damaged.

[0062] 13A and 13B are diagrams (photographs) showing examples of resin rings 204 that failed to be press-fitted and deformed on the membrane housing unit 104. FIG. 13C is a table summarizing examples of the outer diameter dimensions of the resin rings 204 and the success or failure of press-fitting. When the inner diameter R of the membrane housing unit 104 at the position where the purification membrane 201 was fixed was approximately 6.5 mm, and the outer diameter a of the resin ring 204 was 6.9 mm or greater, i.e., when (a-R) was at least 400 μm or greater, press-fit failures like those shown in FIG. 13A were confirmed. This relationship was also observed when the inner diameter b or thickness (height) c of the resin ring 204 was changed, and also when a membrane housing unit 104 (FIG. 9) was equipped with a pedestal instead of the porous holding member 205. At the same time, failures of press-fitting within the range satisfying a-R≧400 μm were confirmed at least when 1 mm≦R≦7 mm. The failure of the press-fitting fixation is thought to be mainly due to the fact that the rigidity of the substrate 102 is greater than that of the spin column method, and the distortion caused when the resin ring 204 is pressed in cannot be absorbed by both the resin ring 204 and the substrate 102 .

[0063] In the case of the spin column method, the container that houses the purification membrane 201 is thin and has low rigidity, so there is no need to consider the dimensional range required for the flow channel device 101 of the present disclosure. As described above, by designing the inner diameter R of the membrane housing section 104 and the resin ring 204 to satisfy, for example, 0≦a−R<400 μm, the flow channel device 101 of the present disclosure can be manufactured stably at a practical level. This dimensional range can be selected based on the physical properties, such as the material, rigidity, and bending moment, of the substrate 102 and the resin ring 204, and is preferably set appropriately.

[0064] (iii) Another issue when using a flow path device 101 with a spin column configuration having a porous retention member 205 is that the eluate 403 is captured by the porous retention member 205, resulting in a reduced volume of recovered liquid. The porous retention member 205 is made of a porous material to hold the purification membrane 201 in a fixed position in the membrane storage section 104 while enabling the recovery of the sample solution 401 that has passed through the purification membrane 201. The porous retention member 205 has a water retention capacity that corresponds to the material, pore size, volume, etc. In the case of the spin column method, the eluate 403 introduced into the column is passed through the purification membrane 201 by centrifugal force (e.g., 20,000 g), so that the eluate 403 retained inside the porous retention member 205 can be largely recovered, and this issue does not occur.

[0065] On the other hand, when centrifugal force is not used and liquid is pumped using a small, general-purpose pneumatic pump, as in the technology disclosed herein, the eluate 403 captured by the porous retaining member 205 cannot be recovered, resulting in a significant reduction in the amount of recovered liquid. Furthermore, when implementing the Boom method purification flow shown in Figures 1 and 2A and 2B, the sample solution 401 and cleaning solution 402 may remain on the porous retaining member 205, potentially contaminating the recovered eluate 403. While some improvement has been seen in this regard (in the case of the Boom method) by using a hydrophobic porous retaining member 205 such as PTFE, increasing the pore size, or thinning the porous retaining member 205 to reduce its volume, these measures have proven insufficient to achieve a satisfactory result in the configuration disclosed herein. Therefore, when implementing the flow path device 101 disclosed herein, it is preferable to use a configuration that does not use a porous retaining member 205.

[0066] (iv) As described above, it is not appropriate to apply a spin column configuration to the flow channel device 101, and it is preferable to adopt a configuration such as the flow channel device 101 according to the present embodiment described above.

[0067] (3) Details of the Liquid Delivery Mechanism and Reagent Retention Unit Here, the detailed configurations of the liquid delivery mechanism 302 and the reagent retention unit 303 in the sample pretreatment system 301 of this embodiment will be described.

[0068] <Configuration of liquid delivery mechanism 302> Fig. 14A is a diagram showing an example of the internal configuration of the liquid delivery mechanism 302. Fig. 14B is a diagram showing an example of the internal configuration of the liquid delivery mechanism 302, which includes a multi-way valve mechanism (V0) that can be opened to the atmosphere.

[0069] 14A , liquid delivery mechanism 302 includes first connection port 3001 and second connection port 3002 that connect to reagent holder 303, liquid delivery power source 3003 that connects to first connection port 3001 and provides liquid delivery pressure, and pressure control mechanism 3004 that adjusts the liquid delivery pressure provided by liquid delivery power source 3003. Liquid delivery power source 3003 and pressure control mechanism 3004 can be configured, for example, by combining a diaphragm pump or DC fan with a constant output (e.g., 100 kPa) with a vacuum regulator, or they may be integrated using a syringe pump or DC fan that can control the amount of discharge and suction.

[0070] 14B , in addition to the configuration shown in FIG. 14A , the liquid delivery mechanism 302 includes a multi-way valve mechanism 3006 (V0) that can be opened to the atmosphere and is located between the pressure control mechanism 3004 and the first connection port 3001. With the configuration of FIG. 14B , the connection between the first connection port 3001 and the liquid delivery power source 3003 can be instantly disconnected by operating the multi-way valve mechanism 3006 at the moment when it is desired to stop the liquid delivery operation, thereby achieving high-speed delivery response. Furthermore, because the liquid delivery power source 3003 can be freely operated in an open-to-air state, the operation of starting liquid delivery after the outputs of the liquid delivery power source 3003 and the pressure control mechanism 3004 have stabilized, or the operation of returning the syringe plunger to a predetermined position when a syringe pump is used as the liquid delivery power source 3003, can be performed while the piping tube 307 between the reagent holder 303 and the liquid delivery power source 3003 is still connected. It is also possible to operate the liquid delivery power source 3003 so that it is constantly driven, and to control the timing of applying pressure to the first connection port 3001 by operating the multi-way valve mechanism 3006. When using a liquid delivery power source 3003 that consumes a lot of power immediately after current application, or when it is desired to minimize the number of times that the liquid delivery power source 3003 and the pressure control mechanism 3004 are switched, the configuration of Fig. 14B can be used. Note that the multi-way valve mechanism 3006 can also achieve the same function by combining multiple on-off valve mechanisms 3005, as shown in Fig. 15.

[0071] The liquid delivery power source 3003, pressure control mechanism 3004, and multi-way valve mechanism 3006 may be connected to the second connection port 3002. In this case, the same operation can be achieved by performing the pressurization and depressurization operations in the opposite direction to when connected to the first connection port 3001. Alternatively, a pair of liquid delivery power source 3003, pressure control mechanism 3004, and multi-way valve mechanism 3006 can be used, each connected to both the first connection port 3001 and the second connection port 3002. This allows access to the flow path device 101 from both connection ports via the reagent holder 303, making it possible to select a liquid delivery route that minimizes the impact of liquid delivery pressure loss due to longer piping lengths. Similarly, a set of liquid delivery power source 3003 and pressure control mechanism 3004 can be used with a multi-way valve mechanism 3006 having a three-way valve function that can switch between the first connection port 3001, the second connection port 3002, and the atmospheric open flow path 3007. This allows pressure to be applied to both the first connection port 3001 and the second connection port 3002, while also allowing the device to be made smaller.

[0072] 16 is a diagram showing an example of the internal configuration of the reagent holding unit 303. The reagent holding unit 303 includes, for example, a first liquid supply port 3101 connected to a first connection port 3001 of the liquid supply mechanism 302, a second liquid supply port 3102 connected to a second connection port 3002, a first device connection port 3103 connected to a first air vent 107 of the flow path device 101, a second device connection port 3104 connected to a second air vent 108, at least one or more reagent tanks 3105 (T1) detachably attached between the first liquid supply port 3101 and the first device connection port 3103 via an opening / closing valve mechanism 3005, and a waste liquid tank 3106 / solution recovery tank 3107 (T2) detachably attached between the second liquid supply port 3102 and the second device connection port 3104. The tank T2 is initially fitted with a waste liquid tank 3106, which is then replaced with a solution recovery tank 3107 after some work has been carried out, and is therefore indicated as 3106 / 3107 in FIG.

[0073] The liquid delivery mechanism 302 and the reagent holding unit 303, and the reagent holding unit 303 and the flow path device 101 may be connected via piping tubes 307, or may be directly connected by piping joints. The first liquid delivery port 3101 and the second liquid delivery port 3102 are pressurized or depressurized under the control of the liquid delivery mechanism 302, and the reagents stored in the reagent tanks 3105 can be transported sequentially to the flow path device 101 and recovered in the waste liquid tank 3106 or the solution recovery tank 3107.

[0074] FIG. 17 is a flowchart illustrating the nucleic acid purification process in the nucleic acid purification system shown in FIG. 16 . First, a reagent tank 3105 containing a sample solution 401 is connected to position T1 in FIG. 16 , and a waste tank 3106 is connected to position T2 (S1701). This connection may be performed automatically by a robot (robot arm) within the system, or by an operator. The liquid delivery mechanism 302 introduces the sample solution 401 (Sample) held in the reagent tank 3105 into the first vent 107 of the flow path device 101, and the sample solution 401 that passes through the purification membrane 201 installed in the membrane housing section 104 of the flow path device 101 and is discharged from the second vent 108 is collected in the waste tank 3106 (S1702). Next, the operator or the robot removes the empty tank from T1 in FIG. 16 and replaces it with the reagent tank 3105 containing the first cleaning solution 402 (S1703). The liquid delivery mechanism 302 introduces the first cleaning liquid 402 (cleaning liquid 1) held in the reagent tank 3105 into the first vent 107 of the flow path device 101, and recovers the first cleaning liquid 402 that has passed through the purification membrane 201 installed in the membrane housing part 104 of the flow path device 101 and been discharged from the second vent 108 into the waste liquid tank 3106 (S1704). The operator or robot removes the empty tank from T1 in Fig. 16 and replaces it with the reagent tank 3105 containing the second cleaning liquid 402 (S1705). The liquid delivery mechanism 302 introduces the second cleaning liquid 402 (cleaning liquid 2) held in the reagent tank 3105 into the first vent 107 of the flow path device 101, and recovers the second cleaning liquid 402 that has passed through the purification membrane 201 installed in the membrane housing part 104 of the flow path device 101 and been discharged from the second vent 108 into the waste liquid tank 3106 (S1706). Similarly, the operator or the robot removes the empty tank from T1 in Fig. 16 and replaces it with the reagent tank 3105 containing the third cleaning liquid 402 (S1707). The liquid delivery mechanism 302 introduces the third cleaning liquid 402 (cleaning liquid 3) held in the reagent tank 3105 into the first vent 107 of the flow path device 101, and recovers the third cleaning liquid 402 that has passed through the purification membrane 201 installed in the membrane housing section 104 of the flow path device 101 and been discharged from the second vent 108 into the waste liquid tank 3106 (S1708).Following the transfer of the third cleaning solution 402, the liquid delivery mechanism 302 blows dry air into the flow path device 101 for three minutes (S1709). Finally, the operator or robot removes the empty tank from T1 in FIG. 16 and replaces it with the reagent tank 3105 containing the eluate 403. At the same time, the operator or robot removes the waste liquid tank 3106 from T2 and replaces it with the solution recovery tank 3107 (S1710). The liquid delivery mechanism 302 introduces the eluate 403 held in the reagent tank 3105 into the first vent 107 of the flow path device 101, and recovers the eluate 403 that has passed through the purification membrane 201 installed in the membrane housing unit 104 of the flow path device 101 and been discharged from the second vent 108 into the solution recovery tank 3107 (S1711).

[0075] The reagent tank 3105, waste liquid tank 3106, and solution recovery tank 3107 are not particularly limited as long as they are commonly used in the relevant technical field. They may be microtubes, conical tubes, blister packs, pouch packs, or other containers that are chemically resistant to the reagents used and can be connected to the liquid delivery mechanism 302 and the flow path device 101 via appropriate connectors. For example, a combination of Microfluidic Reservoirs (Elveflow) and microtubes may be used. Furthermore, the reagent tank 3105 may be sequentially replaced with a tank containing each reagent in advance at position T1 in FIG. 16 to perform the process of FIG. 17 . Furthermore, after each step in FIG. 17 is completed, the reagent may be sequentially added to the empty reagent tank 3105, and the process may proceed to the next step in FIG. 17 . As described above, the tank replacement or reagent addition operation may be performed manually or automatically using a robotic arm or the like.

[0076] Furthermore, the first liquid supply port 3101 may be connected to the second connection port 3002, and the second liquid supply port 3102 may be connected to the first connection port 3001. Similarly, the first device connection port 3103 may be connected to the second air vent port 108, and the second device connection port 3104 may be connected to the first air vent port 107.

[0077] <Modification of Reagent Holding Unit 303> A configuration example of a nucleic acid purification system (reagent holding unit 303) that is more suitable for automation will be described with reference to Figures 18 and 19. Figure 18 is a diagram showing another example (modification) of the internal configuration of the reagent holding unit 303 in the nucleic acid purification system. Figure 19 is a flowchart for explaining the nucleic acid purification process in the nucleic acid purification system shown in Figure 18.

[0078] 18, the reagent holding section 303 includes a first liquid supply port 3101 connected to a first connection port 3001 of the liquid supply mechanism 302, a second liquid supply port 3102 connected to the second connection port 3002, a first device connection port 3103 connected to a first air vent 107 of the flow path device 101, a second device connection port 3104 connected to the second air vent 108, at least one or more reagent tanks 3105 arranged between the first liquid supply port 3101 and the first device connection port 3103 via an opening / closing valve mechanism 3005 and holding a reagent therein, and at least one or more waste liquid tanks 3106 and solution recovery tanks 3107 arranged between the second liquid supply port 3102 and the second device connection port 3104 via a multi-way valve mechanism 3006 and recovering a solution discharged from the flow path device. The liquid delivery mechanism 302 and the reagent holding unit 303, and the reagent holding unit 303 and the flow path device 101 may be connected via piping tubes 307, or may be directly connected by piping joints. The first liquid delivery port 3101 and the second liquid delivery port 3102 are pressurized or depressurized under the control of the liquid delivery mechanism 302, and reagents can be sequentially transported to the flow path device 101 by flow path switching using the on-off valve mechanism 3005 and the multi-way valve mechanism 3006. This eliminates the need for replacement operations of the reagent tank 3105, waste liquid tank 3106, and solution recovery tank 3107, and enables automation of the nucleic acid purification process to be achieved with a simpler system than the configuration of FIG. 16 .

[0079] 19 , first, a control device (not shown; for example, configured by a computer connected to the system) operates the on-off valve mechanism 3005 to open V1 and V6 and close V2 to V5 and V7 to V10. At the same time, the multi-way valve mechanism 3006 (V11, V12) is operated to connect the second liquid supply port 3102 and the second device connection port 3104 to the waste liquid tank 3106 (S1901). The control device operates the liquid supply mechanism 302 to introduce the sample solution 401 (Sample) held in the first reagent tank 3105 into the first vent port 107 of the flow path device 101, and collects the sample solution 401, which has passed through the purification membrane 201 installed in the membrane housing section 104 of the flow path device 101 and been discharged from the second vent port 108, into the waste liquid tank 3106 (S1902). Next, the control device operates the on-off valve mechanism 3005 to open V2 and V7 and close V1, V3 to V6, and V8 to V10 (S1903). The control device operates the liquid delivery mechanism 302 to introduce the first cleaning solution 402 (AW1) held in the second reagent tank 3105 into the first vent 107 of the flow path device 101, and collects the first cleaning solution 402 that has passed through the purification membrane 201 installed in the membrane housing section 104 of the flow path device 101 and been discharged from the second vent 108 into the waste liquid tank 3106 (S1904). After the first cleaning solution 402 has been transported, the control device operates the on-off valve mechanism 3005 to open V3 and V8 and close V1 to V2, V4 to V7, and V9 to V10 (S1905). The control device operates the liquid supply mechanism 302 to introduce the second cleaning solution 402 (AW2) held in the third reagent tank 3105 into the first vent 107 of the flow path device 101, and recovers the second cleaning solution 402 that has passed through the purification membrane 201 installed in the membrane housing part 104 of the flow path device 101 and been discharged from the second vent 108 into the waste liquid tank 3106 (S1906). The control device operates the on-off valve mechanism 3005 to open V4 and V9 and close V1 to V3, V5 to V8, and V10 (S1907).The control device operates the liquid sending mechanism 302 to introduce the third cleaning liquid 402 (EtOH) held in the fourth reagent tank 3105 into the first vent 107 of the flow path device 101, and collects the third cleaning liquid 402 that has passed through the purification membrane 201 installed in the membrane housing part 104 of the flow path device 101 and been discharged from the second vent 108 into the waste liquid tank 3106 (S1908). Following the transport of the third cleaning liquid 402, the control device operates the liquid sending mechanism 302 to blow dry air into the flow path device 101 for three minutes (S1909). Finally, the control device operates the on-off valve mechanism 3005 to open V5 and V10 and close V1 to V4 and V6 to V9, and operates the multi-way valve mechanism 3006 (V11, V12) to connect the second liquid supply port 3102 and the second device connection port 3104 to the solution recovery tank 3107 (S1910). The control device operates the liquid supply mechanism 302 to introduce the eluate 403 (ATE) held in the fifth reagent tank 3105 into the first vent port 107 of the flow path device 101, and recover the eluate 403 that has passed through the purification membrane 201 installed in the membrane housing part 104 of the flow path device 101 and been discharged from the second vent port 108 into the solution recovery tank 3107 (S1911). It is preferable that the solution recovered in the waste liquid tank 3106 or the solution recovery tank 3107 be retained in the waste liquid tank 3106 or the solution recovery tank 3107 without leaking from the second liquid delivery port 3102 or returning to the flow path device 101. For this reason, the waste liquid tank 3106 and the solution recovery tank 3107 should be designed to have a capacity that is sufficiently large relative to the total amount of liquid to be transported. Alternatively, in a system that performs the nucleic acid purification flow of Fig. 17 by pressurization from the first connection port 3001, this can also be achieved by providing the waste liquid tank 3106 and the solution recovery tank 3107 with a filter vent mechanism that allows gas to pass but not liquid to pass, and closing the connection with the second liquid delivery port 3102.

[0080] The on-off valve mechanism 3005 and the multi-port valve mechanism 3006 can be interchangeable, as shown in FIG. 15 . Replacing the multi-port valve mechanism 3006 (V11, V12) in FIG. 16 with the on-off valve mechanism 3005 allows for parallel placement of identical valve mechanisms, which is advantageous in terms of controllability and scalability. On the other hand, replacing the on-off valve mechanism 3005 (V1 to V10) in FIG. 16 with the multi-port valve mechanism 3006 allows for the integration of valve mechanisms, potentially facilitating system miniaturization and common control mechanisms. Furthermore, valve mechanisms do not necessarily need to be provided at both ends of the reagent tank 3105; they may be located only on the upstream side (V1 to V5) or the downstream side (V6 to V10). Providing valve mechanisms at both ends of the reagent tank 3105, as shown in FIG. 16 , reduces the possibility of unintended reagent leakage due to capillary action or changes in the pressure inside the flow channel, thereby achieving more stable liquid transfer. On the other hand, if a valve mechanism is provided on only one side of the reagent tank, the number of valve mechanisms can be reduced, allowing a smaller and simpler system to be constructed. The on-off valve mechanism 3005 and the multi-way valve mechanism 3006 may be, for example, a diaphragm valve, a needle valve, a bellows valve, a gate valve, or the like, as long as they are chemically resistant to the reagents used in the nucleic acid purification process and can allow or stop the passage of fluids and adjust the flow rate, and their operation may be performed manually or by control using an electrical signal or the like.

[0081] (3) Reagent Tank Integrated Nucleic Acid Purification System Applying the Configuration of the Flow Channel Device 101 of the Present Disclosure A sample pretreatment system and a nucleic acid purification method relating to a reagent tank integrated nucleic acid purification device will be described with reference to FIGS. 20 to 22.

[0082] <Configuration Example of Reagent Tank Integrated Nucleic Acid Purification System> FIG. 20 is a diagram showing a system configuration example of a sample pretreatment system 301 in which a reagent tank integrated flow channel device (nucleic acid purification device) 101 is installed. The flow path device (nucleic acid purification device) 101 includes a membrane housing section 104 formed on a substrate 102, a first flow path 105, a second flow path 106, a first vent port 107, a second vent port 108, sealing tape 103 attached to the top and bottom surfaces of the substrate 102, a purification membrane 201 fixed to the membrane housing section 104 by press-fitting a resin ring 204, at least one or more reagent tanks 3105 installed between the first vent port 107 and the first flow path 105 via an opening / closing valve mechanism 3005, a waste liquid tank 3106 connected to the second flow path 106 via the opening / closing valve mechanism 3005 and equipped with a vent filter 3109, and a solution recovery flow path 3108 connected to the second flow path 106 via the opening / closing valve mechanism 3005 and connected to the second vent port 108. The configuration around the membrane housing section 104 is the same as that shown in Figures 9A and 9B and 14A and 14B. The second ventilation port 108 is connected to a solution recovery tank 3107 provided in the reagent holding section 303 .

[0083] FIG. 21A is a diagram showing an example of the top view configuration around the reagent tank 3105. FIG. 21B is a diagram showing an example of the cross-sectional configuration along A-A in FIG. 21A. The reagent tank 3105 is a hollow container having an internal volume slightly larger than the amount of reagent solution (e.g., 800 μL of sample solution, 1000 μL of cleaning solution, and 20 μL of elution solution), in which the reagent solution is sealed, and is affixed to the substrate 102 above the connection flow path 1001, which is provided with a protrusion 1004. When the reagent tank 3105 is crushed by the reagent tank actuator 3110, the protrusion 1004 comes into contact with the sealing member 1003 at the bottom of the reagent tank 3105, destroying the sealing member 1003. This allows the reagent to flow into the connection flow path 1001. In this reagent tank-integrated nucleic acid purification device, the reagent tank 3105 is formed by bending a 50 μm-thick aluminum sheet while providing a reagent sealing port 1005 and a vent hole 1006, and then welding a 10 μm-thick aluminum sheet to the bottom as a sealing member 1003. A micropipette is inserted through the reagent sealing port 1005 to fill the reagent tank 3105 with reagent, and then the reagent sealing port 1005 and the vent hole 1006 are sealed with sealing tape. Finally, the top of the connection channel 1001 provided on the substrate 102 is affixed with double-sided tape. The reagent tank actuator 3110 can be a small linear actuator with a thrust force of 10 N connected to a tapered tip jig.

[0084] The on-off valve mechanism 3005 is composed of a valve section 1002 provided on the flow path device 101 and a valve actuator 3111. The valve section 1002 has a shape that matches the tip of the valve actuator 3111. The connecting flow path 1001 can be sealed by pressing down the valve section 1002 with the valve actuator 3111. The reagent tank-integrated nucleic acid purification device is equipped with a dome-shaped valve section 1002 with a curvature of 1 mm in the connecting flow path 1001, which is 1 mm wide and 1 mm deep. The valve actuator 3111 is a small linear actuator with a propulsion force of 5 N and a spherical tip made of stainless steel with a curvature of 1 mm.

[0085] The material, shape, etc. of the reagent tank 3105 are not particularly limited as long as they are commonly used in the relevant technical field, and a blister pack, pouch pack, etc. can be used. Furthermore, the reagent tank 3105 may be any type that is chemically resistant to the reagent used, can hold the reagent at a predetermined position in the flow path device 101, and can transport the reagent at any timing. Preferably, the flow path device 101 is capable of holding the reagent at a predetermined position even when not installed in the sample pretreatment system 301, and after installation in the sample pretreatment system 301, the reagent tank 3105 is opened by an external force applied from the sample pretreatment system 301. However, the flow path device 101 may be configured not to hold the reagent at a predetermined position when not installed in the sample pretreatment system 301. In this case, for example, after installation in the sample pretreatment system 301 and closing all on-chip valves, the reagent may be introduced through the reagent inlet 1005, and the reagent inlet 1005 and the vent 1006 may be sealed. The reagent tank 3105 does not necessarily have to include the reagent sealing port 1005 and the vent 1006. The sealing member 1003 may be assembled after the reagent is sealed. The shape of the reagent tank 3105 does not have to be hexagonal, but may be, for example, rectangular, triangular, or circular. However, if the liquid delivery direction is diagonally aligned, residual liquid inside the reagent tank 3105 can be minimized. The external force applied to the reagent tank 3105 from the sample pretreatment system 301 does not have to be a mechanical pressing force that deforms the reagent tank 3105, as with the reagent tank actuator 3110. For example, internal flow channel pressure that breaks the seal of the reagent tank 3105 via a flow channel connected to the reagent tank 3105 may be used. In particular, a configuration in which the reagent tank 3105 is opened using internal flow channel pressure can be shared with the liquid delivery mechanism 302 and the piping required for performing the nucleic acid purification flow, which is advantageous in terms of simplifying and miniaturizing the system mechanism.

[0086] The on-off valve mechanism 3005 is not particularly limited as long as it is a commonly used valve in the relevant technical field. It is sufficient if it is chemically resistant to the reagents used, can withstand a liquid delivery pressure (e.g., 100 kPa) or more, can be installed on the flow channel device 101, and can control the liquid delivery direction at any timing. Specifically, examples of the on-off valve mechanism 3005 include a film-deformation valve that achieves a closed state by mechanically deforming a flow channel lid material attached to the surface of the substrate 102 with a pressing force. Other examples include an on-chip diaphragm valve, a valve using magnetic beads, a thermally deformable valve, and a piezoelectric valve. A rotary on-chip valve may also be used in place of the multi-way valve mechanism 3006. The advantages of a film-deformation valve are that it can be operated with a relatively simple structure and can operate stably at low cost. Therefore, a film-deformation valve is a particularly promising option for high-performance sample pretreatment systems that require a complex processing flow, which inevitably requires multiple valve mechanisms, and which also require compactness and low cost. On the other hand, in the case of a conventional membrane fixing technique using a rubber O-ring or gasket ring, pressure must be applied in the fixing direction (toward the membrane surface). Therefore, the flow path lid material must be made of a highly rigid material that does not deform, or if an easily deformable film sheet is attached (see Figure 21B), a pressure actuator must be installed on top. Adding a pressure actuator leads to an increase in the size and cost of the device, while using a highly rigid material for the flow path lid material precludes the use of a film deformation valve, which again raises concerns about the size and cost of the system. The purification membrane fixing method disclosed herein does not require the application of external force from the top surface after the resin ring 204 is pressed in, making it easy to combine with a film deformation valve, which is one of the advantages of the technology disclosed herein.

[0087] The reagent tank actuator 3110 and the valve actuator 3111 do not have to be linear actuators, and for example, a solenoid lock, an XYZ stage, a stepping motor, a DC motor, or the like may be used, or an actuator integrating multiple valve mechanisms may be used to operate them. An arm, a wire coil, or the like may be used to separate the external power source from the operating unit. This allows the mechanisms arranged on the flow channel device 101 to be miniaturized, and the flow channel device 101 can be miniaturized.

[0088] <Nucleic Acid Purification Process in Reagent Tank Integrated Nucleic Acid Purification System> Figure 22 is a flowchart (example) for explaining the nucleic acid purification process executed by the nucleic acid purification system shown in Figure 20. First, the control device (not shown: for example, composed of a computer connected to the system) operates the valve actuator 3111 to open V1 and close V2 to V10, and operates the reagent tank actuator 3110 to break the seal on the reagent tank 3105 (Sample) containing the sample solution 401 (S2201). Next, the control device operates the valve actuator 3111 to open V1, V4, and V8, and close V2 to V3 and V5 to V7. The control device also operates the liquid delivery mechanism 302 to introduce the sample solution 401 into the first flow path 105 of the flow path device 101, and collects the sample solution 401, which has passed through the purification membrane 201 installed in the membrane housing section 104 of the flow path device 101 and been discharged from the second flow path 106, in the waste liquid tank 3106 (S2202). Next, the control device operates the valve actuator 3111 to open V2 and V8 and close V1 and V3 to V7, and further operates the reagent tank actuator 3110 to break the seal of the reagent tank 3105 (wash buffer) containing the washing solution 402 (S2203). The control device then operates the valve actuator 3111 to open V2, V5, and V8 and close V1, V2 to V4, V6, and V8, and further operates the liquid delivery mechanism 302 to introduce the cleaning solution 402 held in the second reagent tank 3105 into the first flow path 105 of the flow path device 101, pass the cleaning solution 402 through the purification membrane 201 installed in the membrane housing part 104 of the flow path device 101, and recover the sample solution 401 discharged from the second flow path 106 in the waste liquid tank 3106 (S2204). Following the delivery of the cleaning solution 402, the control device operates the liquid delivery mechanism 302 to blow dry air into the flow path device 101 for 3 minutes (S2205). Next, the control device operates the valve actuator 3111 to open V3 and close V1-V2 and V4-V8, and further operates the reagent tank actuator 3110 to break the seal on the reagent tank 3105 (Elution buffer) containing the eluate 403 (S2206).Finally, the control device operates the valve actuator 3111 to open V3, V6, and V7 and close V1-2, V4-5, and V8, and further operates the liquid delivery mechanism 302 to introduce the eluate 403 into the first flow path 105 of the flow path device 101, pass it through the purification membrane 201 installed in the membrane housing section 104 of the flow path device 101, and deliver the sample solution 401 discharged from the second flow path 106 to the solution recovery flow path 3108, where it is recovered in the solution recovery tank 3107 connected to the second air vent 108 (S2207).

[0089] Although Figure 22 shows a nucleic acid purification process flow using one type of cleaning solution for the purification membrane 201, by changing the arrangement and combination of the reagent tank 3105 and the opening / closing valve mechanism 3005, it is possible to accommodate various process flows, including the nucleic acid purification flow shown in Figure 2.

[0090] (4) Sample-to-Answer Type DNA Analysis System to which the Configuration of the Flow Channel Device 101 of the Present Disclosure is Applied

[0091] 23 to 28, a sample pretreatment system and a flow path device for a sample-to-answer DNA analysis system will be described. Here, the configuration and processing details of the system will be explained using an example in which personal identification is performed by short tandem repeat (STR) analysis using a capillary electrophoresis device from a swab sample containing several μL of human blood.

[0092] <Configuration Example of Sample-to-Answer DNA Analysis System> Figure 23 is a diagram showing an example of the overall configuration of a sample-to-answer DNA analysis system that is composed of a flow path device 101 that can automatically perform nucleic acid extraction, nucleic acid purification, and nucleic acid amplification / detection from a biological sample, and a sample pretreatment system 301. The configuration of the sample pretreatment system 301 is generally similar to the configuration of the system shown in Figure 3A, and includes a liquid delivery mechanism 302, a reagent holding unit 303, and a chip holding unit 304. The first air vent 107 and the second air vent 108 of the flow path device 101 fixed to the chip holding unit 304 are connected to the reagent holding unit 303 via piping tubes 307. The flow path device 101 includes a nucleic acid extraction unit 1201 having a sample inlet 1202 and dissolving a sample to extract nucleic acids contained in the sample, a nucleic acid purification unit 1101 connected to the nucleic acid extraction unit 1201 and removing impurities from a sample solution 401 discharged from the nucleic acid extraction unit 1201 to recover target nucleic acids, and a nucleic acid amplification unit 1301 connected to the nucleic acid purification unit 1101 and amplifying and detecting the target nucleic acids contained in the eluate 403 discharged from the nucleic acid purification unit 1101.

[0093] 24 is a diagram showing an example of the configuration of the nucleic acid extraction unit 1201 in a sample-to-answer DNA analysis system. The nucleic acid extraction unit 1201 includes a sample inlet 1202 on the substrate 102, a nucleic acid extraction chamber 1203 connected to the sample inlet, at least one reagent tank 3105 that holds reagents for dissolving the sample and performing a nucleic acid purification reaction, at least one opening / closing valve mechanism 3005, a purification unit connection flow path 1204 that connects the nucleic acid extraction chamber 1203 with the nucleic acid purification unit 1101, and a liquid delivery flow path 1205 that connects to the first vent port 107 or the second vent port 108 and enables liquid delivery by the operation of the liquid delivery mechanism 302.

[0094] A sample to be analyzed (e.g., a swab sample (sample 400): for example, a sample (blood) wiped on the tip of a cotton swab) is introduced into the sample inlet 1202. Thereafter, an inlet sealing member 1206 made of polyurethane is attached to the sample inlet 1202. This ensures breathability within the nucleic acid extraction chamber 1203 while preventing the sample and reagent from leaking outside the flow channel device 101.

[0095] The nucleic acid extraction chamber 1203 is designed to have an internal volume of, for example, 4 mL. The temperature control mechanism 3202 controls the nucleic acid extraction unit heat source 3201, and can maintain the nucleic acid extraction chamber 1203, which is in contact with the temperature-regulated nucleic acid extraction unit heat source 3201, at a predetermined temperature. Note that the opening / closing valve mechanism 3005 and the reagent tank 3105 can be similar to those shown in Figures 21A and 21B.

[0096] The inlet sealing member 1206 does not have to be made of polyurethane and can be made of a material that allows air to pass through to a certain extent and prevents leakage of the sample and solution inside the nucleic acid extraction chamber 1203, such as a vent filter, a porous sintered resin filter, or perforated sealing tape. By designing the nucleic acid extraction chamber 1203 as an unsealed structure, the internal pressure of the nucleic acid extraction chamber 1203 can be reduced during the nucleic acid extraction reaction. This allows even a flow channel device 101 with low pressure resistance to be used without damage, thereby reducing the cost of the device. The inlet sealing member 1206 may also be sealed using a non-breathable material (e.g., a rubber member). In this case, providing the nucleic acid extraction chamber 1203 with a filter vent mechanism can achieve the same effect as the inlet sealing member 1206.

[0097] <Nucleic Acid Extraction Process> FIG. 25 is a flowchart for explaining the nucleic acid extraction process in the Sample-to-Answer type DNA analysis system.

[0098] First, the operator inserts a swab sample (sample 400) with blood (e.g., 0.5 μL) attached to the tip into the nucleic acid extraction chamber 1203 through the sample inlet 1202 and seals it with the inlet sealing member 1206 (S2501). Next, in response to an instruction from the operator to start the nucleic acid extraction process, the control device (not shown; for example, configured by a computer connected to the system) operates the valve actuator 3111 (not shown in FIG. 23 ) of the on-off valve mechanism 3005 to open V2 and close V1 and V3-V5, and operates the reagent tank actuator 3110 to break the seal on the reagent tank 3105 containing the nucleic acid extraction solution (lysis buffer, e.g., 300 μL). At the same time, the control device operates the temperature control mechanism 3202 to set the surface temperature of the nucleic acid extraction unit heat source 3201 to 56°C (S2502). When the control device confirms (detects) that the temperature of the nucleic acid extraction chamber 1203 has reached 56°C, it operates the valve actuator 3111 to open V1 and V2 and close V3 to V5, and then operates the liquid delivery mechanism 302 to introduce the nucleic acid extract into the nucleic acid extraction chamber 1203 (S2503). Following the delivery of the nucleic acid extract, the control device controls the system to send air into the nucleic acid extraction chamber 1203, and agitates the nucleic acid extract and sample 400 for 30 minutes by air bubbles and thermal convection (S2504). This dissolves the cell membranes of the blood cells contained in the sample 400, releasing the intracellular nucleic acids into the nucleic acid extract. Next, the control device operates the valve actuator 3111 of the on-off valve mechanism 3005 to open V3 and close V1, V2, and V4, V5, and further operates the reagent tank actuator 3110 to break the seal on the reagent tank 3105 containing the purified reagent (sample buffer, e.g., 500 μL). At the same time, the control device operates the temperature control mechanism 3202 to set the surface temperature of the nucleic acid extraction unit heat source 3201 to 72°C (S2505). Upon confirming (detecting) that the temperature of the nucleic acid extraction chamber 1203 has reached 72°C, the control device operates the valve actuator 3111 to open V3 and V4 and close V1, V2, and V5, and further operates the liquid delivery mechanism 302 to introduce the purified reagent into the nucleic acid extraction chamber 1203 (S2506).Following the transfer of the purification reagent in S2506, the control device controls the system to pump air into the nucleic acid extraction chamber 1203, stirring the sample 400 and solution (nucleic acid extract and purification reagent) for 10 minutes using air bubbles and thermal convection (S2507). The purification reagent is a solution containing chaotropic salts and ethanol, enabling highly efficient DNA adsorption to the silica membrane. The operation of S2507 completely lyses the blood cells remaining in S2504 while simultaneously completing the preparation of a sample solution 401 (a mixture of human genomic DNA in the sample, nucleic acid extract, and purification reagent) suitable for the Boom method in the downstream nucleic acid purification unit 1101. Finally, the control device opens V3-5 and closes V1-2, transferring the sample solution 401 from the purification unit connection channel 1204 to the nucleic acid purification unit 1101 (S2508).

[0099] <Nucleic Acid Purification Process> Figure 26 is a diagram showing an example of the configuration of the nucleic acid purification unit 1101 and its surroundings in a sample-to-answer DNA analysis system. The nucleic acid purification unit 1101 has a configuration generally similar to that of the reagent tank-integrated flow path device (nucleic acid purification device) 101 (see Figure 20). The main differences from the reagent tank-integrated flow path device (nucleic acid purification device) 101 are that the sample solution 401 is transported from a purification unit connection flow path 1204 that connects to a nucleic acid extraction unit 1201, and that a solution recovery flow path 3108 connects to a nucleic acid amplification unit 1301. Even with this nucleic acid purification unit 1101, highly efficient nucleic acid purification can be achieved by the nucleic acid purification process shown in Figure 22.

[0100] <Configuration Example of Nucleic Acid Amplification Unit 1301> Figure 27A is a diagram showing an example of the configuration of the periphery of the nucleic acid amplification unit 1301. Figure 27B is a diagram showing an example of the cross-sectional configuration along A-A in Figure 27A. The nucleic acid amplification unit 1301 includes a nucleic acid amplification chamber 1302 on a substrate 102 connected to a solution recovery flow path 3108 of the nucleic acid purification unit 1101, at least one or more reagent tanks 3105 that hold reagents for performing a nucleic acid amplification reaction, at least one or more opening and closing valve mechanisms 3005, an amplification product recovery flow path 1303 that connects the nucleic acid amplification chamber 1302 to the first vent port 107 or the second vent port 108 and discharges the sample solution after the nucleic acid amplification reaction, and a liquid delivery flow path 1205 that connects to at least one of the first vent port 107 or the second vent port 108 and enables liquid delivery by the operation of the liquid delivery mechanism 302.

[0101] The nucleic acid amplification chamber 1302 can be configured in a linear shape, for example, with dimensions of 5 mm wide, 8 mm long, and 0.5 mm thick. Furthermore, the substrate 102 on the bottom surface of the nucleic acid amplification chamber 1302 is processed to be thinner (e.g., 0.5 mm thick) compared to the peripheral portion. This allows the top and bottom surfaces of the nucleic acid amplification chamber 1302 to come into contact with the nucleic acid amplification unit heat source 3301, thereby rapidly changing the temperature of the sample solution in the nucleic acid amplification chamber. One temperature control mechanism 3302 may be installed for each of the top and bottom nucleic acid amplification unit heat sources 3301. This allows for feedback control of the temperature based on the temperature data from each of the top and bottom surfaces, enabling more precise temperature control. Alternatively, the temperature control mechanism 3302 may be shared with the temperature control mechanism 3202 (temperature control mechanism 3202) of the nucleic acid extraction unit heat source 3201 (see Figure 24). This allows for a more compact device. The on-off valve mechanism 3005 and reagent tank 3105 can employ mechanisms similar to those shown in Figure 20.

[0102] 28 is a flowchart illustrating the nucleic acid amplification process in a sample-to-answer DNA analysis system. The control device (not shown) first operates the valve actuator 3111 to open valves V1 and V2 and close valves V3 and V4, thereby introducing a portion (e.g., 10 μL) of the eluate 403 from the solution recovery channel 3108 into the nucleic acid amplification chamber (S2801). Next, the control device operates the valve actuator 3111 to open valve V3 and close valves V1, V2, and V4, and then operates the reagent tank actuator 3110 to break the seal on the reagent tank 3105 containing the nucleic acid amplification reagent (PCR buffer, e.g., 10 μL of Globalfiler PCR kit reagent) and introduce the eluate into the nucleic acid amplification chamber 1302 (S2802). The control device then operates the valve actuator 3111 to open all of V1 to V4 to remove any air bubbles from the nucleic acid amplification chamber 1302, then closes all of V1 to V4 and stores 20 μL of reaction solution (2 μL of eluate and 18 μL of nucleic acid amplification reagent) in the nucleic acid amplification chamber 1302 (S2803). The control device then operates the temperature control mechanism 3302 to set the surface temperature of the nucleic acid amplification unit heat source 3301 to 95°C, and maintains this temperature for one minute once the nucleic acid extraction chamber 1203 reaches (detects) 95°C (S2804). This activates the polymerase contained in the nucleic acid amplification reagent, allowing the nucleic acid amplification reaction to begin. Next, the control device operates the temperature control mechanism 3302 to change the surface temperature of the nucleic acid amplification unit heat source 3301 to 94°C, and maintains this temperature for 10 seconds once the nucleic acid extraction chamber 1203 reaches 94°C, allowing the DNA denaturation reaction to occur (S2805). Next, the control device operates the temperature control mechanism 3302 to change the surface temperature of the nucleic acid amplification unit heat source 3301 to 59°C, and when the nucleic acid extraction chamber 1203 reaches (detects) 59°C, maintains this temperature for 90 seconds to perform an annealing / DNA extension reaction (S2806). The control device repeats the processes of S2805 and S2806 30 cycles to amplify the target nucleic acid (STR) to a product concentration detectable by the capillary electrophoresis device. After performing the processes of S2805 and S2806 30 cycles, the control device continues to maintain the temperature at 59°C for 10 minutes to perform adenylylation (S2807).Finally, the control device operates the temperature control mechanism 3302 to change the surface temperature of the nucleic acid amplification unit heat source 3301 to 4°C, thereby stopping the nucleic acid amplification reaction (S2808). At the same time, the control device operates the valve actuator 3111 to open V1-V2 and V4 and close V3, and further operates the liquid delivery mechanism 302 to transport the reaction solution from the nucleic acid amplification chamber 1302 to the amplified product recovery channel 1303 (S2809).

[0103] <STR analysis process by electrophoresis> The reaction solution transported to the amplification product recovery channel 1303 is discharged to the outside through the second vent 108 and mixed with an electrophoresis reagent (e.g., formamide solution) at a fixed ratio (e.g., 20 μL of reaction solution to 200 μL of electrophoresis reagent). This mixed solution is used as a sample solution and can be placed in an electrophoresis device for STR analysis.

[0104] Note that, similarly to the nucleic acid extraction unit 1201 to the nucleic acid amplification unit 1301, the electrophoresis reagent may be stored in a reagent tank 3105 and placed on the substrate 102, and the process up to mixing of the reaction solution and the electrophoresis reagent may be performed in the flow channel device 101. Furthermore, the electrophoresis unit may be configured on the flow channel device 101, and the process from sample introduction to electrophoresis analysis may be performed fully automatically.

[0105] (5) Modifications of the membrane containing section 104 and the resin ring 204 Figures 29A to 29E are diagrams showing modified examples of the membrane containing section 104 and the resin ring 204 according to this embodiment. Figure 29A is a diagram showing an example of a configuration of the periphery of the membrane containing section 104 in each modified example as seen from above. Figures 29B to 29E are diagrams showing example cross-sectional configurations of modified examples 1 to 4 taken along line A-A in Figure 29A.

[0106] 29A , in each of the modified examples, resin ring 204 preferably has a continuous circular ring shape when viewed from above. This allows the periphery of purification membrane 201 to be securely fixed to base surface 109 without leakage around the entire periphery, minimizing leakage of the solution. For the same reason, base surface 109 is also preferably a continuous circular ring shape. However, experiments have shown that the effects of the technology disclosed herein can be sufficiently achieved even with a C-shaped base surface 109 that is cut out by approximately 1 / 5 or less of the circumference.

[0107] 29B to 29E, resin ring 204 may be used, which has a circular cross section in the vertical direction of the ring (a doughnut-shaped cross section in the horizontal direction), like a typical O-ring. However, in this case, it is preferable that resin ring 204 and purification membrane 201, and resin ring 204 and the side wall of membrane containment upper section 110, contact each other over as large a surface area as possible. This is because the larger the contact area between resin ring 204 and purification membrane 201, the more likely it is that purification membrane 201 will be prevented from lifting, and because the frictional force acting between resin ring 204 and membrane containment upper section 110 increases in proportion to the contact area.

[0108] One preferred modification is a resin ring 204 whose cross section taken from the vertical direction of the ring is tilted inward, as shown in Fig. 29B. In the shape of the resin ring 204 shown in Fig. 29B, the contact areas between the resin ring 204 and the purification membrane 201 and between the resin ring and the upper part of the membrane containment section 110 are the same as those of the resin ring 204 according to the above-described embodiment (Fig. 9). Therefore, a high purification membrane fixing effect can be obtained, and at the same time, the amount of liquid remaining on the upper surface of the resin ring 204 can be minimized, resulting in a higher yield.

[0109] FIG. 29C shows a modified example using a resin ring 204 with tapered shapes at the top and bottom. As shown in FIG. 29C, the upper taper 2001 of the resin ring 204 is inclined inward toward the center of the purification membrane 201. This, similar to the modified example shown in FIG. 29B, is expected to reduce residual liquid and improve yield. The lower taper 2002 is provided on the outer periphery of the purification membrane 201. This facilitates insertion of the resin ring 204 into the membrane containment upper section 110 during assembly. The resin ring 204 (embodiment and each modified example) according to the present disclosure is characterized by having an inner diameter equal to or larger than the membrane containment upper section 110. This can make positioning and fitting of the resin ring 204 difficult during assembly. Therefore, by providing the lower taper 2002, the outer diameter of the bottom of the resin ring 204 is reduced, making it easier to fit into the membrane containment upper section 110 when fitting begins, facilitating correct press-fitting and fixation.

[0110] Another modified example shown in FIG. 29D relates to a resin ring 204 having a notch 2003 at the connection between the first flow path 105 and the upper membrane housing section 110. The notch 2003 is preferably designed and positioned so that the top surface of the notch 2003 is lower than the bottom surface of the first flow path 105 and the sidewall of the notch 2003 is positioned outside the sidewall surface of the first flow path 105, so as not to block the first flow path 105. A resin ring 204 with a C-shaped top surface (a predetermined percentage of the upper portion of the resin ring 204; a notch is provided at the connection portion with the first flow path 105) and an O-shaped bottom surface (a predetermined percentage of the lower portion of the resin ring 204) as shown in FIG. 29D can reduce the effective volume of the upper membrane housing section 110. This minimizes the amount of air that acts as a damper during liquid transfer, thereby more effectively increasing the liquid transfer pressure. This not only enables the liquid delivery mechanism 302 to be miniaturized by reducing the required pressure, but also improves liquid delivery controllability, enabling more stable automatic nucleic acid purification processing.

[0111] Another modified example shown in FIG. 29E relates to a membrane containment section 104 in which the membrane containment section upper portion 110 and the membrane containment section lower portion 111 are tapered. In this case, it is preferable that the resin ring 204 also has a tapered shape that allows it to be press-fitted into the membrane containment section upper portion 110. The tapered shape of the membrane containment section upper portion 110 and the resin ring 204 has the effect of making it easier to stably fit the resin ring 204 during assembly, similar to the lower taper 2002 shown in FIG. 29C. At the same time, the contact area between the resin ring 204 and the membrane containment section upper portion 110 is larger than when the sidewall of the membrane containment section upper portion 110 is nearly vertical. This results in a higher press-fitting effect. On the other hand, tapering the membrane containment section lower portion 111 reduces the amount of solution remaining in the membrane containment section lower portion 111 while maintaining the effective area of ​​the purification membrane 201, allowing the solution that has passed through the purification membrane to be efficiently transported to the second flow path 106, thereby improving recovery efficiency.

[0112] The characteristic configurations of the modified examples shown in Figures 29B to 29E can also be combined. By appropriately combining them, high purification membrane immobilization strength and recovery efficiency can be achieved. For example, it is preferable to determine an appropriate shape taking into consideration the materials of the substrate 102 and resin ring 204, processing distortion, and the material and size of the purification membrane 201. This membrane immobilization method can also be used in combination with conventional membrane immobilization methods. For example, the small gap between the press-fitted resin ring 204 and the side wall of the membrane storage section upper part 110 can be filled with adhesive. This minimizes defects due to processing errors and surface roughness that occur during material processing, etc., and achieves high-performance nucleic acid purification at a practical level suited to the application.

[0113] (6) Application of the continuous transport method in a system employing the configuration of the flow channel device 101 of the present disclosure (i) Here, a nucleic acid purification process and sample pretreatment system employing the continuous transport method will be described. The composition of the sample solution 401 used in the continuous transport method is as shown in FIG. 2A, and pure ethanol is used as the cleaning solution 402. The sample pretreatment system 301 and the flow channel device 101 are generally identical in configuration to those shown in FIG. 20. FIG. 30A is a diagram showing how the sample solution 401 is transported in the continuous transport method. FIG. 30B is a diagram showing how the cleaning solution 402 is transported in the continuous transport method.

[0114] 30A and 30B, in the continuous transport method, the sample solution 401 is not completely transported to the second flow path 106, and the rear end of the sample solution 401 stops at a liquid transport stop point 1104 located midway through the sample solution flow path 1102. This liquid transport control is what makes it different from that shown in FIG. 20. Note that the liquid transport stop point 1104 only needs to be able to detect that the rear end of the sample solution 401 has reached a predetermined position, and for example, a liquid level detection sensor or a camera can be placed on the upper surface of the liquid transport stop point 1104.

[0115] Next, with reference to Figures 30A and 30B, a brief description of the nucleic acid purification process using the continuous transport method will be given. First, the control device (not shown) transports sample solution 401 from first reagent tank 3105 through purification membrane 201 installed in membrane containment unit 104 to second flow path 106, and stops the transport the moment the rear end of sample solution 401 reaches flow stop point 1104 (Figure 30A). Next, the control device discharges cleaning solution 402 from second reagent tank 3105, and transports sample solution 401 continuously through purification membrane 201 to second flow path 106 (Figure 30B). The subsequent nucleic acid purification process is the same as the steps described in Figure 22.

[0116] (ii) When the purification membrane fixing method using the resin ring 204 according to the present disclosure is applied, the gap between the purification membrane 201 and the membrane housing 104 becomes extremely small, suppressing the passage of liquid and gas. Therefore, it has been experimentally determined that the liquid delivery pressure required for solution transport using the technology of the present disclosure is greater than when the technology of the present disclosure is not implemented. For example, when the configuration shown in Figures 4A and 4B is applied, the maximum is approximately 20 kPa, and when the configuration shown in Figures 9A and 9B is applied, the maximum is approximately 120 kPa. In this case, the highest liquid delivery pressure is required in the process of starting to flow the cleaning solution 402 after the sample solution 401 has been completely drained. This is because, when air reaches the periphery of the purification membrane 201 while the membrane is wet with the sample solution 401, the air cannot pass through the purification membrane 201 until the Laplace pressure is exceeded, and therefore, high pressure is required to allow the cleaning solution 402 to reach the purification membrane 201.

[0117] To suppress such high liquid delivery pressures, the technology disclosed herein can employ a continuous delivery method, which reduces the liquid delivery pressure by continuously delivering two solutions that meet certain conditions without air between them. Experiments have shown that because the sample solution 401 and the cleaning solution 402 are primarily water and ethanol, respectively, applying the continuous delivery method is likely to reduce the liquid delivery pressure. Experiments have also shown that it is preferable for the second liquid (cleaning solution 402) to have a faster evaporation rate than the first liquid (sample solution 401) and a lower surface tension than the first liquid. In fact, when the nucleic acid purification process shown in Figures 30A and 30B was performed, it was confirmed that the maximum liquid delivery pressure was approximately 40 kPa and that high purification efficiency could be achieved even with the flow channel device 101 disclosed herein. Thus, the purification membrane immobilization method using the resin ring 204 disclosed herein achieves high-yield, highly robust nucleic acid purification, while enabling low-pressure liquid delivery using the continuous delivery method, thereby enabling high-performance nucleic acid purification with a compact, low-cost mechanism.

[0118] (7) Summary (i) The flow channel device 101 of this embodiment includes a purification membrane 201 formed on a substrate 102 and capable of recovering nucleic acids, a membrane storage section 104 which is a space formed in the substrate 102 and is connected to a first flow channel 105 and a second flow channel 106 formed on the substrate and which stores the purification membrane 201, and an annular resin ring 204 press-fitted into the membrane storage section 104. The membrane storage section 104 also has a pedestal surface 109 formed between the upper and lower surfaces of the substrate 102 so that the purification membrane 201 can be placed thereon, and is divided by the pedestal surface 109 into a cylindrical membrane storage section upper section 110 and a membrane storage section lower section 111 which has a smaller diameter than the membrane storage section upper section 110. The first flow path 105 and the second flow path 106 include the first flow path 105, which is connected to at least one valve mechanism, formed on the upper surface of the substrate 102, and connected to the sidewall of the membrane containment upper part 110, and the second flow path 106, which is connected to at least one valve mechanism, formed on the lower surface of the substrate 102, and connected to the membrane containment lower part 111. The resin ring 204 has an outer diameter equal to or larger than the inner diameter of the membrane containment upper part 110. The purification membrane 201 is fixed between the base surface 109 and the resin ring 204. The outer diameter of the pressed-in resin ring 204 is smaller than the outer diameter of the resin ring 204 when not contained in the membrane containment part 104, and the inner diameter of the membrane containment upper part 110 is approximately the same as the inner diameter of the membrane containment upper part 110 when the resin ring 204 is not pressed-in into the membrane containment part. Furthermore, at least at the position where the sidewall of the membrane containment upper portion 110 and the pressed-in resin ring 204 are closest to each other, the inner diameter of the membrane containment upper portion 110 and the outer diameter of the pressed-in resin ring 204 are the same. Here, when the resin ring 204 is pressed into the membrane containment portion 104, it undergoes plastic deformation and is held in place by the frictional force between the sidewall of the membrane containment upper portion 110 and the pressed-in location. In other words, when pressed into the membrane containment portion 104, the resin ring 204 does not elastically deform like a well-known O-ring. The resin ring 204 is made of a material with a Young's modulus of 5 MPa or more and 1 GPa or less. The Young's modulus of the material making up the resin ring 204 may be less than the Young's modulus of the material making up the substrate 102. Furthermore, the elastic modulus of the resin ring is 100 kPa or more. Meanwhile, the elastic strain limit value of the resin ring 204 is less than 100%.More specifically, when the inner diameter of the membrane containment upper section 110 in the assembled state of the flow channel device 101 is R' and the outer diameter of the pressed-in resin ring 204 in the assembled state of the flow channel device 101 is a', |R'-a'|<1 μm is satisfied at the position where the side wall of the membrane containment upper section 110 and the pressed-in resin ring 204 are closest to each other. On the other hand, when the inner diameter of the membrane containment upper section 110 in the unassembled state of the flow channel device 101 (the state before the resin ring is pressed-in) is R and the outer diameter of the resin ring (before press-in) 204 in the unassembled state of the flow channel device 101 is a, 0≦a-R<400 μm is satisfied. By configuring the flow channel device 101 in this manner, a low-cost flow channel device (flow channel chip) 101 with a simple configuration can be realized. Furthermore, by utilizing this flow channel device 101, highly efficient purification equivalent to laboratory methods and highly sensitive genetic analysis can be performed in a fully automated manner.

[0119] The inner diameter of the base surface 109 can be made substantially the same as the inner diameter of the resin ring 204. This allows the solution that has come into contact with the purification membrane 201 to pass efficiently through the membrane housing lower part 111, maximizing the amount of solution that can be recovered.

[0120] It is preferable to make the difference between the inner diameter of the membrane housing upper part 110 and the inner diameter of the membrane housing lower part 111, and / or the difference between the outer diameter and inner diameter of the resin ring 204 as small as possible, preferably 2.5 mm or less. This prevents the solution from being trapped and remaining on the upper surface of the resin ring 204, making it possible to recover the solution more efficiently. Note that the difference between the outer diameter and inner diameter of the resin ring 204 may be 1 mm or less.

[0121] Furthermore, the pressed-in resin ring 204 and the side wall of the membrane containment section upper portion 110, and the pressed-in resin ring 204 and the purification membrane 201 are both in surface contact. It is preferable that the respective surfaces be in contact over as large a surface area as possible. As a result, the larger the contact area between the resin ring 204 and the purification membrane 201, the more the floating of the purification membrane 201 can be suppressed, and a higher pressing effect can be obtained.

[0122] Furthermore, the pressed-in resin ring 204 is configured so that the end face opposite to the face where the pressed-in resin ring 204 and the purification membrane 201 come into contact is located lower than the connection between the first flow path 105 and the side wall of the membrane containment upper part 110. This prevents the transported solution from remaining in the gap between the resin ring 204 and the membrane containment part 104. In addition, the flow path resistance can be reduced, allowing trace amounts of solution to be recovered without loss.

[0123] (ii) This embodiment also proposes a nucleic acid purification method for producing nucleic acids using the flow path device 101. As shown in Fig. 17 , for example, this method includes the steps of connecting a sample tank (reagent tank 3105) containing a sample solution (sample solution) to the first flow path 105 of the flow path device 101, connecting the second flow path 106 to a waste liquid tank 3106, operating the liquid delivery mechanism 302 to transport the sample solution from the sample tank to the waste liquid tank 3106 via the first flow path 105, the purification membrane 201, and the second flow path 106, connecting the first flow path 105 to a washing liquid tank (reagent tank 3105 holds washing liquid 402) containing washing liquid for washing the purification membrane 201, and operating the valve mechanism to transport the sample solution from the washing liquid tank to the first flow path 105, the purification membrane 201, and the second flow path 106. 1 and second flow path 106 to waste liquid tank 3106, a step of operating liquid delivery mechanism 302 to blow dry air from first flow path 105 through purification membrane 201 toward second flow path 106, a step of connecting first flow path 105 via a valve mechanism to an elution liquid tank (reagent tank 3105 holds the elution liquid) that contains an elution liquid for eluting nucleic acid, a step of connecting second flow path 106 to solution recovery tank 3107, and a step of operating liquid delivery mechanism 302 to deliver the elution liquid from the elution liquid tank to solution recovery tank 3107 via first flow path 105, purification membrane 201, and second flow path 106. In this manner, nucleic acid can be efficiently purified and highly sensitive genetic analysis can be performed.

[0124] 30A and 30B and 22, this embodiment also proposes another nucleic acid purification method. This method includes the steps of preparing at least a first liquid tank (reagent tank 3105) containing a first liquid (specimen solution: sample solution 401) and a second liquid tank (washing liquid tank) containing a second liquid (washing liquid 402), connecting the first flow path 105 of the flow path device 101 to a first liquid delivery flow path (sample solution flow path 1102) connected to the first liquid tank, connecting the first flow path 105 of the flow path device 101 to a second liquid delivery flow path (washing liquid flow path) connected to the second liquid tank, connecting the second flow path 106 of the flow path device 101 to a waste liquid tank 3106, and operating the liquid delivery mechanism 302 to deliver the first liquid from the first liquid delivery tank to the first flow path 105, the purification membrane 201, and second flow path 106 to waste liquid tank 3106; operating liquid delivery mechanism 302 to stop delivery of the first liquid before the final end of the first liquid reaches the confluence of the first liquid delivery flow path and the first flow path (at liquid delivery stop point 1104) so ​​that the first liquid remains in the first delivery flow path; and operating liquid delivery mechanism 302 while the first liquid remains in the first delivery flow path to start delivery of the second liquid from the second liquid tank to the second liquid delivery flow path, and transporting the second liquid to waste liquid tank 3106 via the confluence of the second liquid delivery flow path and the first flow path, first flow path 105, purification membrane 201, and second flow path 106, and the flow into membrane storage section 104 is carried out continuously without any air being trapped between the first liquid and the second liquid.

[0125] Furthermore, the preparation step of the method further includes the step of preparing a third liquid tank (reagent tank (Elution buffer) 3105) that contains an elution solution for eluting nucleic acids, and further includes the steps of operating liquid delivery mechanism 302 to blow dry air from first flow path 105 through purification membrane 201 toward second flow path 106, and operating liquid delivery mechanism 302 to transport the elution solution from the elution solution tank to solution recovery tank 3107 through first flow path 105, purification membrane 201, and second flow path 106. This reduces the liquid delivery pressure. As a result, the flow path device 101 is not damaged by the transport of the solution.

[0126] (iv) This embodiment proposes a sample pretreatment system 301 including a flow path device 101 according to this embodiment, a nucleic acid extraction unit 1201 connected to the first flow path 105 of the flow path device 101 and extracting nucleic acids from a biological sample, and a nucleic acid amplification unit 1301 connected to the second flow path 106 of the flow path device 101 and amplifying nucleic acids (see Figures 23 to 27A and B).

[0127] (v) The technology according to the present disclosure can achieve various functions by appropriately combining multiple components disclosed in the present embodiment. For example, some components may be omitted from all the components shown in the embodiment, or components from different specific examples may be appropriately combined.

[0128] As described above, the present disclosure has been described using specific examples, which are not intended to be limiting in all respects but are for the purpose of explanation (understanding the technology of the present disclosure). Furthermore, those skilled in the art will appreciate that other implementations of the present disclosure may become apparent from consideration of the present embodiments. It should be noted that the specification and specific examples are merely exemplary, and the scope and spirit of the technology of the present disclosure are defined by the following claims.

[0129] DESCRIPTION OF SYMBOLS 101 Flow path device 102 Substrate 103 Sealing tape 104 Membrane housing 105 First flow path 106 Second flow path 107 First vent 108 Second vent 109 Base surface 110 Upper part of membrane housing (diameter R, depth D) 111 Lower part of membrane housing (diameter r, depth d) 201 Purification membrane (diameter P, thickness Q) 202 O-ring 203 Adhesive 204 Resin ring (outer diameter a, inner diameter b, height c) 205 Porous holding member 301 Sample pretreatment system 302 Liquid delivery mechanism 303 Reagent holding part 304 Chip holding part 305 Cover 306 Fitting 307 Piping tube 400 Sample 401 Sample solution 402 Cleaning solution 403 Elution solution 1001 Connecting flow path 1002 Valve section 1003 Sealing member 1004 Protrusion 1005 Reagent sealing port 1006 Vent 1101 Nucleic acid purification section 1102 Sample solution flow path 1103 Cleaning solution flow path 1104 Liquid delivery stop point 1105 Nucleic acid purification / amplification section 1201 Nucleic acid extraction section 1202 Sample inlet 1203 Nucleic acid extraction chamber 1204 Purification section connecting flow path 1205 Liquid delivery flow path 1206 Inlet sealing member 1301 Nucleic acid amplification section 1302 Nucleic acid amplification chamber 1303 Amplification product recovery flow path 2001 Upper taper 2002 Lower taper 2003 Notch 3001 First connection port 3002 Second connection port 3003 Liquid delivery power source 3004 Pressure control mechanism 3005 Open / close valve mechanism 3006 Multi-way valve mechanism 3007 Atmospheric release channel 3101 First liquid supply port 3102 Second liquid supply port 3103 First device connection port 3104 Second device connection port 3105 Reagent tank 3106 Waste liquid tank 3107 Solution recovery tank 3108 Solution recovery channel 3109 Vent filter 3110 Reagent tank actuator 3111 Valve actuator 3201 Nucleic acid extraction unit heat source 3202, 3302 Temperature control mechanism 3301 Nucleic acid amplification unit heat source

Claims

1. A flow path device having a flow path formed on a substrate, comprising: a purification membrane capable of recovering nucleic acid; a membrane storage section which is a space formed in the substrate and connected to the flow path and stores the purification membrane; and an annular resin ring pressed into the membrane storage section, the membrane storage section having a base surface formed between the upper and lower surfaces of the substrate so that the purification membrane can be installed, and the base surface is used as a boundary to separate the membrane storage section into a cylindrical upper membrane storage section and a lower membrane storage section having a smaller diameter than the upper membrane storage section, the flow path including: a first flow path which is connected to at least one valve mechanism, formed on the upper surface of the substrate, and connected to a side wall of the upper membrane storage section; and a second flow path which is connected to at least one valve mechanism, formed on the lower surface of the substrate, and connected to the lower membrane storage section, the resin ring having an outer diameter equal to or larger than the inner diameter of the upper membrane storage section, the purification membrane being fixed between the base surface and the resin ring, A flow path device in which the outer diameter of the pressed-in resin ring is smaller than the outer diameter of the resin ring when it is not stored in the membrane storage section, and the inner diameter of the upper part of the membrane storage section is approximately the same as the inner diameter of the upper part of the membrane storage section when the resin ring is not pressed into the membrane storage section, and the inner diameter of the upper part of the membrane storage section and the outer diameter of the pressed-in resin ring are the same at least at the position where the side wall of the upper part of the membrane storage section and the pressed-in resin ring are closest to each other.

2. The flow path device according to claim 1, wherein the resin ring is made of a material having a Young's modulus of 5 MPa or more.

3. A flow path device according to claim 2, wherein the resin ring is made of a material having a Young's modulus of 1 GPa or less.

4. A flow path device according to claim 1, wherein the elastic modulus of the resin ring is 100 kPa or more.

5. A flow path device according to claim 1, wherein the resin ring has an elastic strain limit value of less than 100%.

6. A flow path device as claimed in claim 1, wherein, when the inner diameter of the upper part of the membrane storage section when the flow path device is assembled is R' and the outer diameter of the pressed-in resin ring when the flow path device is assembled is a', at the position where the side wall of the upper part of the membrane storage section and the pressed-in resin ring are closest to each other, the relationship |R'-a'|<1 μm is satisfied.

7. A flow path device according to claim 1, wherein, when the inner diameter of the upper part of the membrane housing section in the unassembled state of the flow path device is R and the outer diameter of the resin ring in the unassembled state of the flow path device is a, the relationship 0≦a-R<400 μm is satisfied.

8. The flow path device according to claim 1, wherein the inner diameter of the seat surface is substantially the same as the inner diameter of the resin ring.

9. A flow path device according to claim 1, wherein the difference between the inner diameter of the upper part of the membrane containing section and the inner diameter of the lower part of the membrane containing section is 2.5 mm or less.

10. A flow path device according to claim 9, wherein the difference between the outer diameter and the inner diameter of the resin ring is 1 mm or less.

11. A flow path device according to claim 1, wherein the pressed-in resin ring and the side wall of the upper part of the membrane containment section, and the resin ring and the purification membrane, are in surface contact with each other.

12. A flow path device as claimed in claim 1, wherein the end face of the pressed-in resin ring opposite the surface where the pressed-in resin ring contacts the purification membrane is positioned lower than the connection between the first flow path and the side wall of the upper part of the membrane storage section.

13. A flow path device according to claim 1, wherein the Young's modulus of the material constituting the resin ring is less than the Young's modulus of the material constituting the substrate.

14. A flow path device as claimed in claim 1, wherein a film sheet is attached to the upper and lower surfaces of the substrate, and at least a portion of the openings on the upper or lower surface of the substrate of the first flow path, the second flow path and the membrane storage section are blocked.

15. A flow path device according to claim 1, wherein the pressed-in resin ring is fixed to the seat surface of the membrane housing portion by an adhesive.

16. A flow path device having a flow path formed on a substrate, comprising: a purification membrane capable of recovering nucleic acid; a membrane storage section which is a space formed in the substrate and connected to the flow path and stores the purification membrane; and an annular resin ring pressed into the membrane storage section, wherein the membrane storage section has a base surface formed between the upper and lower surfaces of the substrate so that the purification membrane can be installed, and is divided by the base surface into a cylindrical membrane storage section upper section and a membrane storage section lower section having a smaller diameter than the membrane storage section upper section, wherein the flow path includes a first flow path formed on the upper surface of the substrate and connected to a side wall of the membrane storage section upper section, and a second flow path formed on the lower surface of the substrate and connected to the membrane storage section lower section, wherein the resin ring has an outer diameter equal to or larger than the inner diameter of the membrane storage section upper section, and the purification membrane is fixed between the base surface and the resin ring, A flow path device in which the outer diameter of the pressed-in resin ring is smaller than the outer diameter of the resin ring when it is not stored in the membrane storage section, and the inner diameter of the upper part of the membrane storage section is approximately the same as the inner diameter of the upper part of the membrane storage section when the resin ring is not pressed into the membrane storage section, and the inner diameter of the upper part of the membrane storage section and the outer diameter of the pressed-in resin ring are the same at least at the position where the side wall of the upper part of the membrane storage section and the pressed-in resin ring are closest to each other.

17. A method for purifying nucleic acid using the flow path device of claim 1, comprising the steps of: connecting the first flow path to a sample tank that contains a sample solution; connecting the second flow path to a waste liquid tank; operating a liquid delivery mechanism to transport the sample solution from the sample tank to the waste liquid tank via the first flow path, the purification membrane, and the second flow path; connecting the first flow path via the liquid delivery mechanism to a washing liquid tank that contains a washing liquid for washing the purification membrane; operating the liquid delivery mechanism to transport the washing liquid from the washing liquid tank to the waste liquid tank via the first flow path, the purification membrane, and the second flow path; operating the liquid delivery mechanism to blow dry air from the first flow path through the purification membrane in the direction of the second flow path; connecting the first flow path to an elution liquid tank that contains an elution liquid for eluting the nucleic acid; and connecting the second flow path to a recovery tank. and operating the liquid delivery mechanism to transport the eluate from the eluate tank through the first flow path, the purification membrane, and the second flow path to the recovery tank.

18. A method for purifying nucleic acid, which produces nucleic acid using the flow path device of claim 1, comprising the steps of: preparing at least a first liquid tank for accommodating a first liquid and a second liquid tank for accommodating a second liquid; connecting the first flow path of the flow path device to a first liquid delivery flow path leading to the first liquid tank; connecting the first flow path of the flow path device to a second liquid delivery flow path leading to the second liquid tank; connecting the second flow path of the flow path device to a waste liquid tank; operating a liquid delivery mechanism to transport the first liquid from the first liquid tank through the first flow path, the purification membrane, and the second flow path to the waste liquid tank; operating the liquid delivery mechanism to stop the transportation of the first liquid before a final end of the first liquid reaches a junction of the first liquid delivery flow path and the first flow path, so that the first liquid remains in the first liquid delivery flow path. and operating the liquid delivery mechanism while the first liquid remains in the first liquid delivery flow path to start delivery of the second liquid from the second liquid tank to the second liquid delivery flow path, and transporting the second liquid to the waste liquid tank via a junction of the second liquid delivery flow path and the first flow path, the first flow path, the purification membrane, and the second flow path, wherein the flow into the membrane housing is carried out continuously from the first liquid to the second liquid without trapping air.

19. A method for purifying nucleic acid according to claim 18, wherein the first liquid is a sample solution, and the second liquid is a washing liquid, and the preparing step further includes a step of preparing a third liquid tank for containing an elution liquid for eluting the nucleic acid, and further includes a step of operating the liquid delivery mechanism to blow dry air from the first flow path through the purification membrane in the direction of the second flow path, a step of connecting the second flow path of the flow path device to a recovery tank for recovering the elution liquid, and a step of operating the liquid delivery mechanism to transport the elution liquid from the third liquid tank to the recovery tank via the first flow path, the purification membrane, and the second flow path.

20. A sample pretreatment system comprising: a flow path device according to claim 1; a nucleic acid extraction unit connected to the first flow path of the flow path device and configured to extract nucleic acid from a biological sample; and a nucleic acid amplification unit connected to the second flow path of the flow path device and configured to amplify the nucleic acid.

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