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

Abstract

In order to efficiently perform a process for removing impurities and purifying and recovering nucleic acid from a nucleic acid-containing sample, the present invention proposes a flow path device which is provided with a purification membrane capable of recovering nucleic acid, a membrane housing part that is formed in a space in a substrate, is connected to a flow path, and houses the purification membrane, and an annular resin ring pressed into the membrane housing part, the membrane accommodating part is provided with a pedestal surface which can be used for arranging the purification membrane between the upper surface and the lower surface of the substrate, and is divided into a cylindrical membrane accommodating part upper part and a membrane accommodating part lower part with the small diameter smaller than that of the membrane accommodating part upper part by taking the pedestal surface as a boundary line. The flow path includes a first flow path connected to the at least one valve mechanism, formed on an upper surface of the substrate, and connected to a side wall of an upper portion of the film housing portion, and a second flow path connected to the at least one valve mechanism, formed on a lower surface of the substrate, and connected to a lower portion of the film housing portion. The resin ring has an outer diameter greater than or equal to an inner diameter of the upper portion of the film housing portion. The purification membrane is fixed between the pedestal surface and the resin ring, the outer diameter of the pressed resin ring is smaller than the outer diameter of the resin ring in a state in which the resin ring is not accommodated in the membrane accommodating part, and the inner diameter of the upper part of the membrane accommodating part is substantially the same as the inner diameter of the upper part of the membrane accommodating part in a state in which the resin ring is not accommodated in the membrane accommodating part. The inner diameter of the upper part of the film accommodating part is the same as the outer diameter of the pressed-in resin ring at least at a position where the side wall of the upper part of the film accommodating part is closest to the pressed-in resin ring (referring to Figure 9A and Figure 9B).

Description

Technical Field

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

[0002] With rapid technological advancements, such as improvements in analytical accuracy and the generalization of technology, genetic analysis technology has been utilized in a variety of fields, including in vitro diagnostics, forensics, and environmental measurement. While there are various methods and analysis targets for genetic testing, the typical analytical process consists of collecting a specimen, preparing a solution (sample) to extract genes from the specimen, purifying the sample to remove reaction-inhibiting substances, amplifying the target gene using a gene amplification reaction, detecting the amplified product, and analyzing the detection signal. Generally, collected specimens are transported to specialized facilities equipped with benchtop genetic analysis equipment and specialized instruments, and subsequent processing is performed manually by skilled technicians (laboratory-based methods).

[0003] In recent years, there has been a growing demand for sample-to-answer genetic analysis systems that utilize genetic pretreatment and detection devices and disposable, dedicated cartridges to fully automate everything from sample introduction to measurement and data analysis, with the goal of simplifying, speeding up, and reducing costs. For example, Patent Document 1 proposes a fully automated DNA-based identification 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] The method for purifying nucleic acid utilizes organic phase solidification method or uses the solid phase extraction method of porous membrane, magnetic beads. Especially, solid phase extraction method is widely used because many commercially available test kits and purification efficiency are also good. Solid phase extraction method is a method for combining nucleic acid with a solid carrier and recovering it. The carrier uses a porous membrane made of silicon dioxide, glass fiber, magnetic beads coated with silicon dioxide, etc. Generally speaking, when carrying out purification treatment with manual operation, the centrifugal column method of placing solution in the porous membrane arranged on the centrifugal column shown in patent document 2 is mostly adopted, and the solution is carried by centrifugal operation. This is because it can be implemented by general laboratory equipment, and the operation is relatively simple.

[0005] On the other hand, when applied to automated systems, a magnetic bead method is mostly used, which enables solution exchange by aggregating magnetic beads on a magnet without the need for centrifugation. For example, non-patent document 1 discloses a method for automating gene extraction and purification steps using a magnetic bead method. Furthermore, non-patent document 2 discloses a nucleic acid purification method that is not limited to the magnetic bead method but uses a flow chip. In particular, the Boom method is widely used as a method for efficiently and robustly purifying nucleic acids using a simple process. The Boom method is a DNA purification technology that utilizes the effect of silica firmly binding to DNA in the presence of a chaotropic salt. The Boom method is applied to a large number of dissolution purification kits for the centrifugal column method and the magnetic bead method, as well as nucleic acid purification on a flow chip.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent No. 5815572

[0009] Patent Document 2: Japanese Patent Application Laid-Open No. 2014-030397

[0010] Patent Document 3: Japanese Patent Application No. 2002-512688

[0011] Patent Document 4: Japanese Patent Application Laid-Open No. 2005-257647

[0012] Patent Document 5: Japanese Patent Application Laid-Open No. 2016-63475

[0013] Patent Document 6: Japanese Patent Application Laid-Open No. 2007-111653

[0014] Patent Document 7: Japanese Patent Application Publication No. 2019-144164

[0015] Patent Document 8: Japanese Patent Application Laid-Open No. 2010-257691

[0016] Patent Document 9: Japanese Patent Application Laid-Open No. 2002-39984

[0017] Patent Document 10: International Publication No. 2022 / 030605

[0018] Non-patent literature

[0019] Non-patent document 1: "Microfluidic Devices for Nucleic Acid(NA)Isolation, Isothermal NA Amplification, and Real-Time Detection," Methods Mol. Biol. 1256, 15-40 (2015).

[0020] Non-patent document 2: "Magnetic particles for integrated nucleic acid purification, amplification and detection without pipetting," TrendsAnalyt.Chem. 127: 115912 (2020).

[0021] Non-patent document 3: “Purification of Nucleic Acids in Microfluidic Devices,” Anal. Chem. 80, 6472-6479 (2008) Summary of the Invention

[0022] Problems to be solved by the invention

[0023] In addition to requiring a magnet and a driving circuit for the magnet to recover the beads, the magnetic bead method also requires a mechanism for accurately transporting the beads during the multi-stage analysis process. Therefore, there are issues with the complexity of the structure of the device and the flow path chip, and the increased cost. For example, in the case of a microcolumn or overall structure that is provided on the flow path to form a binding carrier, as shown in Non-Patent Document 1, the flow path chip needs to be specially processed, and thus the manufacturing cost of the flow path chip increases.

[0024] Patent Document 1, Patent Document 3, and Non-Patent Document 3, mentioned above, disclose examples of nucleic acid purification within a flow channel chip containing a membrane-like carrier. However, none of these documents explore the application of high-sensitivity analysis, which allows for sensitive examination of even small amounts of samples (e.g., a level suitable for forensic specimens (e.g., remains left at crime scenes)). While it is well known that the amount and purity of nucleic acids recovered from a sample significantly influence the accuracy and sensitivity of the analysis, there are no known examples that address the purification efficiency of these techniques.

[0025] One of the problems with using a membrane-like carrier on a flow chip to implement efficient nucleic acid purification is that the purification efficiency is low compared to laboratory methods and flow chip purification methods using carriers other than membranes. This is a new topic that has been clarified for the first time through the research of the inventors of this application. In addition, the main cause of this topic is the leakage of the solution outside the membrane, which has also been clarified for the first time through the research of the inventors of this application. In addition, as described below, the discovery process and details of this topic will be clarified through the records in the embodiment and the accompanying drawings.

[0026] As one solution to this problem, for example, sealing and securing the purification membrane to prevent solution leakage is a possible solution. This approach, not limited to nucleic acid purification applications, is also a method for securing a thin film within a predetermined container so that liquids or gases can contact it without leakage. Patent Documents 4 to 8 exemplify membrane securing methods using gaskets such as O-rings and C-rings.

[0027] Patent document 4 discloses the following structure: an outer container sequentially accommodates a reinforcing plate, a breathable membrane, and an O-ring (rubber seal), and the O-ring is pressed toward the membrane direction by fitting an inner container from the upper part of the O-ring. When the structure of patent document 4 is applied to the above-mentioned high-sensitivity analysis purposes (for example, the inspection of forensic samples), the solution is attracted to the gap between the inner container and the outer container, or the gap between the reinforcing plate and the outer container, and leakage occurs. The solution absorbed into the gap cannot be recovered. This is because patent document 4 is intended to allow the passage of gas components and does not consider allowing the liquid to pass in the membrane direction. At the same time, when the structure of patent document 4 is intended to be set on the substrate of the flow path chip, it is necessary to assemble an outer container accommodating a membrane and an annular member, an inner container pressing the annular member, a lower cover body for setting the flow path, and at least four layers of substrates of an upper cover body pressing the lower cover body, so the chip is large and costly.

[0028] Patent document 5 shows the following structure: a C-ring is embedded in the groove of the embedded component, and on this basis, it is engaged with the outer container so that the solution does not leak out of the container and the liquid is continuously fed to the separation membrane. This is also the general method of using a gasket ring. When the structure of patent document 5 is applied to the above-mentioned high-sensitivity analysis (for example, the inspection of forensic samples), it can prevent the liquid from leaking out of the container. On the other hand, the liquid invades the gap between the outer container and the annular part, and the solution absorbed by the gap cannot be recovered. This is because patent document 5 is intended to continuously feed liquid and does not consider making a trace amount of solution flow and recovering it in the later stage. Moreover, like patent document 4, when the structure of patent document 5 is to be applied to a substrate-shaped chip device, the components of the chip become complicated, and it is still large and costly.

[0029] Patent document 6 shows the following structure: a filter is accommodated between two chip substrates formed with a flow path and a filter storage portion, and the filter is fixed by joining the chip substrates. The structure of Patent Document 6 must be a structure in which a flow path is provided in two chip substrates. Therefore, this structure cannot be applied to a flow path chip formed by laminating and joining thin and soft materials such as a film on a chip substrate. Therefore, it is considered to construct a diaphragm valve on a chip as shown by way of example in Patent Document 7. However, in this case, in order to set the valve, it is necessary to further set other bonding layers, and the layer structure of the chip becomes complicated. At the same time, in order to precisely control the bonding state, it is necessary to strictly control the bonding conditions and positioning accuracy. Therefore, the flow path shape that can be formed and the chip material are greatly restricted, and the manufacturing cost becomes high.

[0030] Patent Document 8 and Patent Document 9 disclose the following structure: by sequentially accommodating a breathable membrane, a sealing annular member, and a fixing member and pressurizing and deforming the annular member, the electrolyte contained in the device will not leak to the outside. This enables trace amounts of solution to be filled in the device without leaking. However, this structure is not intended for liquid delivery, so a flow path cannot be set. Furthermore, it is clear that even if a flow path is set in order to apply the technology to the above-mentioned high-sensitivity analysis purposes (for example, the inspection of forensic samples), unrecoverable liquid residue will still be generated.

[0031] Patent document 10 shows the following structure: a nucleic acid extraction membrane is set in a membrane storage container formed on a flow path chip, and the membrane is fixed by pressing a rubber O-ring with a cutout provided in a manner that does not block the flow path, so that a certain amount of liquid passes through the membrane. Through research by the present inventors, it is clear that when the structure of patent document 10 is applied to the above-mentioned high-sensitivity analysis application (for example, the examination of forensic samples), the purification efficiency is low and the amount of recovered liquid is significantly reduced. Furthermore, through research by the present inventors, it is clear that the sealing and fixing force of the membrane in the structure of patent document 10 is insufficient for the application of high-sensitivity analysis, and the phenomenon of recovered liquid volume is caused by liquid residue generated by the cutout. Because the O-ring of patent document 8 is elastically deformed, the cutout portion is more easily deformed than the surrounding area and deforms unevenly. At this time, if the cutout is small, the flow path is narrower, thereby increasing the flow path resistance and impairing the liquid supply controllability. On the other hand, if the cutout is large, the cutout portion deforms in a more significantly flattened manner, so that the flow path becomes too narrow. At the same time, liquid residue is likely to form in the cutout. Furthermore, due to the O-shaped cross-section of the annular member, liquid easily intrudes into the gap between the annular member and the membrane housing, increasing the amount of unrecoverable solution. The example structure illustrated in Patent Document 10 assumes the transport of at least several hundred μL of liquid. In contrast, the aforementioned high-sensitivity analysis requires the passage of trace amounts of liquid, typically tens of μL, through the membrane before recovery. Therefore, even the example structure illustrated in Patent Document 10 does not envision the aforementioned high-sensitivity analysis application, and the expected effect cannot be achieved.

[0032] In summary, there are many membrane sealing and fixation methods available today. However, all of these methods are limited to the concept of gas circulation, continuous liquid delivery, or liquid collection, and do not envision the collection of trace amounts of liquid. Specifically, the technical significance of these known membrane fixation methods is to prevent gas or liquid from leaking from flow paths or containers and invading areas that are otherwise inaccessible. In contrast, in high-sensitivity analysis applications, it is crucial that trace amounts of liquid flow through the membrane without loss, allowing for high recovery rates in later stages.

[0033] Furthermore, as another method for keeping a thin film-like object in a predetermined storage portion in contact with liquid or gas without leakage, there is a membrane fixing method using a hard ring-shaped part as an example shown in Patent Document 2. Patent Document 2 shows the following structure: the membrane is fixed by pressing a circular porous component, a membrane, and a hollow component made of resin into a spin column made of resin in sequence. This is the structure of a general spin column, which enables trace amounts of liquid to pass through the membrane without loss and can be recovered at a high recovery rate. However, the research of the present inventors has made it clear that when the structure of Patent Document 2 is intended to be applied to the purpose of high-sensitivity analysis, the pressed-in ring-shaped part or the flow path chip is damaged or deformed and cannot be used. It can also be seen that this is because, unlike the spin column, the flow path chip has a larger rigidity, and thus the deformation of the ring-shaped part when pressed into it cannot be absorbed by both the ring-shaped part and the flow path chip.

[0034] Another issue when applying the structure of Patent Document 2 for high-sensitivity analysis is that solution can remain inside the circular porous retaining member that serves as the membrane holder, reducing recovery rates. This phenomenon has been shown to occur when centrifugation is not performed, compared to solution transport using centrifugation, a prerequisite for implementing Patent Document 2. Furthermore, another issue when applying the structure of Patent Document 2 for high-sensitivity analysis is that, despite measures being taken to address these issues, even the standard Boom method, a highly efficient nucleic acid purification method, can sometimes exhibit low purification efficiency, sometimes hindering subsequent PCR reactions. This has been shown to be caused by sample clogging and residual inhibitors due to insufficient liquid feed pressure. When performing a series of purification processes using a chip that securely secures the purification membrane to ensure loss-free passage of the sample solution, a high liquid feed pressure (approximately greater than 200 kPa) is required. On the other hand, with typical sample-to-answer flow chips, achieving sufficient liquid feed pressure is currently difficult due to pressure drop in the flow path and the pressure resistance of the valves within the flow path. To achieve the same level of liquid delivery pressure as centrifugal solution transport using a combination of known technologies, improvements in the chip's pressure resistance and a higher output from the liquid delivery pressure source are necessary. Consequently, the chip and system costs, as well as the system's size, are unavoidable, making it impossible to achieve a fully automated, highly sensitive genetic analysis system using a simple, low-cost device and flow chip.

[0035] In view of such circumstances, the present disclosure proposes a technology capable of efficiently performing the process of removing impurities and purifying and recovering nucleic acids from a sample containing nucleic acid using a device and a flow chip having a simple structure and low cost.

[0036] Solutions to Problems

[0037] In order to solve the above-mentioned problems, the present disclosure proposes a flow path device, in which a flow path is formed on a substrate, wherein the device comprises: a purification membrane capable of recovering nucleic acids; a membrane storage portion, which is a space formed on the substrate, connected to the flow path, and storing the purification membrane; and an annular resin ring, which is pressed into the membrane storage portion, the membrane storage portion having a seat surface formed between the upper surface and the lower surface of the substrate so as to be capable of setting the purification membrane, and the membrane storage portion is divided into a cylindrical upper portion of the membrane storage portion and a lower portion of the membrane storage portion having a diameter smaller than that of the upper portion of the membrane storage portion by the seat surface as a boundary, the flow path including a first flow path and a second flow path, the first flow path being connected to at least one valve mechanism and formed on the upper surface of the substrate. The second flow path is connected to at least one valve mechanism and is formed on the lower surface of the substrate and is connected to the lower part of the membrane storing portion. The resin ring has an outer diameter greater than the inner diameter of the upper part of the membrane storing portion. The purification membrane is fixed between the seat surface and the resin ring. The outer diameter of the resin ring after being pressed in is smaller than the outer diameter of the resin ring in a state not being stored in the membrane storing portion. Moreover, the inner diameter of the upper part of the membrane storing portion is approximately the same as the inner diameter of the upper part of the membrane storing portion in a state not being stored in the membrane storing portion. At least at a position where the side wall of the upper part of the membrane storing portion is closest to the pressed-in resin ring, the inner diameter of the upper part of the membrane storing portion is the same as the outer diameter of the pressed-in resin ring.

[0038] Other features related to the present disclosure will become apparent from the description and drawings of this specification. Furthermore, the present disclosure is achieved and realized by elements, combinations of multiple elements, the detailed description below, and the appended claims.

[0039] The descriptions in this specification are merely typical examples and do not limit the claims or application examples of the present disclosure in any sense.

[0040] The effects of the invention are as follows.

[0041] According to the technology disclosed herein, it is possible to efficiently perform the process of removing impurities from a sample containing nucleic acid and purifying and recovering nucleic acid using a device and a flow chip having a simple structure and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a diagram showing the treatment steps of a nucleic acid purification method based on the standard Boom method.

[0043] Figure 2A It is shown in Figure 1 The diagram shows the treatment steps and the composition of the sample solution used in this investigation.

[0044] Figure 2B It is shown in Figure 1 Figure 2 shows the reagent and solution transport methods used in the purification treatment process.

[0045] Figure 3AThis is a diagram showing the use of flow path equipment to implement Figure 1 A conceptual diagram of a configuration example of a sample pre-processing system 301 for a processing step.

[0046] Figure 3B Shows the state where the flow path device is installed Figure 3A Figure 2 shows the structure of the AA section.

[0047] Figure 4A It is a top view of a flow channel device (Example 1) 101 to which a known membrane installation method is applied.

[0048] Figure 4B It shows Figure 4A Figure 2. AA cross-sectional structure of the PDMS PDMS.

[0049] Figure 5A It is a top view of a flow channel device (Example 2) 101 to which a known membrane installation method is applied.

[0050] Figure 5B It shows Figure 5A Figure 2. AA cross-sectional structure of the PDMS PDMS.

[0051] Figure 6A It is a top view of a flow channel device (Example 3) 101 to which a known membrane installation method is applied.

[0052] Figure 6B It shows Figure 6A Figure 2. AA cross-sectional structure of the PDMS PDMS.

[0053] Figure 7A Graphs showing the results of quantitative evaluation of nucleic acid purification efficiency and recovered liquid volume using a spin column and a flow channel device of a known structure.

[0054] Figure 7B It is shown that real-time PCR Figure 7A Figure 1 shows the purification efficiency calculated by quantifying the amount of DNA contained in the eluate recovered (DNA recovery efficiency: dark-colored bar graph (left side) for each method), and the DNA leakage rate calculated by quantifying the amount of DNA contained in the sample solution (hereinafter referred to as the waste liquid) after passing through the purification membrane (light-colored bar graph (right side) for each method).

[0055] Figure 8 This is a diagram (photograph) showing a phenomenon in which a liquid accumulation portion of the eluate 403 is formed near the cutout portion (considered as liquid being trapped on the cutout portion) in the flow channel device 101 (an example) having a membrane fixing structure using an O-ring after the eluate is recovered.

[0056] Figure 9A It is a top view of the flow channel device 101 according to this embodiment.

[0057] Figure 9B It shows Figure 9A FIG. 1 is a diagram showing an example of a cross-sectional structure of AA.

[0058] Figure 10A This figure shows a state where the purification membrane 201 is installed from the membrane housing upper portion 110 onto the pedestal surface 109 .

[0059] Figure 10B This is a diagram showing a state where the resin ring 204 is press-fitted into the film storage upper portion 110 .

[0060] Figure 10C This is a diagram showing the purification membrane 201 fixed between the pedestal surface 109 and the resin ring.

[0061] Figure 11A Is shown using Figure 3A 9 shows the results of a nucleic acid purification experiment conducted using the sample pretreatment system shown and the flow channel device 101 of FIG. 9 and quantitatively evaluating the amount of recovered eluate.

[0062] Figure 11B Is shown using Figure 3A 9 and 101. A nucleic acid purification experiment was conducted using the sample pretreatment system shown in FIG. 9 and the flow channel device 101, and the DNA purification efficiency was quantitatively evaluated.

[0063] Figure 12A 1 is a diagram showing the upper surface structure of a flow channel device 101 to which a spin column structure is applied.

[0064] Figure 12B It shows Figure 12A Figure 2. AA cross-sectional structure of the PDMS PDMS.

[0065] Figure 13A This is a diagram (enlarged photograph) showing an example of the resin ring 204 that has failed in press-fitting and has been deformed on the film housing portion 104 .

[0066] Figure 13B This is a diagram (photograph) showing an example of the resin ring 204 that has failed in press-fitting and has been deformed on the film housing portion 104 .

[0067] Figure 13C This is a table summarizing examples of the outer diameter of the resin ring 204 and the success or failure of press-fit fixation.

[0068] Figure 14A 302 is a diagram showing an example of the internal structure of the liquid feeding mechanism 302.

[0069] Figure 14B This is a diagram showing an example of the internal structure of the liquid feeding mechanism 302 including a multi-way valve mechanism (V0) that can be opened to the atmosphere.

[0070] Figure 15This is a diagram showing a configuration example in which a plurality of on-off valve mechanisms 3005 are combined to achieve a function equivalent to that of the multi-way valve mechanism 3006 .

[0071] Figure 16 303 is a diagram showing an example of the internal structure of the reagent holding section 303 .

[0072] Figure 17 Is used to illustrate Figure 16 Flowchart of the nucleic acid purification process in the nucleic acid purification system shown.

[0073] Figure 18 This is a diagram showing another internal configuration example (modification example) of the reagent holding unit 303 in the nucleic acid purification system.

[0074] Figure 19 Is used to illustrate Figure 18 Flowchart of the nucleic acid purification process in the nucleic acid purification system shown.

[0075] Figure 20 1 is a diagram showing a system configuration example of a sample pretreatment system 301 provided with a reagent tank-integrated flow channel device (nucleic acid purification device) 101 .

[0076] Figure 21A 31 is a diagram showing an example of the upper surface structure of the reagent tank 3105 and its surroundings.

[0077] Figure 21B It shows Figure 21A Figure 2 is a diagram showing an example of a cross-sectional structure at AA.

[0078] Figure 22 Is used to illustrate the Figure 20 Flowchart (example) of the nucleic acid purification process performed by the nucleic acid purification system shown.

[0079] Figure 23 This diagram shows an example of the overall configuration of a Sample-to-Answer type DNA analysis system consisting of a flow device 101 and a sample pretreatment system 301 that can automatically perform nucleic acid extraction, nucleic acid purification, nucleic acid amplification, and detection from a biological sample.

[0080] Figure 24 1 is a diagram showing a configuration example of the nucleic acid extraction unit 1201 in a Sample-to-Answer type DNA analysis system.

[0081] Figure 25 This is a flowchart for explaining the nucleic acid extraction process in the Sample-to-Answer type DNA analysis system.

[0082] Figure 2611 is a diagram showing an example of the configuration of the periphery of the nucleic acid purification unit 1101 in a sample-to-answer type DNA analysis system.

[0083] Figure 27A 1301 is a diagram showing a configuration example of the periphery of the nucleic acid amplification unit 1301.

[0084] Figure 27B It shows Figure 27A FIG. 1 is a diagram showing an example of a cross-sectional structure of AA.

[0085] Figure 28 This is a flowchart for explaining the nucleic acid amplification process in the Sample-to-Answer type DNA analysis system.

[0086] Figure 29A 1 and 2 are diagrams showing configuration examples of the periphery of the film storage section 104 in each modification example as viewed from above.

[0087] Figure 29B This is a diagram showing the modification example 1. Figure 29A FIG. 1 is a diagram showing an example of a cross-sectional structure of AA.

[0088] Figure 29C This is a diagram showing the modification example 2. Figure 29A FIG. 1 is a diagram showing an example of a cross-sectional structure of AA.

[0089] Figure 29D This is a diagram showing the modification example 3. Figure 29A FIG. 1 is a diagram showing an example of a cross-sectional structure of AA.

[0090] Figure 29E This is a diagram showing the modification example 4. Figure 29A FIG. 1 is a diagram showing an example of a cross-sectional structure of AA.

[0091] Figure 30A This is a diagram showing how a sample solution 401 is transported in the continuous transport method.

[0092] Figure 30B This is a diagram showing how the cleaning liquid 402 is conveyed in the continuous conveying method. DETAILED DESCRIPTION

[0093] The embodiment of the present disclosure proposes the following technology: in a flow path device (flow path chip), a trace amount of reaction solution can be made to pass through a purification membrane without leakage and can be efficiently recovered, thereby enabling efficient nucleic acid purification. In this specification, first, the subject clarified for the first time in the research of the present disclosure is described, and then the characteristics of the sample pretreatment system (sample pretreatment device) of the embodiment are described. Next, the description of the modified example of the present embodiment is moved to. In the drawings of this specification, sometimes functionally identical elements are represented by the same reference numerals. In addition, the drawings show specific embodiments and installation examples based on the principles of the present disclosure, but these specific embodiments and installation examples are used to understand the present disclosure, not to interpret the present disclosure in a limiting sense. Moreover, in the present embodiment, although it has been described in sufficient detail for those skilled in the art to implement the present disclosure, other installations and methods can also be performed, and it is necessary to understand that, without departing from the scope and spirit of the technical idea of ​​the present disclosure, changes in structure and construction, and replacement of various elements can be performed. Therefore, the following description cannot be interpreted as limiting.

[0094] (1) Further examination of issues in nucleic acid purification (detailed study)

[0095] <Nucleic acid purification process>

[0096] Figure 1 : This is a diagram showing the processing 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). In the case of a nucleic acid purification method based on the Boom method, a chaotropic agent such as guanidine salt is added to the sample solution after the biological sample has been subjected to cell lysis treatment. Next, a cleaning solution is passed through the purification membrane to rinse impurities attached to the purification membrane (step II). Generally speaking, a solution containing ethanol is used as the cleaning solution, and cleaning is sometimes performed multiple times. Furthermore, the purification membrane is dried to remove the cleaning solution components (step III). Finally, through the above process, the purification membrane is bound to the DNA, and impurities and cleaning solution components are removed, and the DNA eluate is passed through the purification membrane and recovered, thereby recovering the target DNA (step IV).

[0097] Figure 2A It is shown in Figure 1 The processing steps and the composition of the sample solution used in the investigation are shown in FIG. Figure 2B It is shown in Figure 1The diagram shows the reagents and solution transport methods used in the purification process. Here, a human genome sample (2800 M) was used as the sample, and the standard QIAamp DNA Investigator Kit (Qiagen) was used for dissolution and purification reagents as a laboratory method. In the case of the spin column method implemented in the laboratory, each solution was transported by centrifugation.

[0098] Sample pretreatment system for nucleic acid purification

[0099] Figure 3A This is a diagram showing the use of flow path equipment to implement Figure 1 The schematic diagram shows a conceptual example of the structure of a sample pretreatment system 301 for a sample pretreatment process. The sample pretreatment system 301 includes a liquid delivery mechanism 302, a reagent holding unit 303, a chip holding unit 304 made of PEEK (polyetheretherketone), a PEEK cover 305 with an accessory 306, and a pipe 307. The liquid delivery mechanism 302 is composed of a liquid delivery power source and a pressure control mechanism that can apply solution transport pressure at any time, and is connected to the reagent holding unit 303 via the pipe 307. The reagent holding unit 303 includes an area for holding a solution internally and an area for recovering the transported solution, and is connected to the flow path device 101 via the pipe 307, respectively, and can transport and recover any solution according to the pressurization or decompression action controlled by the liquid delivery mechanism 302. The specific internal system structure example of the liquid delivery mechanism 302 and the reagent holding unit 303 is described below.

[0100] Figure 3B This shows the state where the flow path device is installed. Figure 3A 1 shows the structure of the AA section in FIG. The flow path device 101 is arranged so that it can be held at a predetermined position between the chip holding portion 304 and the cover 305. When the flow path device 101 is installed, the first vent 107 and the second vent 108 provided in the flow path device 101 are connected to the fitting 306 provided in the cover 305.

[0101] The liquid delivery power source of the liquid delivery mechanism 302 can apply sufficient pressure to transport the solution while not exceeding the chip's withstand pressure. For example, a diaphragm pump or syringe pump capable of controlling the applied pressure within a range of 0.1 kPa to 250 kPa can be used. While air pressure is used as the liquid delivery pressure source in this example, mechanical compression, centrifugal force, and the like can also be used.

[0102] The chip holder 304 and cover 305 do not need to be made of PEEK; any material having sufficient rigidity to maintain the flow path device 101 is not particularly limited. In addition to PEEK, other materials can be selected, such as resin materials such as polycarbonate (PC) and polymethyl methacrylate (PMMA), or metal materials such as aluminum and stainless steel. Considering the possibility of leakage of surfactants and salts contained in the sample solution, and solutions such as ethanol contained in the cleaning solution, chemical resistance to these solutions is preferred.

[0103] <Flow path devices for nucleic acid purification>

[0104] Next, the flow channel device 101 used here (at the time of this examination) will be described. Figure 4A 、 Figure 5A as well as Figure 6A 1 is a top view of a flow channel device (Examples 1 to 3) 101 to which a known membrane installation method is applied. Figure 4B 、 Figure 5B as well as Figure 6B It shows Figure 4A 、 Figure 5A as well as Figure 6A AA cross-sectional structure in each figure.

[0105] All flow channel devices 101 are composed of a substrate 102 forming a flow channel structure, polyolefin (PO) sealing tape 103 adhered to the upper and lower surfaces of the substrate 102, and a purification membrane 201 placed in a membrane housing 104. First, a coaxial, cylindrical membrane housing 104 with an upper diameter of 6 mm and a depth of 1 mm and a lower diameter of 4 mm and a depth of 2 mm is formed by cutting on a PC substrate 102 measuring 20 mm in length, 50 mm in width, and 4 mm in thickness. The membrane housing 104 is formed, along with first and second flow channels 105 and 1 mm in width and 1 mm in depth, and first and second vents 107 and 108 with diameters of 1 mm. The first vent 107 is located at the endpoint of the first flow channel 105, and the second vent 108 is located at the endpoint of the second flow channel 106. These vents are formed as through-holes in the substrate 102, with the upper surface open. The first flow path 105 is formed on the upper surface of the substrate 102 and connected to the side wall of the film storage upper portion 110. The second flow path 106 is formed on the lower surface of the substrate 102 and connected to the side wall of the film storage lower portion 111.

[0106] Figure 4A and Figure 4BThe structure of a flow channel device 101 without a membrane-fixing structure is shown, namely, a structure in which a purification membrane 201 is placed statically on a seating surface 109. Before sealing tape 103 is attached to the upper surface of substrate 102, a purification membrane 201 (silica membrane filter, GF / F, Whatman) with a diameter of 6 mm is pressed from the upper portion 110 of the membrane housing toward the seating surface 109. After confirming that the outer periphery of the purification membrane 201 can be placed without lifting, sealing tape 103 is attached to the upper surface of substrate 102, completing the flow channel device 101 without a membrane-fixing structure.

[0107] Figure 5A and Figure 5B The structure of a flow channel device 101 with a membrane-fixing structure using a notched O-ring is shown. Before sealing tape 103 is attached to the upper surface of substrate 102, a 6 mm diameter purification membrane 201 (silica membrane filter, GF / F, Whatman) is pressed from the membrane housing upper portion 110 toward the base surface 109. Furthermore, a notched rubber O-ring 202 (outer diameter 6 mm x inner diameter 4 mm) is inserted so that the notch aligns with the first flow channel 105 to prevent clogging. After confirming that the purification membrane 201 and O-ring 202 can be positioned without lifting, sealing tape 103 is attached to the upper surface of substrate 102. A 10 mm square PMMA block is then attached to the sealing tape 103 above the membrane housing 104 to flatten the O-ring. This completes flow channel device 101 with a membrane-fixing structure using an O-ring.

[0108] Figure 6A and Figure 6B The structure of a flow channel device 101 with a membrane-fixing structure using an adhesive is shown. Before attaching sealing tape 103 to the upper surface of substrate 102, double-sided tape (#9969, 3M) is attached to the seating surface 109. Furthermore, a purification membrane 201 (silica membrane filter, GF / F, Whatman) with a diameter of 6 mm is pressed toward the seating surface 109 from the upper portion 110 of the membrane housing, thereby adhering to the seating surface 109. After confirming that the outer periphery of the purification membrane 201 can be installed without lifting, sealing tape 103 is attached to the upper surface of substrate 102, completing the flow channel device 101 with a membrane-fixing structure using an adhesive.

[0109] Experimental Results

[0110] use Figure 3A and Figure 3B The sample pretreatment system shown and Figure 3A and Figures 3B to 5A and Figure 5B A nucleic acid purification experiment was carried out using the flow channel device 101. Figure 7A and Figure 7B: is a graph showing the results of quantitative evaluation of the amount of recovered eluate and DNA purification efficiency based on nucleic acid purification experiments. Figure 7A As shown in the figure, the average amount of eluate finally recovered in the centrifugal column method is 17 μL, while the average amount of eluate recovered in the chip device is less than 10 μL. In particular, in the case of the flow path device 101 having a membrane fixing structure using an O-ring with a cutout, the amount of recovered liquid is reduced to less than 1 / 10 of the amount of input liquid. Figure 8 This is a diagram (photograph) showing a phenomenon in which a liquid accumulation portion of the eluate 403 is formed near the cutout portion (considered as liquid being trapped on the cutout portion) in the flow channel device 101 (an example) having a membrane fixing structure using an O-ring after the eluate is recovered.

[0111] Figure 7B In the figure, the dark bar graphs (left side) of each method indicate the real-time PCR results. Figure 7A The amount of DNA contained in the eluate recovered was quantified and the purification efficiency (DNA recovery efficiency) was calculated. Figure 7B In the graph, the light-colored (right) bar graphs for each method show the DNA leakage rate calculated by quantifying the amount of DNA contained in the sample solution (hereinafter referred to as waste liquid) after passing through the purification membrane. The DNA leakage rate is evaluated by adding the waste liquid to a new spin column and performing the purification process of this embodiment, and then quantifying the DNA content by real-time PCR. Figure 7B As shown, the purification efficiency of the spin column method averaged approximately 50%, with DNA leakage being 0% in all six cases tested. In contrast, the purification efficiency of the chip device was less than half that of the spin column method, with a higher DNA leakage rate. Furthermore, in the flow channel device 101 with a membrane fixed using an adhesive, there were cases where PCR inhibitors were suspected to have been contaminated in the recovered eluate.

[0112] The reason for the reduced amount of recovered liquid in the spin column method on the chip device is believed to be due not only to the residual liquid observed on the ring in the flow channel device 101, which utilizes an O-ring membrane fixation structure (which differs from the press-fit method described below in the present disclosure, where it is simply the insertion of an elastomer), but also to the influence of the different liquid delivery methods. 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 material, density, and delivery pressure. The purification membrane 201 used in this study was a silica membrane with a similar density in both the spin column method and the chip device. Therefore, it is not believed to contribute to the difference in recovered liquid volume observed in this study. Meanwhile, the delivery pressure differed between the spin column method and the chip device due to the different solution delivery methods. In the spin column method, centrifugal force is used to propel the solution introduced into the column through the purification membrane and into a collection tube. During this process, 20,000 g (equivalent to a maximum delivery pressure of approximately 100 to 150 kPa under the conditions of this study) was always applied to the solution. On the other hand, the chip device used in this study uses pneumatic liquid delivery, and the maximum eluent delivery pressure is around 50 kPa, which is relatively low. Therefore, it is believed that in the flow channel device method, the sample solution retained in the membrane is difficult to be discharged into the flow channel, resulting in a reduction in the amount of recovered liquid.

[0113] Regarding purification efficiency, the waste liquid contained almost no DNA in the centrifugal column method. This suggests that the sample solution passed through the purification membrane without leakage and that the binding efficiency between the purification membrane and DNA was extremely high. On the other hand, in the case of the flow path device 101 without a membrane fixing structure, there was no mechanism to fix the purification membrane. Therefore, it is believed that the membrane floated and leaked from the peripheral portion. The flow path device 101 with a membrane fixing structure utilizing an O-ring was able to suppress membrane floatation compared to the flow path device 101 without a membrane fixing structure, resulting in a lower DNA leakage rate and a tendency for improvement in purification efficiency. However, when compared with the centrifugal column method, the purification efficiency was lower, suggesting that the ability to suppress DNA leakage, that is, the membrane fixing force, was insufficient.

[0114] High DNA leakage rates and significantly lower purification efficiency were also observed in flow path devices 101 with a membrane-fixing structure utilizing adhesives. This is believed to be due to insufficient bonding, resulting in unexpected gaps, or clogging of the purification membrane by the adhesive. No PCR hindrance occurred in flow path devices other than those using adhesive 203. Therefore, contamination by adhesive 203 is suspected to be the cause of PCR hindrance. The adhesive 203 used in this study was confirmed to be chemically resistant to purification reagents. However, this adhesive 203 is not biocompatible. Therefore, small amounts of eluted components, which are not considered a problem in other applications, could potentially hinder PCR analysis. Furthermore, since the flow path device 101 used in this study was assembled manually, the bonding state could not be reproducibly controlled, resulting in a high likelihood of adhesive components leaking from the base or unevenly affixing. It is also believed that PCR-inhibiting substances could easily mix into the solution due to contact with the solution. To address this issue, it is necessary to more precisely manage the device manufacturing process and use adhesive components that do not contain PCR-inhibiting substances. However, considering the size of the membrane storage section 104 and the base surface 109 (the part that becomes the adhesive) of the flow path device 101 used in this study, stable laboratory testing is expected to be difficult. Furthermore, because the purification reagent used in this study contains a highly concentrated solution of ethanol as an organic solvent, the types of adhesives and double-sided tapes that can be used are significantly limited, which is also a major concern.

[0115] (2) Implementation of the present disclosure

[0116] The sample pretreatment system 301 and the flow channel device 101 of the present embodiment, which serve as solutions to the problems discovered in the above-mentioned investigation (study), will be described.

[0117] <System and flow path equipment>

[0118] Sample pre-treatment system 301 and Figure 3A Same system as shown. Figure 9A : is a top view of the flow channel device 101 of this embodiment. Figure 9B It shows Figure 9AFigure 1 shows a cross-sectional structure taken along line AA of FIG. The flow path device 101 includes a membrane housing 104 formed on a substrate 102, a first flow path 105, a second flow path 106, a first vent 107, a second vent 108, sealing tape (film sheet) 103 adhered to the upper and lower surfaces of the substrate 102, and a purification membrane 201 disposed in the membrane housing 104. The membrane housing 104 consists of an upper membrane housing portion 110 and a lower membrane housing portion 111, each having different inner diameters, separated by the pedestal 109, to form a seating surface 109 between the upper and lower surfaces of the substrate 102 on which the purification membrane 201 can be mounted. The inner diameter of the upper membrane housing portion 110 is approximately the same as that of the purification membrane 201, while the inner diameter of the lower membrane housing portion 111 is smaller than that of the purification membrane 201 and the upper membrane housing portion 110. First vent 107 is located at the endpoint of first flow path 105. Second vent 108 is located at the endpoint of second flow path 106. Both first vent 107 and second vent 108 are formed, for example, as through holes in substrate 102, with the upper surface open. First flow path 105 is formed on the upper surface of substrate 102 and connected to the sidewall of membrane storage upper portion 110. Second flow path 106 is formed on the lower surface of substrate 102 and connected to the sidewall of membrane storage lower portion 111.

[0119] Specifically, the flow path device 101 of this embodiment is formed by cutting a PC substrate 102 with a length of 20× a width of 50× a thickness of 4 mm to form a coaxial different-diameter cylindrical membrane storage portion 104 with an upper portion having a diameter of 6× a depth of 1 mm and a lower portion having a diameter of 4× a depth of 2 mm, a first flow path 105 and a second flow path 106 with a width of 1× a depth of 1 mm, and a first air vent 107 and a second air vent 108 with a diameter of 1 mm.

[0120] The material of the substrate 102 and the sealing tape 103 is not particularly limited as long as it uses materials commonly used in this technical field. For example, as a material with a relatively low DNA adsorption capacity, PC, polypropylene (PP), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate, and polyurethane can be used. In addition, by implementing negatively charged modification on the surface, the adsorption capacity can also be suppressed. As other materials, for example, glass materials such as glass, quartz glass, fused quartz, synthetic quartz, alumina, sapphire, ceramics, forsterite, and photosensitive glass, plastics such as polyester resin, polystyrene, polyethylene resin, polydimethylsiloxane (PDMS), nylon, polymethyl methacrylate resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, and vinyl chloride resin, or any combination of the above materials can be cited. A sealing tape (film sheet) 103 made of the above-mentioned material is pasted on the upper and lower parts of the substrate 102 on which the first flow path 105, the second flow path 106, and the membrane storage part 104 are formed (bonded by an adhesive that does not hinder PCR), and at least a part of the opening of the flow path, etc. (at least the part other than the first vent 107 and the second vent 108 is sealed), thereby forming a flow path device 101.

[0121] <Method for fixing the purification membrane>

[0122] Figures 10A to 10C This is a diagram for explaining the installation method of the purification membrane 201. Before the sealing tape 103 is attached to the upper surface of the substrate 102, the purification membrane 201 (diameter P, thickness Q) is installed from the upper portion 110 of the membrane storage portion (diameter R, depth D) onto the base surface 109. Figure 10A ). Furthermore, a resin ring 204 having an outer diameter a, an inner diameter b, and a height c (where a>R) is pressed in, and the purification membrane 201 is fixed between the resin ring and the seat surface 109 ( Figure 10B and Figure 10C After confirming that the inner diameter of the membrane storage upper portion 110 is the same as the outer diameter of the resin ring 204 (R'=a': R' represents the diameter of the membrane storage upper portion 110 after the resin ring 204 is pressed in; a' represents the outer diameter of the resin ring 204 after the membrane storage upper portion 110 is pressed in), and that the purification membrane 201 and the resin ring 204 can be set without floating, the sealing tape 103 is attached to the upper surface of the substrate 102 to form the flow path device 101. In addition, the height of the resin ring 204 in the assembled state ( Figure 10C), preferably at a height that does not block the first flow channel 105. Furthermore, it is preferred that the upper surface of the resin ring 204 (the end surface not in contact with the purification membrane 201) be located below the bottom surface of the first flow channel 105, at least around the connection between the membrane housing upper portion 110 and the first flow channel 105 (chip thickness T - (depth of the first flow channel 105) ≥ Q + c). In other words, the upper surface of the resin ring 204 is preferably located below the bottom surface of the first flow channel 105. Furthermore, the side surfaces of the resin ring 204 preferably contact the side surfaces of the membrane housing 104 over as large an area as possible. Therefore, the height of the resin ring 204 is preferably determined so that the upper surface of the resin ring 204 is at the same height as the bottom surface of the first flow channel 105. This prevents the transported solution from remaining in the gap between the resin ring 204 and the membrane housing 104, reduces flow resistance, and enables the recovery of trace amounts of solution without loss.

[0123] In the technology disclosed herein, "press-fitting" refers to a state of engagement equivalent to pushing into a press-fitting state based on the mechanical fitting accuracy standard. That is, after the press-fitting is completed, the membrane housing 104 and the resin ring 204 cannot move relative to each other, resulting in a state in which they can be disassembled when a strong force is applied, or cannot be disassembled without damaging the components. In the case of a membrane fixing structure using an O-ring as shown in Patent Document 10 or the aforementioned research (investigation of the subject), the O-ring 202 elastically deforms (its shape returns to normal when the external force is removed). Therefore, after being assembled to the upper portion 110 of the membrane housing, the O-ring returns in a direction away from the membrane in order to repel the external force of assembly. At the same time, friction acts between the outer periphery of the O-ring 202 and the sidewalls of the upper portion 110 of the membrane housing. Therefore, once accommodated, the O-ring 202 will not fly out, but the force pressing down on the purification membrane is weak. Therefore, this does not correspond to the "press-fitting" state in the technology disclosed herein.

[0124] On the other hand, when the resin ring 204 is inserted (pressed) into the membrane housing upper portion 110, it plastically deforms (it remains deformed even after the external force is removed and does not return to its original shape) and is held in position by friction with the sidewalls of the membrane housing upper portion 110. Consequently, a force pressing down the purification membrane 201 is maintained within the membrane housing 104. By appropriately setting the dimensional range relative to the membrane housing upper portion 110, the resin ring 204 can be held in a fitted state, from being pushed in to being pressed in. This achieves a high membrane-fixing force. In the flow path device 101, by ensuring that R'=a' at least at the position where the side wall of the membrane storage portion upper portion 110 and the resin ring 204 are closest in the assembled state, and more strictly, by precisely observing the flow path device 101 in the assembled state, and ensuring that |R'-a'| is less than 1 μm (below the resolution of an optical microscope) at least at the position where the side wall of the membrane storage portion upper portion 110 and the resin ring 204 are closest, it is possible to confirm whether the membrane fixing structure formed by the press-fitting of the resin ring 204 is achieved. Measurement of the non-assembled state ( Figure 10B ) and assembled state ( Figure 10C ) and the outer diameter of the resin ring 204 under the membrane storage portion, by confirming that R / R'(≈1)>a' / a holds, the implementation of the technology disclosed in the present invention can also be confirmed. That is, in the unassembled state and the assembled state, the upper part 110 of the membrane storage portion has a large rigidity and is therefore basically not deformed. The resin ring 204 having an outer diameter a larger than the inner diameter R of the upper part 110 of the membrane storage portion is arranged in a shrinking manner, which is an important feature of the technology disclosed in the present invention. In addition, a known example of a membrane fixing method using a pressed-in annular member made of resin is the structure of a centrifugal column shown in Patent Document 2. However, when the inner diameters of the centrifugal column in the unassembled state and the assembled state are respectively set to S and S' and the outer diameters of the annular member are respectively set to A and A', S / S'<A' / A(≈1) holds, which becomes a relationship opposite to that of the technology disclosed in the present invention. This is because, in the case of the structure of the centrifugal column, the centrifugal column is a thin-walled container with relatively low rigidity and can be deformed by the pressing of the annular member. 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, A = approximately 7 to 7.2 mm, and S' = A' ≈ A holds.

[0125] In this embodiment, specifically, in the flow channel device 101 , a purification membrane 201 (silica membrane filter, GF / F, Whatman) with a diameter of 6 mm and a thickness of 0.4 mm and a PE resin ring 204 with an outer diameter of 6.2 mm, an inner diameter of 4 mm, and a height of 0.5 mm are provided in the membrane housing 104 .

[0126] The purification membrane 201 can be any membrane capable of retaining particles larger than 100 μm. Furthermore, the membrane thickness is preferably at least 1 μm. Furthermore, since finer mesh sizes allow for more efficient DNA recovery, the purification membrane 201 is preferably capable of retaining particles larger than 10 μm, more preferably larger than 1 μm, and even more preferably larger than 0.1 μm. Membranes other than silica membranes can be used, such as those made from solid substrates primarily composed of DNA-adsorbing cellulose, carboxylated particles, or ion exchange resins. If the volume of the purification membrane is too small, the amount of biomolecules it can adsorb decreases. On the other hand, if the volume is too large, there is a concern about the possibility of unintended molecular adsorption during purification or subsequent steps, and a decrease in solution transport efficiency. Therefore, the membrane size should be appropriately set, but there is no specific size limit.

[0127] Resin ring 204 may be made of a material other than PE. Preferably, it is a material that does not elastically deform, such as polyester resin, polystyrene, polyethylene resin, nylon, polymethyl methacrylate resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, or vinyl chloride resin. Furthermore, it does not need to be made of resin. Specific physical properties include a Young's modulus of 5 MPa to 1 GPa, an elastic modulus of 100 kPa or more, and an elastic strain limit of less than 100%.

[0128] The outer diameter a of the resin ring 204 can be any size sufficient to fit within the membrane-housing upper portion 110 and be press-fitted and secured. Specifically, 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 satisfy the conditions 0 ≤ a - R < 400 μm or (a - R) / R ≤ 0.2, as clarified by the inventors' research. Details of the outer diameter a of the resin ring 204 are described below.

[0129] The inner diameter b of the resin ring 204 can be any diameter sufficient for solution passage, but is preferably approximately the same as the inner diameter r of the lower membrane housing. This allows the solution, after contact with the purification membrane 201, to efficiently pass through the lower membrane housing 111, maximizing the amount of recoverable solution. Furthermore, the difference between the outer diameter a and the inner diameter b of the resin ring 204 is preferably as small as possible. Specifically, it is preferably approximately 2.5 mm or less. This prevents solution from being trapped and remaining on the upper surface of the resin ring 204, enabling more efficient solution recovery.

[0130] Experimental Results

[0131] Figure 11A and Figure 11B Is shown using Figure 3AThe results of the nucleic acid purification experiment performed with the sample pretreatment system and the flow path device 101 of FIG9 and the quantitative evaluation of the amount of recovered eluate and the DNA purification efficiency are shown in FIG9. In addition, in this embodiment, as in the above-mentioned "Further consideration of the issues in nucleic acid purification", Figure 1 The sample solutions and treatment steps shown were performed.

[0132] like Figure 11A As shown, the average amount of recovered eluate in the chip device of the disclosed technology is 13 μL. Compared with the spin column method, this amount of recovered eluate is slightly less, but compared with existing chip devices, it achieves a good recovery efficiency (over 50%). Figure 11B The dark bar graphs (left side) of each method are the results of real-time PCR on Figure 11A The amount of DNA contained in the eluate recovered was quantified and the purification efficiency (DNA recovery efficiency) was calculated. Figure 11B The light-colored bar graphs (right side) of 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) after passing through the purification membrane. Figure 11B As shown, when the chip device of this embodiment is used, the problem of high DNA leakage rate, which is a problem of existing chip devices, is solved, and the purification efficiency is obtained at the same level as the centrifugal column method. In addition, in the purification process of this embodiment, the required solution handling pressure is a maximum of 65kPa, which is the same level as the case of existing chip devices. In summary, according to this embodiment, a chip device with a simple structure and a small device are used to achieve firm membrane fixation without sample leakage and stable liquid delivery of trace solutions, which can achieve efficient purification with the same performance as the centrifugal column method. The effects of the above technology are also the same in the following embodiments and their derivatives.

[0133] (3) Examination of Issues in Flow Devices Using a Centrifugal Column Structure

[0134] like Figure 11B As shown, efficient purification can also be achieved using a spin column structure. Therefore, here, research and investigation are conducted on the subject of a flow path device 101 employing a spin column structure, the only known example of membrane fixation achieved by press-fitting a resin ring, to further clarify the advantages of the disclosed flow path device 101 (a structure that does not use a porous retaining member 205) shown in FIG9 .

[0135] Figure 12A 1 is a diagram showing the upper surface structure of a flow channel device 101 to which a spin column structure is applied. Figure 12B It shows Figure 12AFigure AA cross-sectional structure in FIG. The flow channel device 101 used in this study comprises a membrane housing 104 with a porous retaining 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 of a = 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and 7.1) and an inner diameter of 5 mm and a height of 0.5 mm. The flow channel device 101, which utilizes a spin column structure, is formed by cutting a PC substrate 102 having a diameter of 20 mm in length, 50 mm in width, and 5 mm in thickness. The membrane housing 104 is tapered, with a diameter of 7.1 mm at the top and 5 mm at the bottom, continuously changing in diameter. The first and second flow channels 105 and 106 have a width of 1 mm and a depth of 1 mm, and first and second vents 107 and 108 have a diameter of 1 mm. The first vent 107 is located at the end of the first flow channel 105. Second vent 108 is located at the endpoint of second flow path 106. Both first vent 107 and second vent 108 are formed as through-holes in substrate 102, with the upper surface open. First flow path 105 is formed on the upper surface of substrate 102 and connected to the side wall of membrane storage portion 104. Furthermore, second flow path 106 is formed on the lower surface of substrate 102 and connected to the side wall of membrane storage portion 104.

[0136] The porous retaining member 205 holds the purification membrane 201 at a fixed position within the membrane housing 104. It deforms to flatten toward the center of the circle when pressed into the spin column and flow path device 101. First, the porous retaining member 205 is pressed horizontally into the membrane housing 104 (operation sequence 1). The purification membrane 201 is placed on the secured porous retaining member 205 (operation sequence 2), and the resin ring 204 is pressed horizontally into the membrane housing 104 (operation sequence 3). The purification membrane securing steps 1 through 3 revealed the following issues.

[0137] (i) The first challenge in the purification membrane fixation procedure using a flow path device 101 based on a spin column structure having a porous retaining member 205 is that, in procedure 1, controlling the fixed position of the porous retaining member 205 is difficult. The porous retaining member 205 is easily deformed by the stress from the sidewalls of the membrane housing 104 during insertion. Therefore, if pressure is continued from above even after reaching the predetermined fixed position, the porous retaining member 205 will be positioned at the connection between the membrane housing 104 and the second flow path 106, increasing flow path resistance. Eventually, the porous retaining member 205 will reach the bottom of the membrane housing 104, completely blocking 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 the pressure application from above is stopped before reaching the predetermined fixing position, the resin ring 204 will be located at the connection between the membrane housing 104 and the first flow channel 105 after the purification membrane 201 and the resin ring 204 are fixed, thereby increasing the flow channel resistance. If the sample solution 401 is introduced from the first flow channel 105 in this state, the sample solution 401 may not efficiently contact the purification membrane 201, or the sample solution 401 may flow into unintended voids.

[0138] When a flow path is provided on a substrate 102 and a purification membrane 201 is installed, as in the flow path device 101 of the present disclosure (see FIG9 ), generally speaking, the thinner the substrate 102, the more likely it is that the flow path device will be simpler, more compact, and less expensive. Therefore, it is preferable to provide the flow path in the direction of the surface of the purification membrane 201. On the other hand, in the spin column method, there is no incentive to provide the flow path in the direction of the surface of the purification membrane, and thus the disadvantages described in the first issue do not occur. In other words, the spin column method does not suffer from the disadvantages associated with using a porous retaining member 205.

[0139] (ii) A second problem with the purification membrane fixation procedure when using a flow device 101 based on a spin column structure having a porous retaining member 205 is that, in the aforementioned procedure 3, the resin ring 204 cannot be press-fitted and fixed into the membrane housing 104 using the same dimensional control as the spin column method. As described in the structure of the flow device 101 of this embodiment ( FIG. 9 ), the resin ring 204 deforms due to stress from the sidewalls of the membrane housing 104 when pressed into the membrane housing 104. Since the resin ring 204 is not an elastic material like rubber, if the resin ring 204 is too large relative to the membrane housing 104, the resin ring 204 may not fit into the membrane housing 104 and may be crushed, or cracks may form in the substrate 102 surrounding the membrane housing 104, causing damage.

[0140] Figure 13A and Figure 13BThis is a diagram (photograph) showing an example of the resin ring 204 that has failed in press-fitting and has been deformed on the film housing portion 104 . Figure 13C This table summarizes the outer diameter of the resin ring 204 and the success or failure of press-fit fixation. When the inner diameter R of the membrane fixing portion 104 at the fixing position of the purification membrane 201 is about 6.5 mm and the outer diameter a of the resin ring 204 is 6.9 mm or more, that is, when (a-R) is at least 400 μm, it is confirmed that Figure 13A This relationship shows the same results when the inner diameter b and thickness (height) c of the resin ring 204 are changed, and when the membrane storage part 104 (Figure 9) is provided with a pedestal instead of the porous holding part 205. At the same time, at least in the case of 1mm≤R≤7mm, the phenomenon of failure of press-fitting within the range of a-R≥400μm was confirmed. It is believed that the main reason for the failure of press-fitting is that the rigidity of the substrate 102 is greater than that of the spin column method, and the deformation after the resin ring 204 is pressed in cannot be absorbed by both the resin ring 204 and the substrate 102.

[0141] In the spin column method, since the container housing the purification membrane 201 is thin-walled and relatively rigid, the size range required for the flow channel device 101 of the present disclosure need not be considered. As described above, by designing the inner diameter R of the membrane housing 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 stably manufactured at a practical level. This size range can be selected based on the physical properties of the substrate 102 and resin ring 204, such as their material, rigidity, and bending moment, and is preferably set appropriately.

[0142] (iii) Another issue with using a flow device 101 based on a spin column structure having a porous retaining member 205 is that the eluent 403 is trapped by the porous retaining member 205, resulting in a reduced amount of recovered liquid. To maintain the purification membrane 201 at a fixed position within the membrane housing 104 and to enable recovery of the sample solution 401 after it has passed through the membrane 201, the porous retaining member 205 is constructed from a porous material. The porous retaining member 205 has a water-retention capacity commensurate with its material, pore size, and volume. In the spin column method, the eluent 403 introduced into the column is forced through the purification membrane 201 using centrifugal force (e.g., 20,000 g). Therefore, the eluent 403 retained within the porous retaining member 205 can be largely recovered, obviating this issue.

[0143] On the other hand, when the liquid is delivered by a small general-purpose air pressure pump without using centrifugal force, for example, as in the technology disclosed in the present invention, the eluate 403 captured by the porous holding member 205 cannot be recovered, and the amount of recovered liquid is greatly reduced. Figure 1 、 Figure 2A and Figure 2BIn the case of a purification process of the Boom method, which shows an example, the sample solution 401 and the cleaning solution 402 remain in the porous holding member 205, thereby mixing the above components into the recovered eluent 403, which may be contaminated. It can be seen that this phenomenon can be improved to a certain extent by using a hydrophobic porous holding member 205 such as PTFE, or increasing the pore size, or making the porous holding member 205 thinner and reducing the volume (in the case of the Boom method), but in the case of the structure disclosed in the present invention, sufficient improvement cannot be obtained by the above methods. Therefore, when implementing the flow path device 101 disclosed in the present invention, it is preferably set to a structure that does not use the porous holding member 205.

[0144] (iv) As described above, it is understood that it is inappropriate to apply a spin column structure to the flow channel device 101 , and it is preferable to adopt the structure of the flow channel device 101 according to the present embodiment described above.

[0145] (3) Details of the liquid delivery mechanism and reagent holding unit

[0146] Here, the detailed structures of the liquid delivery mechanism 302 and the reagent holding unit 303 in the sample pretreatment system 301 of this embodiment will be described.

[0147] <Structure of Liquid Supply Mechanism 302>

[0148] Figure 14A 302 is a diagram showing an example of the internal structure of the liquid feeding mechanism 302. Figure 14B This is a diagram showing an example of the internal structure of the liquid feeding mechanism 302 including a multi-way valve mechanism (V0) that can be opened to the atmosphere.

[0149] according to Figure 14A The liquid delivery mechanism 302 includes a first connection port 3001 and a second connection port 3002 connected to the reagent holding portion 303, a liquid delivery power source 3003 connected to the first connection port 3001 and providing liquid delivery pressure, and a pressure control mechanism 3004 for regulating the liquid delivery pressure provided by the liquid delivery power source 3003. The liquid delivery power source 3003 and the pressure control mechanism 3004 can be configured by, for example, a combination of a diaphragm pump with a constant output (e.g., 100 kPa), a DC fan, and a vacuum regulator. Alternatively, they can be integrated using a syringe pump and a DC fan capable of controlling the discharge and suction volumes.

[0150] according to Figure 14B , except for the liquid delivery mechanism 302 Figure 14A In addition to the structure shown, a multi-way valve mechanism 3006 (V0) capable of opening to the atmosphere is provided between the pressure control mechanism 3004 and the first connection port 3001. Figure 14BIn the case of a structure of , the multi-way valve mechanism 3006 is operated at the moment when the liquid feeding action is to be stopped, and the connection between the first connection port 3001 and the liquid feeding power source 3003 can be instantly disconnected, thereby obtaining high-speed delivery responsiveness. Furthermore, since the liquid feeding power source 3003 can be made to work freely in an atmosphere-open state, the action of starting liquid feeding after the output of the liquid feeding power source 3003 and the pressure control mechanism 3004 are stabilized can be implemented in a state where the piping 307 between the reagent holding portion 303 is connected, and the action of returning the plunger of the syringe to a predetermined position when the liquid feeding power source 3003 uses a syringe pump can be implemented. It can also be used as follows: the liquid feeding power source 3003 can be driven at all times, and the timing of applying pressure to the first connection port 3001 can be controlled by the action of the multi-way valve mechanism 3006. In the case of using a liquid feeding power source 3003 that consumes a large amount of power just after current is applied, when it is desired to minimize the number of switching operations of the liquid feeding power source 3003 and the pressure control mechanism 3004, Figure 14B In addition, Figure 15 As shown, the multi-way valve mechanism 3006 can also achieve the same function by combining multiple on-off valve mechanisms 3005.

[0151] The liquid feeding power source 3003, the pressure control mechanism 3004, and the multi-way valve mechanism 3006 can also be connected to the second connection port 3002. In this case, by performing the pressurization and decompression actions opposite to the situation of being connected to the first connection port 3001, it is possible to achieve equivalent actions. In addition, it is also possible to use a pair of liquid feeding power sources 3003, the pressure control mechanism 3004, and the multi-way valve mechanism 3006 to be connected to the first connection port 3001 and the second connection port 3002, respectively. Thus, it is possible to access the fluid path device 101 from the two connection ports via the reagent holding portion 303, and it is possible to select the liquid feeding path that is suppressed to the minimum due to the influence of the liquid feeding loss caused by the elongated pipe length. Similarly, it is also possible to use the multi-way valve mechanism 3006 that has the function of switching a group of liquid feeding power sources 3003 and the pressure control mechanism 3004 to the three-way valve of the first connection port 3001, the second connection port 3002, or the atmosphere to open the flow path 3007. This allows pressure to be applied to both the first connection port 3001 and the second connection port 3002 , and also allows for miniaturization of the device.

[0152] <Structure of the Reagent Holding Unit 303>

[0153] Figure 163 is a diagram showing an example of the internal structure of the reagent holding portion 303. The reagent holding portion 303 includes, for example, a first liquid delivery port 3101 connected to the first connection port 3001 of the liquid delivery mechanism 302, a second liquid delivery port 3102 connected to the second connection port 3002, a first device connection port 3103 connected to the first vent port 107 of the flow path device 101, a second device connection port 3104 connected to the second vent port 108, at least one reagent tank 3105 (T1) that can be loaded and unloaded between the first liquid delivery port 3101 and the first device connection port 3103 via an opening and closing valve mechanism 3005, and a waste liquid tank 3106 / solution recovery tank 3107 (T2) that can be loaded and unloaded between the second liquid delivery port 3102 and the second device connection port 3104. In addition, as for the tank T2, the waste liquid tank 3106 is initially installed, and after the operation is performed midway, it is replaced with the solution recovery tank 3107, so Figure 16 It is expressed as 3106 / 3107.

[0154] The liquid delivery mechanism 302 and the reagent holding unit 303, as well as the reagent holding unit 303 and the fluidic device 101, can be connected separately via piping 307 or directly connected via piping joints. The liquid delivery mechanism 302 controls the first liquid delivery port 3101 and the second liquid delivery port 3102 to pressurize or depressurize the reagents stored in the reagent tank 3105, and sequentially transports the reagents to the fluidic device 101, where they can be recovered in the waste liquid tank 3106 or the solution recovery tank 3107.

[0155] Figure 17 Is used to illustrate Figure 16 Flowchart of nucleic acid purification process in the nucleic acid purification system shown in FIG. Figure 16 The reagent tank 3105 containing the sample solution 401 is connected to the position T1 in the system, and the waste liquid tank 3106 is connected to the position T2 (S1701). This connection can be automatically performed by a robot (robot arm) in the system, or it can be performed by an operator. The liquid delivery mechanism 302 introduces the sample solution 401 (Sample) held by the reagent tank 3105 into the first vent 107 of the flow path device 101, and discharges it from the second vent 108 through the purification membrane 201 provided in the membrane storage portion 104 of the flow path device 101, and recovers the discharged sample solution 401 to the waste liquid tank 3106 (S1702). Then, the operator or robot starts Figure 16Remove the empty tank T1 and replace it with the reagent tank 3105 containing the first cleaning liquid 402 (S1703). The liquid feeding 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 discharges it from the second vent 108 through the purification membrane 201 provided in the membrane storage section 104 of the flow path device 101, and recovers the discharged first cleaning liquid 402 into the waste liquid tank 3106 (S1704). The operator or robot Figure 16 The empty tank T1 is removed and replaced with the reagent tank 3105 containing the second cleaning liquid 402 (S1705). The liquid feeding 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 discharges it from the second vent 108 through the purification membrane 201 provided in the membrane storage portion 104 of the flow path device 101, and recovers the discharged second cleaning liquid 402 into the waste liquid tank 3106 (S1706). Similarly, the operator or robot Figure 16 Remove the empty tank T1 and replace it with the reagent tank 3105 containing the third cleaning liquid 402 (S1707). The liquid feeding mechanism 302 introduces the third cleaning liquid 402 (cleaning liquid 3) held by the reagent tank 3105 into the first vent 107 of the flow path device 101, and discharges it from the second vent 108 through the purification membrane 201 provided in the membrane storage portion 104 of the flow path device 101, and recovers the discharged third cleaning liquid 402 to the waste liquid tank 3106 (S1708). After the transportation of the third cleaning liquid 402, the liquid feeding mechanism 302 delivers dry air to the flow path device 101 for three minutes (S1709). Finally, the operator or robot from Figure 16 The empty tank in T1 is removed and replaced with the reagent tank 3105 containing the eluent 403. Simultaneously, 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 eluent 403 held in the reagent tank 3105 into the first vent 107 of the flow device 101, where it is then discharged from the second vent 108 through the purification membrane 201 disposed in the membrane housing 104 of the flow device 101. The discharged eluent 403 is then recovered in the solution recovery tank 3107 (S1711).

[0156] In addition, the reagent tank 3105, the waste liquid tank 3106, and the solution recovery tank 3107 are components commonly used in this technical field and are not particularly limited. As long as the microtube, conical tube, blister package, or bag package is resistant to the reagents used and can be connected to the liquid delivery mechanism 302 and the flow path device 101 through an appropriate connector, it can be used. For example, a microfluidic reservoir (Microfluidic Reservoirs) (Elveflοw Company) can be used in combination with a microtube. In addition, the reagent tank 3105 can also replace the tanks that have previously stored each reagent in sequence. Figure 16 The location of T1 and implementation Figure 17 And, Figure 17 After each process is completed, the reagent can be added to the empty reagent tank 3105 in sequence and moved to Figure 17 As described above, the replacement of the tank or the addition of the reagent can be performed manually or automatically using a robot arm or the like.

[0157] 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 vent 108, and the second device connection port 3104 may be connected to the first vent 107.

[0158] <Modification of the Reagent Holding Unit 303>

[0159] Reference Figure 18 and Figure 19 , a structural example of a nucleic acid purification system (reagent holding unit 303) that is more suitable for automation is described. Figure 18 This is a diagram showing another internal configuration example (modification example) of the reagent holding unit 303 in the nucleic acid purification system. Figure 19 Is used to illustrate Figure 18 Flowchart of the nucleic acid purification process in the nucleic acid purification system shown.

[0160] Figure 18In the embodiment, the reagent holding portion 303 includes a first liquid delivery port 3101 connected to the first connection port 3001 of the liquid delivery mechanism 302, a second liquid delivery port 3102 connected to the second connection port 3002, a first device connection port 3103 connected to the first vent port 107 of the flow device 101, a second device connection port 3104 connected to the second vent port 108, at least one reagent tank 3105 disposed between the first liquid delivery port 3101 and the first device connection port 3103 via an on-off valve mechanism 3005 and holding the reagent therein, and at least one waste liquid tank 3106 and a solution recovery tank 3107 disposed between the second liquid delivery port 3102 and the second device connection port 3104 via a multi-way valve mechanism 3006 and recovering the solution discharged from the flow device. The liquid delivery mechanism 302 and the reagent holding portion 303, as well as the reagent holding portion 303 and the flow device 101, may be connected via piping 307, or may be directly connected via piping joints. The first liquid delivery port 3101 and the second liquid delivery port 3102 are pressurized or depressurized by the control of the liquid delivery mechanism 302, and the reagents can be sequentially transported to the flow path device 101 by switching the flow path based on the opening and closing valve mechanism 3005 and the multi-way valve mechanism 3006. Therefore, there is no need to replace the reagent tank 3105, the waste liquid tank 3106, and the solution recovery tank 3107. Figure 16 Compared with the structure of the PCR product, the automation of nucleic acid purification processing can be achieved with a simple system.

[0161] Figure 19In the present invention, the control device (not shown; for example, a computer connected to the system) first operates the on-off valve mechanism 3005, opening V1 and V6 and closing V2 to V5 and V7 to V10. Simultaneously, the multi-way valve mechanism 3006 (V11, V12) is operated to connect the second liquid delivery port 3102 and the second device connection port 3104 to the waste liquid tank 3106 (S1901). The control device activates the liquid delivery mechanism 302, introducing the sample solution 401 (Sample) held in the first reagent tank 3105 into the first vent 107 of the flow device 101. The sample solution 401 passes through the purification membrane 201 provided in the membrane housing 104 of the flow device 101 and is discharged from the second vent 108. The discharged sample solution 401 is then recovered in the waste liquid tank 3106 (S1902). Next, the control device operates the on-off valve mechanism 3005 to open V2 and V7, and to close V1, V3 to V6, and V8 to V10 (S1903). The control device operates the liquid delivery mechanism 302 to introduce the first cleaning liquid 402 (AW1) held in the second reagent tank 3105 into the first vent 107 of the flow device 101. The first cleaning liquid 402 passes through the purification membrane 201 disposed in the membrane housing 104 of the flow device 101 and is discharged from the second vent 108. The discharged first cleaning liquid 402 is then recovered in the waste liquid tank 3106 (S1904). After the transfer of the first cleaning liquid 402 is completed, the control device operates the on-off valve mechanism 3005 to open V3 and V8, and to close V1 to V2, V4 to V7, and V9 to V10 (S1905). The control device activates the liquid delivery mechanism 302, introducing the second cleaning liquid 402 (AW2) held in the third reagent tank 3105 into the first vent 107 of the flow device 101. The second cleaning liquid 402 passes through the purification membrane 201 disposed in the membrane housing 104 of the flow device 101 and is discharged from the second vent 108. The discharged second cleaning liquid 402 is then recovered in the waste liquid tank 3106 (S1906). The control device operates the on-off valve mechanism 3005, opening V4 and V9 and closing V1 to V3, V5 to V8, and V10 (S1907). The control device activates the liquid delivery mechanism 302, introducing the third cleaning liquid 402 (EtOH) held in the fourth reagent tank 3105 into the first vent 107 of the flow device 101. The third cleaning liquid 402 passes through the purification membrane 201 disposed in the membrane housing 104 of the flow device 101 and is discharged from the second vent 108. The discharged third cleaning liquid 402 is then recovered in the waste liquid tank 3106 (S1908). After the third cleaning liquid 402 is transported, the control device activates the liquid delivery mechanism 302 to supply dry air to the flow device 101 for three minutes (S1909).Finally, the control device operates the on-off valve mechanism 3005 to open V5 and V10, closes V1 to V4, and V6 to V9, and operates the multi-way valve mechanism 3006 (V11, V12) to connect the second liquid delivery port 3102 and the second device connection port 3104 to the solution recovery tank 3107 (S1910). The control device activates the liquid delivery mechanism 302 to introduce the eluent 403 (ATE) held in the fifth reagent tank 3105 into the first vent 107 of the flow device 101. The eluent 403 passes through the purification membrane 201 provided in the membrane housing 104 of the flow device 101 and is discharged from the second vent 108. The discharged eluent 403 is then recovered in the solution recovery tank 3107 (S1911). Furthermore, it is preferred that the solution recovered in the waste liquid tank 3106 or the solution recovery tank 3107 does not leak out of the second liquid delivery port 3102 or return to the flow channel device 101, but is retained in the waste liquid tank 3106 and the solution recovery tank 3107. Therefore, the waste liquid tank 3106 and the solution recovery tank 3107 are preferably designed to have a sufficiently large capacity relative to the total amount of liquid transported by each. Alternatively, this can be achieved by utilizing a pressurizing action from the first connection port 3001. Figure 17 In the system of the nucleic acid purification process, a filter ventilation mechanism that allows the passage of gas but not liquid is set in the waste liquid tank 3106 and the solution recovery tank 3107. This can also be achieved by setting the connection with the second liquid delivery port 3102 to a closed state.

[0162] like Figure 15 As shown, the opening and closing valve mechanism 3005 and the multi-way valve mechanism 3006 can be replaced with each other. Figure 16 The multi-way valve mechanism 3006 (V11, V12) can be used in parallel with the same valve mechanism, which is excellent in terms of controllability, expansibility, etc. On the other hand, if Figure 16 If the opening and closing valve mechanism 3005 (V1 to V10) is replaced with a multi-way valve mechanism 3006, the valve mechanism can be centralized, which may easily realize the miniaturization of the system and the universalization of the control mechanism. In addition, the valve mechanism may not necessarily be set at both ends of the reagent tank 3105, but may be configured only on the upstream side (V1 to V5) or the downstream side (V6 to V10). Figure 16As shown, when valve mechanisms are provided at both ends of the reagent tank 3105, the possibility of unexpected leakage of the reagent due to capillary phenomena, changes in the internal pressure of the flow path, etc. can be reduced, and a more stable liquid delivery can be achieved. On the other hand, if the valve mechanism is provided only on one side of the reagent tank, the number of valve mechanisms can be reduced, and a smaller and simpler system can be constructed. The opening and closing valve mechanism 3005 and the multi-way valve mechanism 3006 can be, for example, a diaphragm valve, a needle valve, a bellows valve, a gate valve, etc., which have chemical resistance to the reagents used in the nucleic acid purification process and can allow the fluid to pass through, stop the fluid, or adjust the flow rate. The action can be implemented manually or by control such as an electrical signal.

[0163] (3) Reagent tank-integrated nucleic acid purification system using the structure of the flow channel device 101 disclosed herein

[0164] Reference Figures 20 to 22 , describes the sample pretreatment system and nucleic acid purification method of the reagent tank integrated nucleic acid purification equipment.

[0165] <Configuration example of a nucleic acid purification system with integrated reagent tank>

[0166] Figure 20 This diagram shows an example of the system configuration of a sample pretreatment system 301 equipped with a reagent tank-integrated flow channel device (nucleic acid purification device) 101. This flow channel device (nucleic acid purification device) 101 includes a membrane housing 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 103 adhered to the upper and lower surfaces of the substrate 102, a purification membrane 201 secured to the membrane housing 104 by press-fitting a resin ring 204, at least one reagent tank 3105 disposed between the first vent 107 and the first flow channel 105 via an on-off valve mechanism 3005, a waste liquid tank 3106 connected to the second flow channel 106 via the on-off valve mechanism 3005 and equipped with a vent filter 3109, and a solution recovery flow channel 3108 connected to the second flow channel 106 via the on-off valve mechanism 3005 and to the second vent 108. The structure around the film storage unit 104 and Figure 9A and Figure 9B 、 Figure 14A and Figure 14B The second vent 108 is connected to the solution recovery tank 3107 provided in the reagent holding unit 303 .

[0167] Figure 21A 31 is a diagram showing an example of the upper surface structure of the reagent tank 3105 and its surroundings. Figure 21B It shows Figure 21AFIG. 31 shows an example of a cross-sectional structure at AA of the reagent tank. The reagent tank 3105 is sealed with a reagent solution in a hollow container having an internal volume slightly larger than the amount of the reagent solution (e.g., 800 μL of sample solution, 1000 μL of cleaning solution, and 20 μL of eluent), and is adhered to the substrate 102 on the upper portion of the connecting flow path 1001 provided with a protrusion 1004. If the reagent tank actuator 3110 flattens the reagent tank 3105, the protrusion 1004 contacts the sealing component 1003 at the bottom of the reagent tank 3105, destroying the sealing component 1003. As a result, the reagent can flow into the connecting flow path 1001. In this reagent tank integrated nucleic acid purification device, the reagent tank 3105 is formed by providing a reagent sealing port 1005 and a vent 1006 on an aluminum sheet with a thickness of 50 μm while performing a bending process, so that an aluminum sheet with a thickness of 10 μm is welded to the bottom to form the sealing component 1003. Next, a micropipette is inserted through the reagent sealing port 1005. After the reagent tank 3105 is filled with the reagent, the reagent sealing port 1005 and the vent 1006 are sealed with sealing tape. Finally, double-sided tape is used to adhere the upper portion of the connecting flow path 1001 provided on the substrate 102. The reagent tank actuator 3110 can use a small linear actuator with a thrust force of 10N connected to a tapered tip fixture.

[0168] The on-off valve mechanism 3005 is composed of a valve portion 1002 and a valve actuator 3111 provided on the flow path device 101. The valve portion 1002 is shaped to match the tip of the valve actuator 3111. By pressing down the valve portion 1002 with the valve actuator 3111, the connecting flow path 1001 can be sealed. This reagent tank-integrated nucleic acid purification device has a curvature in the connecting flow path 1001 with a width of 1 mm and a depth of 1 mm. The dome-shaped valve portion 1002 is used as the valve actuator 3111. A spherical small linear actuator with a propulsion force of 5N.

[0169] The reagent tank 3105 can be any component commonly used in the art, and its material and shape are not particularly limited. Blister packaging, bag packaging, etc. can be used. Furthermore, the reagent tank 3105 can be any component that is chemically resistant to the reagent being used, capable of retaining the reagent at a predetermined location within the flow path device 101, and capable of transporting the reagent at any time. Preferably, the flow path device 101 is configured to retain the reagent at a predetermined location even when not installed in the sample pretreatment system 301. After installation in the sample pretreatment system 301, the reagent tank 3105 is unsealed by external force applied from the sample pretreatment system 301. Alternatively, the flow path device 101 can be configured to be unable to retain the reagent at a predetermined location 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 is introduced through the reagent sealing port 1005, and the reagent sealing port 1005 and the vent 1006 are sealed. Furthermore, the reagent tank 3105 does not necessarily need to have the reagent sealing port 1005 and the vent 1006. The sealing component 1003 can also be assembled after the reagent is sealed. Furthermore, the shape of the reagent tank 3105 can be, for example, a quadrilateral, triangle, or circle, rather than a hexagon. Specifically, when the reagent tank 3105 is configured such that the liquid delivery direction is diagonally aligned, residual liquid within the reagent tank 3105 can be minimized. Furthermore, the external force applied to the reagent tank 3105 by the sample pretreatment system 301 need not be a mechanical downward force, such as that used by the reagent tank actuator 3110, which deforms the reagent tank 3105. For example, the pressure within the flow path connected to the reagent tank 3105 can be used to rupture the seal of the reagent tank 3105. In particular, in the case of a structure that uses the pressure within the flow path to unseal the reagent tank 3105, since it can be used in conjunction with the liquid delivery mechanism 302 and the piping flow paths required to implement the nucleic acid purification process, it is superior in terms of simplifying and miniaturizing the system's structure.

[0170] The opening and closing valve mechanism 3005 is not particularly limited as long as it is a component commonly used in this technical field, as long as it has chemical resistance for the reagent used and a pressure resistance of more than the liquid feeding pressure (for example, 100kPa), can be set on the flow path device 101, and can control the liquid feeding direction at any time. Specifically, as the opening and closing valve mechanism 3005, in addition to the thin film deformable valve that achieves a closed state by deforming the flow path cover material attached to the surface of the substrate 102 using mechanical downward pressure, on-chip diaphragm valves, valves using magnetic beads, thermal deformation valves, piezoelectric valves, etc. can be cited. In addition, a rotary on-chip valve can also be used and replaced with a multi-way valve mechanism 3006. The advantage of the thin film deformable valve is that it can be operated through a relatively simple structure and can be operated stably at a low cost. Therefore, in a high-performance sample pretreatment system that requires a complicated processing flow and inevitably requires multiple valve mechanisms and requires small size and low cost, the thin film deformable valve is a particularly promising option. On the other hand, in the case of the method using a rubber O-ring or a gasket ring, which is one of the existing membrane fixing technologies, pressure must be applied in the fixing direction (the membrane surface direction). Therefore, the flow channel cover material needs to be made of a rigid material that does not deform, or when attaching a thin film that can be easily deformed (see Figure 21B ) requires an actuator for applying pressure on the upper portion. Adding a pressurizing actuator would increase the size and cost of the device. On the other hand, if the flow path cover material is made of a relatively rigid material, a thin film deformable valve cannot be used, thus still raising concerns about the system's size and cost. Because the purification membrane fixing method disclosed herein does not require the application of external force from the upper surface after the resin ring 204 is pressed in, it can be easily combined with a thin film deformable valve, which is one of the benefits of the technology disclosed herein.

[0171] The reagent tank actuator 3110 and valve actuator 3111 do not need to be linear actuators. For example, electromagnetic locks, XYZ worktables, stepper motors, DC motors, etc. can be used. Alternatively, they can be operated by actuators that integrate multiple valve mechanisms. Alternatively, arms, metal coils, etc. can be used to separate the external power source from the active part. This allows the mechanism configured on the flow path device 101 to be miniaturized, allowing the flow path device 101 to be miniaturized.

[0172] <Nucleic acid purification in a reagent tank-integrated nucleic acid purification system>

[0173] Figure 22 Is used to illustrate the Figure 20The following is a flowchart (example) of a nucleic acid purification process performed by a nucleic acid purification system. First, a control device (not shown; for example, a computer connected to the system) operates valve actuator 3111 to open valve V1 and close valves V2 through V10. It also operates reagent tank actuator 3110 to break the seal of reagent tank 3105 (sample) containing sample solution 401 (S2201). Next, the control device operates valve actuator 3111 to open valves V1, V4, and V8, and close valves V2 through V3, and V5 through V7. Furthermore, the control device activates liquid delivery mechanism 302 to introduce sample solution 401 into first flow channel 105 of flow device 101. The sample solution 401 passes through purification membrane 201 disposed in membrane housing 104 of flow device 101 and is discharged from second flow channel 106. The discharged sample solution 401 is then recovered in waste liquid tank 3106 (S2202). Next, the control device operates valve actuator 3111 to open valves V2 and V8 and close valves V1, V3, and V7. Furthermore, the control device operates reagent tank actuator 3110 to break the seal of reagent tank 3105 (washing buffer) containing washing solution 402 (S2203). The control device then operates valve actuator 3111 to open valves V2, V5, and V8 and close valves V1, V2, V4, V6, and V8. Furthermore, the control device activates liquid delivery mechanism 302 to introduce washing solution 402 held in second reagent tank 3105 into first flow channel 105 of flow channel device 101. The washing solution 402 passes through purification membrane 201 provided in membrane housing 104 of flow channel device 101 and is discharged from second flow channel 106. The discharged sample solution 401 is then recovered in waste liquid tank 3106 (S2204). After the cleaning solution 402 is transported, the control device activates the liquid delivery mechanism 302 to deliver dry air to the flow channel 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. Furthermore, the control device operates the reagent tank actuator 3110 to break the seal of the reagent tank 3105 (elution buffer) containing the eluent 403 (S2206). Finally, the control device operates the valve actuator 3111 to open valves V3, V6, and V7, and to close valves V1 to V2, V4 to V5, and V8. Furthermore, the control device operates the liquid delivery mechanism 302 to introduce the eluent 403 into the first flow channel 105 of the flow channel device 101. The eluent 403 passes through the purification membrane 201 provided in the membrane housing 104 of the flow channel device 101 and is discharged from the second flow channel 106. The discharged sample solution 401 is then transported to the solution recovery flow channel 3108 and recovered in the solution recovery tank 3107 connected to the second vent 108 (S2207).

[0174] also, Figure 222 shows a nucleic acid purification process flow in which only one cleaning solution for the purification membrane 201 is used. However, by changing the arrangement and combination of the reagent tank 3105 and the opening and closing valve mechanism 3005, various process flows including the nucleic acid purification process shown in FIG. 2 can be handled.

[0175] (4) Sample-to-Answer DNA Analysis System Using the Structure of the Flow Channel Device 101 of the Present Disclosure

[0176] Reference Figures 23 to 28 This section describes the sample pretreatment system and flow path equipment of a sample-to-answer DNA analysis system. This section describes the system's structure and processing details, using the example of human identification using STR (short tandem repeat) analysis using a capillary electrophoresis device, using a swab sample containing a few μL of human blood.

[0177] <Sample-to-Answer DNA Analysis System Configuration Example>

[0178] Figure 23 This figure shows an example of the overall structure of a sample-to-answer type DNA analysis system consisting of a flow device 101 and a sample pre-processing system 301 that can automatically perform nucleic acid extraction, nucleic acid purification, nucleic acid amplification, and detection from a biological sample. Figure 3A The illustrated system has a substantially similar structure, comprising a liquid delivery mechanism 302, a reagent holder 303, and a chip holder 304. The first and second vents 107, 108 of the flow device 101, fixed to the chip holder 304, are connected to the reagent holder 303 via piping 307. The flow device 101 comprises a nucleic acid extraction unit 1201 having a sample inlet 1202 and dissolving the sample to extract the nucleic acid contained therein; a nucleic acid purification unit 1101 connected to the nucleic acid extraction unit 1201 and removing impurities from the sample solution 401 discharged from the nucleic acid extraction unit 1201 to recover the target nucleic acid; and a nucleic acid amplification unit 1301 connected to the nucleic acid purification unit 1101 and amplifying and detecting the target nucleic acid contained in the eluate 403 discharged from the nucleic acid purification unit 1101.

[0179] <Nucleic acid extraction unit 1201 Nucleic acid extraction process>

[0180] Figure 24This diagram shows an example 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 base plate 102, a nucleic acid extraction chamber 1203 connected to the sample inlet, at least one reagent tank 3105 holding reagents used for sample dissolution and nucleic acid purification reactions, at least one on-off valve mechanism 3005, a purification unit connection channel 1204 connecting the nucleic acid extraction chamber 1203 to the nucleic acid purification unit 1101, and a liquid supply channel 1205 connected to the first vent 107 or the second vent 108 and capable of supplying liquid through the operation of the liquid supply mechanism 302.

[0181] A sample to be analyzed (e.g., a swab sample (sample 400): for example, a cotton swab coated with the sample (blood)) is introduced into the sample inlet 1202. A polyurethane inlet seal 1206 is then attached to the sample inlet 1202. This ensures air permeability within the nucleic acid extraction chamber 1203 and prevents the sample and reagents from leaking outside the flow device 101.

[0182] Furthermore, the nucleic acid extraction chamber 1203 is set to have an internal volume of 4 mL, for example. The temperature control mechanism 3202 controls the nucleic acid extraction heat source 3201, and can maintain the nucleic acid extraction chamber 1203 in contact with the temperature-regulated nucleic acid extraction heat source 3201 at a predetermined temperature. Figure 21A and Figure 21B The same institution as shown.

[0183] The introduction port sealing component 1206 may also not be made of polyurethane. For example, a ventilation filter, a porous resin sintered filter, an open-pore sealing tape, etc. can be used to allow air to pass through to a certain extent and to prevent the sample and solution inside the nucleic acid extraction chamber 1203 from leaking out. By making the nucleic acid extraction chamber 1203 an unsealed structure, the internal pressure of the nucleic acid extraction chamber 1203 can be reduced during the nucleic acid extraction reaction. Therefore, even if the flow path device 101 has a smaller pressure resistance, it can be used without damage, which can achieve low cost of the equipment. In addition, the introduction port sealing component 1206 may also be sealed using a material that does not have air permeability (such as a rubber component). In this case, by arranging a filter ventilation mechanism in the nucleic acid extraction chamber 1203, the same effect as the introduction port sealing component 1206 can be obtained.

[0184] <Nucleic acid extraction and processing>

[0185] Figure 25 This is a flowchart for explaining the nucleic acid extraction process in the sample-to-answer type DNA analysis system.

[0186] 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 from the sample introduction port 1202 and seals the introduction port with the sealing member 1206 (S2501). Next, in response to the operator's instruction to start the nucleic acid extraction process, the control device (not shown: for example, a computer connected to the system) operates the valve actuator 3111 of the opening and closing valve mechanism 3005 ( Figure 23(not shown), V2 is opened, V1 and V3 to V5 are closed, and the reagent tank actuator 3110 is operated to destroy the seal of the reagent tank 3105 containing the nucleic acid extract (dissolution buffer, for example, 300 μL). At the same time, the control device operates the temperature control mechanism 3202 to make the surface temperature of the heat source 3201 of the nucleic acid extraction part 56°C (S2502). When the control device confirms (detects) that the nucleic acid extraction chamber 1203 is 56°C, it operates the valve actuator 3111 to open V1 and V2 and close V3 to V5. Furthermore, it activates the liquid feeding mechanism 302 to introduce the nucleic acid extract into the nucleic acid extraction chamber 1203 (S2503). After the nucleic acid extract is transported, the control device controls the system by feeding air into the nucleic acid extraction chamber 1203, and stirs the nucleic acid extract and the sample 400 for 30 minutes by using bubbles and thermal convection (S2504). As a result, the cell membranes of the blood cells contained in sample 400 are dissolved, releasing the nucleic acids within the cells into the nucleic acid extract. Next, the control device operates valve actuator 3111 of the on-off valve mechanism 3005, opening V3 and closing V1, V2, V4, and V5. Furthermore, the control device operates reagent tank actuator 3110 to break the seal of reagent tank 3105 containing the purification reagent (sample buffer, e.g., 500 μL). Simultaneously, the control device operates temperature control mechanism 3202 to set the surface temperature of nucleic acid extraction heat source 3201 to 72°C (S2505). Upon confirming (detecting) that the nucleic acid extraction chamber 1203 is at 72°C, the control device operates valve actuator 3111 to open V3 and V4 and close V1, V2, and V5. Furthermore, the control device activates liquid delivery mechanism 302 to introduce the purification reagent into nucleic acid extraction chamber 1203 (S2506). After the purification reagent is transferred in S2506, the control device controls the system by introducing air into the nucleic acid extraction chamber 1203. This system uses air bubbles and thermal convection to agitate the sample 400 and the solution (nucleic acid extract and purification reagent) for 10 minutes (S2507). The purification reagent is a solution containing chaotropic salts and ethanol, which enables efficient DNA adsorption on the silica membrane. The operation in S2507 completely lyses the remaining blood cells in S2504 and simultaneously completes the preparation of the sample solution 401 (a mixed solution of human genomic DNA in the sample, nucleic acid extract, and purification reagent) for the subsequent Boom method in the nucleic acid purification unit 1101. Finally, the control device opens V3 to V5 and closes V1 and V2, transferring the sample solution 401 from the purification unit connecting flow path 1204 to the nucleic acid purification unit 1101 (S2508).

[0187] <Nucleic acid purification treatment>

[0188] Figure 26This figure shows an example of the structure of the nucleic acid purification unit 1101 and its surroundings in the sample-to-answer type DNA analysis system. The nucleic acid purification unit 1101 has a flow path device (nucleic acid purification device) 101 (see FIG. 1 ) integrated with a reagent tank. Figure 20 ) has a structure roughly the same as that of the flow path device (nucleic acid purification device) 101 integrated with the reagent tank. The main difference is that the sample solution 401 is transported from the purification part connection flow path 1204 connected to the nucleic acid extraction part 1201, and the solution recovery flow path 3108 is connected to the nucleic acid amplification part 1301. In the nucleic acid purification part 1101, Figure 22 The nucleic acid purification process shown also enables efficient nucleic acid purification.

[0189] <Configuration Example of Nucleic Acid Amplification Unit 1301>

[0190] Figure 27A 1301 is a diagram showing a configuration example of the periphery of the nucleic acid amplification unit 1301. Figure 27B It shows Figure 27A The nucleic acid amplification unit 1301 includes, on the substrate 102, a nucleic acid amplification chamber 1302 connected to the solution recovery channel 3108 of the nucleic acid purification unit 1101; at least one reagent tank 3105 for holding reagents for performing a nucleic acid amplification reaction; at least one opening and closing valve mechanism 3005; an amplification product recovery channel 1303 connecting the nucleic acid amplification chamber 1302 to the first vent 107 or the second vent 108 and discharging the sample solution after the nucleic acid amplification reaction; and a liquid delivery channel 1205 connected to at least one of the first vent 107 and the second vent 108 and capable of delivering liquid through the operation of the liquid delivery mechanism 302.

[0191] The nucleic acid amplification chamber 1302 can be composed of a straight line shape of, for example, 5 mm in width × 8 mm in length × 0.5 mm in thickness. Furthermore, the substrate 102 on the lower surface of the nucleic acid amplification chamber 1302 is processed to be thinner than the peripheral portion (for example, 0.5 mm in thickness). Thus, the upper and lower surfaces of the nucleic acid amplification chamber 1302 can be brought into contact with the nucleic acid amplification section heat source 3301, and the temperature of the sample solution in the nucleic acid amplification chamber can be changed at high speed. In addition, a temperature control mechanism 3302 can also be provided on each of the upper and lower nucleic acid amplification section heat sources 3301. Thus, feedback control of temperature regulation can be performed based on the temperature data of each of the upper and lower surfaces, thereby enabling more precise temperature control. On the other hand, the temperature control mechanism 3302 can also be connected to the nucleic acid extraction section heat source 3201 (see Figure 24 ) of the temperature control mechanism (temperature control mechanism 3202). Thus, the device can be miniaturized. In addition, the opening and closing valve mechanism 3005 and the reagent tank 3105 can be used in the same Figure 20 The same mechanism as shown in .

[0192] <Nucleic acid amplification treatment>

[0193] Figure 28This is a flowchart for illustrating the nucleic acid amplification process in a sample-to-answer DNA analysis system. The control device (not shown) first operates valve actuator 3111, opening valves V1 and V2 and closing valves V3 and V4, thereby introducing a portion (e.g., 10 μL) of eluent 403 from solution recovery flow path 3108 into the nucleic acid amplification chamber (S2801). Next, the control device operates valve actuator 3111, opening valve V3 and closing valves V1, V2, and V4. Furthermore, the control device operates reagent tank actuator 3110, breaking the seal of reagent tank 3105 containing nucleic acid amplification reagent (PCR buffer, e.g., 10 μL of reagent from the Globalfiler PCR kit) and introducing the reagent into nucleic acid amplification chamber 1302 (S2802). Next, the control device operates valve actuator 3111, opening valves V1 through V4 simultaneously to expel air bubbles from nucleic acid amplification chamber 1302. It then closes valves V1 through V4, allowing 20 μL of reaction solution (2 μL of eluent and 18 μL of nucleic acid amplification reagent) to be placed in nucleic acid amplification chamber 1302 (S2803). Here, the control device operates temperature control mechanism 3302 to set the surface temperature of the nucleic acid amplification unit heat source 3301 to 95°C. After the nucleic acid extraction chamber 1203 reaches (or detects) 95°C, the temperature is maintained for one minute (S2804). This activates the polymerase contained in the nucleic acid amplification reagent, enabling the initiation of the nucleic acid amplification reaction. Next, the control device operates temperature control mechanism 3302 to change the surface temperature of the nucleic acid amplification unit heat source 3301 to 94°C. After the nucleic acid extraction chamber 1203 reaches (or detects) 95°C, the temperature is maintained for 10 seconds, after which the DNA denaturation reaction proceeds (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. After reaching (or detecting) 59°C in the nucleic acid extraction chamber 1203, the temperature is maintained for 90 seconds, followed by annealing and DNA extension reactions (S2806). The control device repeats S2805 and S2806 30 times to amplify the target nucleic acid (STR) to a product concentration that can be detected by capillary electrophoresis. After performing S2805 and S2806 30 times, the control device continues to maintain the temperature at 59°C for 10 minutes to perform adenylation (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 and terminate 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. Furthermore, the liquid delivery mechanism 302 is activated to transport the reaction solution from the nucleic acid amplification chamber 1302 to the amplification product recovery flow path 1303 (S2809).

[0194] STR analysis by electrophoresis

[0195] The reaction solution transported to the amplification product recovery flow path 1303 is discharged to the outside through the second vent 108 and mixed with an electrophoresis reagent (e.g., formamide solution) at a predetermined ratio (e.g., 200 μL of electrophoresis reagent per 20 μL of reaction solution). This mixed solution, used as the sample solution, can be placed in an electrophoresis apparatus for STR analysis.

[0196] Alternatively, the electrophoresis reagent can be stored in the reagent tank 3105 from the nucleic acid extraction unit 1201 and placed on the base plate 102 in the same manner as the nucleic acid amplification unit 1301, and the reaction solution and the electrophoresis reagent can be mixed by the flow device 101. Furthermore, the electrophoresis unit can be formed in the flow device 101, and the process from sample introduction to electrophoresis analysis can be fully automated.

[0197] (5) Modifications of the Film Storage Section 104 and the Resin Ring 204

[0198] Figures 29A to 29E It is a diagram showing a modified example of the film storage portion 104 and the resin ring 204 of the present embodiment. Figure 29A 1 and 2 are diagrams showing configuration examples of the periphery of the film storage section 104 in each modification example as viewed from above. Figures 29B to 29E The following are examples of modifications 1 to 4: Figure 29A FIG. 1 is a diagram showing an example of a cross-sectional structure of AA.

[0199] like Figure 29A As shown, in each of the modified examples, the resin ring 204 preferably has a seamless annular shape when viewed from above. This allows the periphery of the purification membrane 201 to be securely fixed to the seating surface 109 throughout its entire circumference, minimizing solution leakage. For similar reasons, the seating surface 109 is also preferably a seamless annular shape. However, experiments have shown that a C-shaped seating surface 109 with a cutout of approximately 1 / 5 or less of its circumference can also fully achieve the effects of the disclosed technology.

[0200] exist Figures 29B to 29E In the illustrated modification, a resin ring 204 may be used, similar to a typical O-ring, with a circular cross-section when viewed vertically (a donut-shaped cross-section when viewed horizontally). In this case, the resin ring 204 and the purification membrane 201, as well as the sidewalls of the upper membrane-housing portion 110, are preferably in contact over as large a surface area as possible. This is because a larger contact area between the resin ring 204 and the purification membrane 201 can more effectively suppress lifting of the purification membrane 201, and the frictional force acting between the resin ring 204 and the upper membrane-housing portion 110 increases in direct proportion to the contact area.

[0201] One of the preferred variations is as follows Figure 29B As shown, the cross section of the resin ring 204 is tilted inward when viewed from the vertical direction of the annular member. Figure 29B In the illustrated shape of the resin ring 204, the contact areas between the resin ring 204 and the purification membrane 201, and between the resin ring and the membrane housing upper portion 110, are similar to those of the resin ring 204 of the aforementioned embodiment ( FIG. 9 ). This achieves a high purification membrane fixation effect while minimizing residual liquid on the upper surface of the resin ring 204, thereby achieving a higher recovery rate.

[0202] Figure 29C A modified example using a resin ring 204 with a tapered top and bottom is shown. Figure 29C As shown, in the resin ring 204, the upper cone 2001 is provided to be inclined inward toward the center of the purification membrane 201. Figure 29B The same modification examples can be expected to suppress liquid residue and improve the recovery rate. The lower conical member 2002 is provided on the outer peripheral side of the purification membrane 201. As a result, the resin ring 204 can be easily inserted into the upper portion 110 of the membrane storage portion during assembly. The resin ring 204 (embodiment and each modification example) disclosed herein is characterized in that it has an inner diameter greater than the upper portion 110 of the membrane storage portion. Therefore, there are cases where it is difficult to position the resin ring 204 and it is difficult to embed it during assembly. Therefore, by providing the lower conical member 2002, the outer diameter of the bottom surface of the resin ring 204 becomes smaller, so that it is easy to accommodate the upper portion 110 of the membrane storage portion at the beginning of embedding, and it is easy to correctly implement press-in fixation.

[0203] Figure 29D Another variation shown relates to a resin ring 204 having a cutout 2003 at the connection between the first flow channel 105 and the upper portion 110 of the membrane storage unit. The cutout 2003 is preferably designed and arranged so as not to block the first flow channel 105: the upper surface of the cutout 2003 is located lower than the bottom surface of the first flow channel 105, and the sidewalls of the cutout 2003 are located outside the sidewalls of the first flow channel 105. Figure 29D The resin ring 204, with its C-shaped top surface (predetermined proportions at the upper portion of the resin ring 204; a cutout is provided at the connection with the first flow path 105) and O-shaped bottom surface (predetermined proportions at the lower portion of the resin ring 204), reduces the effective volume of the membrane housing upper portion 110. This minimizes the amount of air that acts as a damper during liquid delivery, allowing for more efficient increases in liquid delivery pressure. This not only allows for miniaturization of the liquid delivery mechanism 302 by reducing the required pressure, but also improves liquid delivery controllability, enabling automated and more stable nucleic acid purification processing.

[0204] Figure 29EAnother modification shown relates to a film receiving portion 104 in which the film receiving portion upper portion 110 and the film receiving portion lower portion 111 are tapered. In this case, the resin ring 204 is also preferably tapered so as to be press-fitted into the film receiving portion upper portion 110. The film receiving portion upper portion 110 and the resin ring 204 are tapered, thereby Figure 29C The lower tapered member 2002 shown in the figure facilitates and stably inserts the resin ring 204 during assembly. Furthermore, compared to a case where the sidewalls of the membrane-housing upper portion 110 are nearly vertical, the contact area between the resin ring 204 and the membrane-housing upper portion 110 is increased, thereby achieving a higher pressing effect. Furthermore, when the membrane-housing lower portion 111 is tapered, the effective area of ​​the purification membrane 201 is ensured, and the amount of solution remaining in the membrane-housing lower portion 111 is reduced. This allows the solution that has passed through the purification membrane to be efficiently transported to the second flow path 106, further improving recovery efficiency.

[0205] also, Figures 29B to 29E The characteristic structures of the modified examples shown can also be combined separately. By appropriate combination, higher purification membrane fixing force and recovery efficiency can be obtained. For example, it is preferable to determine the appropriate shape by considering the material and processing deformation of the substrate 102 and the resin ring 204, the material and size of the purification membrane 201, etc. In addition, the membrane fixing method can also be used in combination with the existing membrane fixing method. For example, the tiny gap between the pressed-in resin ring 204 and the side wall of the upper part 110 of the membrane storage part can also be filled with an adhesive. In this way, the defects caused by processing errors and surface roughness generated in material processing can be minimized, and high-performance nucleic acid purification can be achieved at a practical level according to the application.

[0206] (6) Application of the continuous transport method in a system using the structure of the flow path device 101 disclosed herein (i) Here, a nucleic acid purification process and a sample pretreatment system using the continuous transport method are described. The composition of the sample solution 401 used in the continuous transport method is as follows: Figure 2A As shown in FIG, pure ethanol is used as the cleaning liquid 402. In addition, the sample pre-treatment system 301 and the flow path device 101 are roughly the same as those in FIG. Figure 20 The structure shown in is the same. Figure 30A This is a diagram showing how a sample solution 401 is transported in the continuous transport method. Figure 30B This is a diagram showing how the cleaning liquid 402 is conveyed in the continuous conveying method.

[0207] In the application of continuous handling method, such as Figure 30A and Figure 30B As shown in FIG, the sample solution 401 is not completely transported to the second flow path 106, but stops at the liquid delivery stop portion 1104 where the rear end of the sample solution 401 is located midway in the sample solution flow path 1102. Such liquid delivery control is similar to Figure 20 Furthermore, the liquid supply stop portion 1104 only needs to be able to detect that the rear end of the sample solution 401 has reached a predetermined position. For example, a liquid level detection sensor or a camera can be disposed on the upper surface of the liquid supply stop portion 1104 .

[0208] Next, refer to Figure 30A and Figure 30B The nucleic acid purification process based on the continuous transport method is briefly described. First, the control device (not shown) transports the sample solution 401 from the first reagent tank 3105 through the purification membrane 201 provided in the membrane storage unit 104 to the second flow path 106. The liquid delivery is stopped at the moment the rear end of the sample solution 401 reaches the liquid delivery stop portion 1104 ( Figure 30A Next, the control device discharges the cleaning solution 402 from the second reagent tank 3105 and allows it to continuously pass through the purification membrane 201 with the sample solution 401 and be transported to the second flow path 106 ( Figure 30B ). The subsequent nucleic acid purification process is the same as in Figure 22 The process is the same as described in .

[0209] (ii) When the purification membrane fixing method using the resin ring 204 disclosed in the present invention is applied, the gap between the purification membrane 201 and the membrane storage portion 104 becomes extremely small, thereby suppressing the leakage of liquid and gas. Therefore, it was found through experiments that the liquid delivery pressure required for the solution transport in the technology disclosed in the present invention is greater than that in the case where the technology disclosed in the present invention is not implemented. For example, when the resin ring 204 disclosed in the present invention is applied, the gap between the purification membrane 201 and the membrane storage portion 104 becomes extremely small, thereby suppressing the leakage of liquid and gas. Figure 4A and Figure 4B In the case of the structure shown, the maximum is 20kPa. Figure 9A and Figure 9B In the case of a structure with a maximum pressure of approximately 120 kPa, the highest pressure required is in the process where the cleaning solution 402 begins flowing after the sample solution 401 has completely flowed out. This is because if air reaches the periphery of the purification membrane 201 while it is wet with the sample solution 401, the air cannot pass through the purification membrane 201 until the Laplace pressure is exceeded. Therefore, a higher pressure is required before the cleaning solution 402 reaches the purification membrane 201.

[0210] In order to suppress such a high liquid delivery pressure, the technology disclosed in the present invention can apply a continuous transport method: continuously transporting two solutions that meet certain conditions without trapping air, thereby reducing the liquid delivery pressure. Since the main component of the above-mentioned sample solution 401 is water, and the main component of the cleaning solution 402 is ethanol, it has been found through experiments that the liquid delivery pressure reduction effect can be easily obtained by applying the continuous transport method. Moreover, it has been found through experiments that it is preferable that "the evaporation rate of the second liquid (cleaning liquid 402) is faster than that of the first liquid (sample solution 401), and the surface tension of the second liquid is lower than that of the first liquid." In fact, it has been confirmed that: implementation Figure 30A and Figure 30B During the nucleic acid purification process shown, even with the flow channel device 101 of the present disclosure, the maximum liquid delivery pressure is approximately 40 kPa, achieving high purification efficiency. Thus, the purification membrane immobilization method using the resin ring 204 of the present disclosure achieves high recovery rates and robust nucleic acid purification. Furthermore, low-pressure liquid delivery can be achieved using a continuous transport method, enabling high-performance nucleic acid purification using a compact, low-cost mechanism.

[0211] (7) Summary

[0212] (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 housing 104, which serves as a space formed in the substrate 102 and is connected to the first flow channel 105 and the second flow channel 106 formed in the substrate and houses the purification membrane 201; and an annular resin ring 204 pressed into the membrane housing 104. The membrane housing 104 includes a seating surface 109 formed so that the purification membrane 201 can be placed between the upper and lower surfaces of the substrate 102. The membrane housing 104 is divided, with the seating surface 109 as the boundary, into a cylindrical membrane housing upper portion 110 and a membrane housing lower portion 111 having a smaller diameter than the membrane housing upper portion 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 and formed on the upper surface of the substrate 102 and connected to the sidewall of the upper membrane housing portion 110; and the second flow path 106, which is connected to at least one valve mechanism and formed on the lower surface of the substrate 102 and connected to the lower membrane housing portion 111. The resin ring 204 has an outer diameter greater than the inner diameter of the upper membrane housing portion 110. Furthermore, the purification membrane 201 is fixed between the pedestal surface 109 and the resin ring 204. Furthermore, the outer diameter of the pressed-in resin ring 204 is smaller than the outer diameter of the resin ring 204 when not housed in the membrane housing portion 104. Furthermore, the inner diameter of the upper membrane housing portion 110 is substantially the same as the inner diameter of the upper membrane housing portion 110 when the resin ring 204 is not pressed into the membrane housing portion. Furthermore, at least at the position where the side wall of the upper portion 110 of the film storage section is closest to the pressed-in resin ring 204, the inner diameter of the upper portion 110 of the film storage section is the same as the outer diameter of the pressed-in resin ring 204. Here, the resin ring 204 plastically deforms when pressed into the film storage section 104 and is maintained in the pressed-in position by the friction between it and the side wall of the upper portion 110 of the film storage section. In other words, the resin ring 204 does not elastically deform when pressed in, unlike a known O-ring. The resin ring 204 is made of a material having a Young's modulus of 5 MPa or more and 1 GPa or less. In addition, the Young's modulus of the material constituting the resin ring 204 may also be smaller than the Young's modulus of the material constituting the substrate 102. Furthermore, the elastic modulus of the resin ring is greater than 100 kPa. On the other hand, the elastic strain limit value of the resin ring 204 is less than 100%. More specifically, when the inner diameter of the membrane-housing upper portion 110 in the assembled state of the flow path device 101 is R', and the outer diameter of the resin ring 204 after being pressed into the assembled state of the flow path device 101 is a', at the position where the side wall of the membrane-housing upper portion 110 and the pressed-in resin ring 204 are closest, |R'-a'| < 1 μm. On the other hand, when the inner diameter of the membrane-housing upper portion 110 in the unassembled state of the flow path device 101 (before the resin ring is pressed into) is R, and the outer diameter of the resin ring 204 in the unassembled state of the flow path device 101 (before the resin ring is pressed into) is a, 0 ≤ a-R < 400 μm.By configuring the flow channel device 101 in this manner, a simple and low-cost flow channel device (flow channel chip) 101 can be realized. Furthermore, the flow channel device 101 can fully automatically perform efficient purification equivalent to laboratory methods and enable highly sensitive gene analysis.

[0213] The inner diameter of the seat surface 109 can be 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 efficiently pass through the membrane housing lower portion 111, maximizing the amount of recoverable solution.

[0214] The difference between the inner diameter of the upper membrane-housing portion 110 and the inner diameter of the lower membrane-housing portion 111, and / or the difference between the outer diameter and inner diameter of the resin ring 204, is preferably as small as possible, desirably 2.5 mm or less. This prevents the solution from being trapped and remaining on the upper surface of the resin ring 204, allowing for more efficient solution recovery. Alternatively, the difference between the outer diameter and inner diameter of the resin ring 204 may be 1 mm or less.

[0215] Furthermore, the pressed-in resin ring 204 is in surface contact with the sidewall of the membrane housing upper portion 110, and with the purification membrane 201. Preferably, each of these contacts is made with as large a surface as possible. Thus, the larger the contact area between the resin ring 204 and the purification membrane 201, the more effectively it suppresses the purification membrane 201 from floating, achieving a higher pressing effect.

[0216] Furthermore, the pressed-in resin ring 204 is configured so that the end surface opposite the surface in contact with the purification membrane 201 is located below the connection between the first flow channel 105 and the side wall of the membrane housing upper portion 110. This prevents the transported solution from remaining in the gap between the resin ring 204 and the membrane housing 104. Furthermore, the flow channel resistance can be reduced, allowing for the lossless recovery of minute amounts of solution.

[0217] (ii) This embodiment also proposes a method for purifying nucleic acid using the flow device 101 to generate nucleic acid. Figure 17As shown, the method includes the steps of: connecting a sample tank (reagent tank 3105) containing a sample solution (specimen solution) to the first flow channel 105 of the flow channel device 101; connecting the second flow channel 106 to the waste liquid tank 3106; operating the liquid feeding mechanism 302 to transport the sample solution from the sample tank to the waste liquid tank 3106 via the first flow channel 105, the purification membrane 201, and the second flow channel 106; connecting a cleaning liquid tank (cleaning liquid 402 is held in the reagent tank 3105) containing a cleaning liquid for cleaning the purification membrane 201 to the first flow channel 105; and operating the valve mechanism to transport the cleaning liquid 402 from the cleaning liquid tank via the first flow channel 105. 5. The steps of transporting the eluate from the first flow channel 105 to the waste liquid tank 3106 via the purification membrane 201 and the second flow channel 106; operating the liquid feeding mechanism 302 to supply dry air from the first flow channel 105 to the second flow channel 106 via the purification membrane 201; connecting the first flow channel 105 to an eluent tank containing an eluent for eluting nucleic acids (the eluent is held in the reagent tank 3105) via a valve mechanism; connecting the second flow channel 106 to the solution recovery tank 3107; and operating the liquid feeding mechanism 302 to transport the eluent from the eluent tank via the first flow channel 105, the purification membrane 201, and the second flow channel 106 to the solution recovery tank 3107. This allows efficient purification of nucleic acids and high-sensitivity genetic analysis.

[0218] (iii) This embodiment also provides another method for purifying nucleic acid. Figure 30A and Figure 30B as well as Figure 22As shown, the method includes: a step of preparing at least a first liquid tank (reagent tank 3105) containing a first liquid (sample solution: sample solution 401) and a second liquid tank (cleaning liquid tank) containing a second liquid (cleaning liquid 402); a step of 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; a step of connecting the first flow path 105 of the flow path device 101 to a second liquid delivery flow path (cleaning liquid flow path) connected to the second liquid tank; a step of connecting the second flow path 106 of the flow path device 101 to a waste liquid tank 3106; a step of operating the liquid delivery mechanism 302 to deliver the first liquid from the first liquid delivery tank via the first flow path 105, the purification membrane 201, and the second flow path 1102. The liquid feeding mechanism 302 is operated to stop the feeding of the first liquid before the terminal end of the first liquid reaches the confluence point of the first liquid delivery channel and the first channel (at the liquid feeding stop portion 1104), thereby causing the first liquid to remain in the first delivery channel; and the liquid feeding mechanism 302 is operated to start feeding the second liquid from the second liquid tank to the second liquid delivery channel while the first liquid remains in the first delivery channel, thereby feeding the second liquid to the waste liquid tank 3106 via the confluence point of the second liquid delivery channel and the first channel, the first channel 105, the purification membrane 201, and the second channel 106, thereby continuously flowing the first liquid and the second liquid into the membrane storage section 104 without trapping air between the first liquid and the second liquid.

[0219] Furthermore, the preparatory steps of this method include preparing a third liquid tank (reagent tank (elution buffer) 3105) containing an eluent for eluting nucleic acids, operating the liquid delivery mechanism 302 to deliver dry air from the first flow channel 105 through the purification membrane 201 toward the second flow channel 106, and operating the liquid delivery mechanism 302 to transport the eluent from the eluent tank through the first flow channel 105, the purification membrane 201, and the second flow channel 106 to the solution recovery tank 3107. This reduces the liquid delivery pressure, thereby preventing damage to the flow channel device 101 due to the transport of the solution.

[0220] (iv) This embodiment proposes a sample pretreatment system 301, which includes the flow path device 101 of this embodiment, a nucleic acid extraction unit 1201 connected to the first flow path 105 of the flow path device 101 and extracting nucleic acid 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 acid (see Figures 23 to 27A and Figure 27B ).

[0221] (v) The technology disclosed herein can also form various functions by appropriately combining the multiple components disclosed in the embodiments. For example, some components can be deleted from all the components shown in the embodiments, or components of different specific examples can be appropriately combined.

[0222] As described above, the present disclosure is described using specific examples. However, these specific examples are intended only to illustrate (understanding the technology of the present disclosure) and are not intended to limit all aspects. Furthermore, other embodiments of the present disclosure will be apparent to those with ordinary knowledge in this technical field based on an examination of the present embodiment. Furthermore, the description and specific examples are merely typical examples, and the technical scope and gist of the present disclosure are set forth in the claims.

[0223] Explanation of symbols

[0224] 101—Flow path device, 102—Substrate, 103—Sealing tape, 104—Membrane storage unit, 105—First flow path, 106—Second flow path, 107—First vent, 108—Second vent, 109—Base surface, 110—Upper portion of membrane storage unit (diameter R, depth D), 111—Lower portion of membrane storage unit (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 retaining member, 301—Sample pretreatment System, 302—liquid delivery mechanism, 303—reagent holding part, 304—chip holding part, 305—cover, 306—accessories, 307—piping, 400—sample, 401—sample solution, 402—cleaning liquid, 403—elution liquid, 1001—connecting flow path, 1002—valve part, 1003—sealing component, 1004—protrusion, 1005—reagent sealing port, 1006—vent, 1101—nucleic acid purification part, 1102—sample solution flow path, 1103—cleaning liquid flow path, 1104—liquid delivery stop part, 1105—nucleic acid Purification and amplification section, 1201—nucleic acid extraction section, 1202—sample introduction port, 1203—nucleic acid extraction chamber, 1204—purification section connecting flow path, 1205—liquid delivery flow path, 1206—introduction port sealing component, 1301—nucleic acid amplification section, 1302—nucleic acid amplification chamber, 1303—amplification product recovery flow path, 2001—upper conical part, 2002—lower conical part, 2003—cut part, 3001—first connection port, 3002—second connection port, 3003—liquid delivery power source, 3004—pressure control mechanism, 3005— Opening and closing valve mechanism, 3006—multi-way valve mechanism, 3007—atmospheric open flow path, 3101—first liquid delivery port, 3102—second liquid delivery 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 flow path, 3109—vent filter, 3110—reagent tank actuator, 3111—valve actuator, 3201—heat source for nucleic acid extraction section, 3202, 3302—temperature control mechanism, 3301—heat source for nucleic acid amplification section.

Claims

1. A flow path device having a flow path formed on a substrate, characterized in that: have: purification membranes, which are capable of recovering nucleic acids; a membrane housing portion formed in a space of the substrate and connected to the flow path and housing the purification membrane; and The annular resin ring is pressed into the film receiving portion. The membrane storage section has a seating surface formed between the upper and lower surfaces of the substrate so as to be capable of placing the purification membrane, and is divided into a cylindrical membrane storage section upper portion and a membrane storage section lower portion having a smaller diameter than the membrane storage section upper portion, with the seating surface serving as a boundary. The flow path includes a first flow path and a second flow path, the first flow path is connected to at least one valve mechanism and is formed on the upper surface of the substrate and is connected to the side wall of the upper portion of the film storage portion, the second flow path is connected to at least one valve mechanism and is formed on the lower surface of the substrate and is connected to the lower portion of the film storage portion, The resin ring has an outer diameter greater than the inner diameter of the upper portion of the film storage portion. The purification membrane is fixed between the base surface and the resin ring. The outer diameter of the resin ring after being pressed in is smaller than the outer diameter of the resin ring when not housed in the film housing portion, and the inner diameter of the upper portion of the film housing portion is substantially the same as the inner diameter of the upper portion of the film housing portion when the resin ring is not pressed in the film housing portion. At least at a position where the side wall of the upper portion of the film housing portion is closest to the pressed-in resin ring, the inner diameter of the upper portion of the film housing portion is equal to the outer diameter of the pressed-in resin ring.

2. The flow channel device according to claim 1, characterized in that The resin ring is made of a material having a Young's modulus of 5 MPa or more.

3. The flow channel device according to claim 2, characterized in that The resin ring is made of a material having a Young's modulus of 1 GPa or less.

4. The flow channel device according to claim 1, characterized in that The resin ring has an elastic modulus of 100 kPa or more.

5. The flow channel device according to claim 1, characterized in that The elastic strain limit value of the resin ring is less than 100%.

6. The flow channel device according to claim 1, characterized in that When the inner diameter of the upper part of the above-mentioned membrane storage part in the state after the above-mentioned flow path device is assembled is set to R', and the outer diameter of the above-mentioned pressed resin ring in the state after the above-mentioned flow path device is assembled is set to a', at the position where the side wall of the upper part of the above-mentioned membrane storage part and the above-mentioned pressed resin ring are closest, |R'-a'|<1μm.

7. The flow channel device according to claim 1, characterized in that When the inner diameter of the upper portion of the membrane housing in the unassembled state of the flow channel device is R and the outer diameter of the resin ring in the unassembled state of the flow channel device is a, 0≤a-R<400 μm is satisfied.

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

9. The flow channel device according to claim 1, characterized in that The difference between the inner diameter of the upper portion of the film storage section and the inner diameter of the lower portion of the film storage section is 2.5 mm or less.

10. The flow channel device according to claim 9, characterized in that The difference between the outer diameter and the inner diameter of the resin ring is 1 mm or less.

11. The flow channel device according to claim 1, wherein: The pressed resin ring is in surface contact with the side wall of the upper portion of the membrane housing portion, and the resin ring is in surface contact with the purification membrane.

12. The flow channel device according to claim 1, wherein: An end surface of the pressed-in resin ring opposite to a surface in contact with the purification membrane is located below a connection portion between the first flow channel and a side wall of an upper portion of the membrane housing section.

13. The flow channel device according to claim 1, wherein The Young's modulus of the material constituting the resin ring is smaller than the Young's modulus of the material constituting the substrate.

14. The flow channel device according to claim 1, wherein The film sheet is attached to the upper and lower surfaces of the substrate and is configured to block at least a portion of the first flow path, the second flow path, and the opening of the film housing portion on the upper or lower surface of the substrate.

15. The flow channel device according to claim 1, wherein The press-fitted resin ring is fixed to the seating surface of the film storage portion by an adhesive.

16. A flow path device having a flow path formed on a substrate, characterized in that: have: purification membranes, which enable the recovery of nucleic acids; a membrane housing portion formed in a space of the substrate and connected to the flow path and housing the purification membrane; and The annular resin ring is pressed into the film receiving portion. The membrane storage section has a seating surface formed between the upper and lower surfaces of the substrate so as to be capable of placing the purification membrane, and is divided into a cylindrical membrane storage section upper portion and a membrane storage section lower portion having a smaller diameter than the membrane storage section, with the seating surface as a boundary. The flow path includes a first flow path and a second flow path, wherein the first flow path is formed on the upper surface of the substrate and connected to the side wall of the upper portion of the film storage portion, and the second flow path is formed on the lower surface of the substrate and connected to the lower portion of the film storage portion. The resin ring has an outer diameter greater than the inner diameter of the upper portion of the film storage portion. The purification membrane is fixed between the base surface and the resin ring. The outer diameter of the resin ring after being pressed in is smaller than the outer diameter of the resin ring when not housed in the film housing portion, and the inner diameter of the upper portion of the film housing portion is substantially the same as the inner diameter of the upper portion of the film housing portion when the resin ring is not pressed in the film housing portion. At least at a position where the side wall of the upper portion of the film housing portion is closest to the pressed-in resin ring, the inner diameter of the upper portion of the film housing portion is equal to the outer diameter of the pressed-in resin ring.

17. A method for purifying nucleic acid, comprising producing nucleic acid using the flow channel device according to claim 1, wherein: include: connecting the first flow path to a sample tank containing a sample solution; The step of connecting the second flow path to the waste liquid tank; a step of operating a liquid feeding mechanism to transport the sample solution from the sample tank through the first flow path, the purification membrane, and the second flow path to the waste liquid tank; a step of connecting the first flow path to a cleaning liquid tank containing a cleaning liquid for cleaning the purification membrane via the liquid feeding mechanism; a step of operating the liquid feeding mechanism to transport the cleaning liquid from the cleaning liquid tank through the first flow path, the purification membrane, and the second flow path to the waste liquid tank; a step of operating the liquid feeding mechanism to feed dry air from the first flow path through the purification membrane toward the second flow path; a step of connecting an elution liquid tank containing an elution liquid for eluting the nucleic acid to the first flow path; The step of connecting the second flow path to a recovery tank; and A step of operating the liquid feeding mechanism to convey the eluent from the eluent tank via the first flow path, the purification membrane, and the second flow path to the recovery tank.

18. A method for purifying nucleic acid, comprising producing nucleic acid using the flow channel device according to claim 1, wherein: include: a step of preparing at least a first liquid tank for accommodating a first liquid and a second liquid tank for accommodating a second liquid; a step of connecting the first flow path of the flow path device to a first liquid delivery flow path connected to the first liquid tank; a step of connecting the first flow path of the flow path device to a second liquid delivery flow path connected to the second liquid tank; The step of connecting the second flow path of the flow path device to a waste liquid tank; a step of operating a liquid feeding 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; A step of operating the liquid delivery mechanism to stop conveying the first liquid before the terminal end of the first liquid reaches a junction of the first liquid delivery channel and the first channel, thereby causing the first liquid to remain in the first liquid delivery channel; and The step of operating the liquid delivery mechanism while the first liquid remains in the first liquid delivery channel to start delivering the second liquid from the second liquid tank to the second liquid delivery channel, and conveying the second liquid to the waste liquid tank via the confluence of the second liquid delivery channel and the first channel, the first channel, the purification membrane, and the second channel. The first liquid and the second liquid are continuously flowed into the film housing portion without trapping air.

19. The method for purifying nucleic acid according to claim 18, wherein: The first liquid is a sample solution. The second liquid is a cleaning liquid. The preparation step further includes preparing a third liquid tank for accommodating an eluent for eluting the nucleic acid. Also includes: a step of operating the liquid feeding mechanism to feed dry air from the first flow path through the purification membrane toward the second flow path; The step of connecting the second flow path of the flow path device to a recovery tank for recovering the eluent; and A step of operating the liquid feeding mechanism to convey the eluate 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 pre-processing system, characterized in that: have: The flow path device of claim 1; a nucleic acid extraction unit connected to the first flow channel of the flow channel device and configured to extract nucleic acid from a biological sample; and The nucleic acid amplification unit is connected to the second flow channel of the flow channel device and amplifies the nucleic acid.

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