Flow path device, nucleic acid cleaning process and sample preparation system

Abstract

The present disclosure proposes a flow path device comprising the following for performing a treatment to remove impurities from a sample containing a nucleic acid and a treatment to purify and collect the nucleic acid with high efficiency: a purification membrane capable of collecting nucleic acid, a membrane storage section which is a space formed on the substrate, connected to the flow path and which receives the purification membrane, and a resin ring which is pressed into the membrane storage section, wherein the membrane storage section has a base surface formed between an upper and a lower surface of the substrate, such that the purification membrane is installed thereon, wherein the membrane storage section is divided into a cylindrical upper part of the membrane storage section and a lower part of the membrane storage section.which has a smaller diameter than the upper part of the membrane storage section, is subdivided, the base surface between being sandwich-shaped as a boundary, the flow path comprising a first flow path connected to at least one valve mechanism on the upper surface of the substrate and connected to a side wall of the upper part of the membrane storage section, and a second flow path connected to at least one valve mechanism on the lower surface of the substrate and connected to the lower part of the membrane storage section, the resin ring having an outer diameter greater than or equal to the inner diameter of the upper part of the membrane storage section, the cleaning membrane being fixed between the base surface and the resin ring, the outer diameter of the pressed-in resin ring being smaller than the outer diameter of the resin ring in a state,in which the resin ring is not included in the membrane storage section, and the inner diameter of the upper part of the membrane storage section is substantially the same as the inner diameter of the upper part of the membrane storage section in the state in which the resin ring is not pressed into the membrane storage section, and the inner diameter of the upper part of the membrane storage section and the outer diameter of the pressed-in resin ring are the same at least at a position where the side wall of the upper part of the membrane storage section and the pressed-in resin ring are closest to each other.

Description

Technical field

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

[0002] Genetic analysis technologies have been applied in various fields, including in vitro diagnostics, forensic science, and environmental monitoring, with rapid technological advances such as improved analytical accuracy and generalization of the technologies. Although there are various genetic testing procedures and analytical targets, the typical analytical process consists of a sample collection step, an extraction step to prepare a solution (sample) obtained by extracting a gene from the sample, a purification step to remove any reaction-inhibiting substances present in the sample, a step to amplify a target gene through a gene amplification reaction, a step to detect an amplification product, and a step to analyze a detection signal.Generally, the sample taken is transported to a special facility equipped with a tabletop gene analysis device and special instruments, and the subsequent processing is carried out manually by experienced specialist technicians (laboratory procedure).

[0003] In recent years, there has been a growing demand for a sample-to-response gene analysis system that enables fully automated workflows from sample introduction to measurement and data analysis using a gene pretreatment / detection device and a disposable, specialized cartridge. This simplification, acceleration, cost reduction, and other benefits are key to streamlining and accelerating the process. For example, PTL 1 proposes a fully automated DNA typing system that integrates a sample introduction port, nucleic acid extraction section, purification section, PCR section, and detection section into a single cartridge.

[0004] A solid-phase extraction process, using either a porous membrane or magnetic beads, is employed to purify nucleic acids. Solid-phase extraction, in particular, has become widely used due to the availability of commercially available kits and satisfactory purification efficiency. This process involves collecting nucleic acids by binding them to a solid support. Supports include porous silica or glass fiber membranes, silica-coated magnetic beads, or similar materials. Generally, in manual purification procedures, a spin-column method is commonly used. This involves applying a solution to a porous membrane mounted on a spin column (as shown in PTL 2) and then centrifuging the solution.This means that the spin column method can be carried out with general laboratory equipment and its procedures are relatively simple.

[0005] On the other hand, in applications within automated systems, a magnetic bead method is frequently used, where solution exchange can be achieved without centrifugation by a treatment that causes magnetic beads to accumulate on a magnet. For example, NPL 1 discloses a method for automating a gene extraction and purification step using the magnetic bead method. Furthermore, NPL 2 discloses not only the magnetic bead method but also a nucleic acid purification method using a flow path chip. In particular, the Boom method has been widely used as a technique capable of performing highly efficient and sample-robust nucleic acid purification through a simple treatment. This Boom method is a DNA purification technology that utilizes the strong DNA-binding effect of silica in the presence of a chaotropic salt.The Boom method has been applied to many spin column and magnetic bead lysis purification kits and nucleic acid purification on flow path chips. List of citations from patent literature PTL 1: JP 5815572 PTL 2: JP 2014-030397 A PTL 3: JP 2002-512688 T PTL 4: JP 2005-257647 A PTL 5: JP 2016-63475 A PTL 6: JP 2007-111653 A PTL 7: JP 2019-144164 A PTL 8: JP 2010-257691 A PTL 9: JP 2002-39984 A PTL 10: WO 2022 / 030605 A Non-patented literature NPL 1: “Microfluidic Devices for Nucleic Acid (NA) Isolation, Isothermal NA Amplification, and Real-Time Detection”, Methods Mol. Biol. 1256, 15-40(2015). NPL 2: “Magnetic particles for integrated nucleic acid purification, amplification and detection without pipetting”, Trends Analyt. Chem. 127:115912(2020). NPL 3: “Purification of Nucleic Acids in Microfluidic Devices”, Anal. Chem. 80, 6472-6479(2008) Summary of the invention: Technical problem

[0006] The magnetic bead method requires, in addition to a magnet for collecting beads and a driver circuit for the magnet, a mechanism to transport the beads with high accuracy in order to implement a multi-stage analysis workflow. This leads to the problem that the configurations of the apparatus and the flow path chip become more complex, resulting in increased costs. In a case where a micropillar or monolithic structure serving as a bonding carrier is provided on a flow path, as described, for example, in NPL 1, the flow path chip requires special machining, resulting in increased manufacturing costs for the flow path chip.

[0007] PTL 1, PTL 3, and NPL 3, described above, reveal examples of nucleic acid purification implemented in flow-path chips embedding membrane-like supports. However, applications to high-sensitivity analysis at a level (for example, a standard that can be used for the inspection of forensic samples (such as items left at a crime scene)) where even a small amount of a sample can be inspected with high sensitivity were not considered in any of these documents. This is further supported by the absence of known examples relating to the purification efficiency of these technologies, despite the fact that it is generally understood that the quantity and purity of nucleic acids collected from a sample significantly affect the accuracy and sensitivity of the analysis.

[0008] One of the problems in implementing highly efficient nucleic acid purification using a membrane-like support on a flow-path chip is that the purification efficiency is lower than that achieved by a laboratory setup and a flow-path chip purification scheme using a support other than a membrane. This is a novel problem that has become clear for the first time through the investigations of the inventors of the present application. Furthermore, the investigations of the inventors of the present application have revealed for the first time that a major cause of the problem is leakage of a solution from the membrane. It should be noted that the process and details that led to the discovery of this problem will become clear from the description in the embodiment and the accompanying drawings, which will be described later.

[0009] One solution to the aforementioned problem is, for example, to secure a cleaning membrane with a seal to prevent leakage of the solution. Membrane fastening methods using seals such as O-rings or C-rings, as exemplified in PTL 4 to PTL 8, are conceivable not only for cleaning nucleic acids, but also as a method for holding a thin, film-like object in a predetermined storage section by contact, without leakage of liquid or gas.

[0010] PTL 4 discloses a structure in which a reinforcing plate, a gas-permeable membrane, and an O-ring (rubber seal) are housed in that order within an outer container, and the O-ring is pressed in the membrane direction by fitting an inner container from the upper part of the O-ring. If the configuration from PTL 4 is applied to a highly sensitive analysis (for example, inspection of forensic specimens) as described above, a solution may be drawn into a gap between the inner and outer containers or between the reinforcing plate and the outer container, causing liquid leakage, and the solution absorbed in the gap cannot be collected. This is because PTL 4 anticipates the passage of gas components and does not account for the passage of a liquid in the membrane direction.At the same time, if an attempt is made to provide the PTL 4 configuration on a substrate of a flow path chip, substrates of at least four layers must be assembled, namely an outer container that holds a membrane and a ring, an inner container that presses against the ring, a bottom lid that is provided with a flow path, and an upper lid that presses against the bottom lid, which leads to an increase in the size and cost of the chip.

[0011] PTL 5 shows a configuration in which a C-ring is fitted into a groove of an inner connector and then fitted into an outer container to continuously feed a liquid to a separation membrane without causing leakage of solution from the container. This is also a common method using a sealing ring. If the configuration from PTL 5 is applied to a highly sensitive analysis (for example, inspection of forensic specimens), as described above, while leakage of liquid from the containers can be prevented, the liquid enters the space between the outer container and the ring, and the solution absorbed in the space cannot be collected.This is because PTL 5 expects a continuous liquid supply and does not account for the fact that a small amount of liquid flows and is collected at a later stage. Additionally, if an attempt is made to apply the PTL 5 configuration to a substrate-shaped chip device, the chip components become more complex, leading to increased size and cost, similar to PTL 4.

[0012] PTL 6 discloses a configuration in which a filter is embedded between two chip substrates, on which flow path and filter receiving sections are formed, and the chip substrates are bonded together to fix the filter. A structure in which flow paths are provided in the two chip substrates is essential for the configuration of PTL 6. Therefore, it cannot be applied to a flow path chip formed by attaching or bonding a thin and soft material, such as a film, to a chip substrate. Therefore, the formation of an on-chip membrane valve or the like, as described in PTL 7, is conceivable. However, in this case, it is necessary to provide an additional bonding layer to support the valve, thus complicating the layered structure of the chip.At the same time, precise control of the bond state requires strict control of bond conditions and positioning accuracy. Therefore, there are significant limitations on the shape of the flow path that can be formed and on the chip material, leading to increased manufacturing costs.

[0013] PTL 8 and PTL 9 disclose configurations to prevent electrolytes contained within devices from leaking outwards by incorporating gas-permeable membranes, sealing rings, and fasteners in that order, and by applying pressure deformation to the rings. This allows the devices to be filled with small quantities of solution without leakage. However, because the configurations do not anticipate any liquid feed operations, no flow paths can be provided. Furthermore, even if flow paths were provided to apply the technologies to highly sensitive analysis (e.g., forensic specimen inspection), as described above, it is evident that residual liquid would still occur that cannot be collected.

[0014] PTL 10 shows a configuration in which a nucleic acid extraction membrane is installed in a membrane receiving vessel formed on a flow path chip. A rubber O-ring, provided with a notch to avoid obstructing the flow path, is pressed onto the O-ring to secure the membrane, and a specific amount of liquid is directed to pass through the membrane. The inventors' investigations have shown that when the configuration of PTL 10 is applied to a highly sensitive analysis (for example, forensic examination of samples), as described above, the purification efficiency is low and the amount of collected liquid decreases significantly.In addition, the investigations of the present inventors have shown that the sealing clamping force acting on the membrane in the PTL 10 configuration is insufficient for the application of a highly sensitive analysis, and that the phenomenon relating to the amount of collected liquid is due to the generation of liquid residue as a result of the indentation. Because the O-ring made of PTL 8 is elastically deformed, the indentation section deforms more easily than its periphery and deforms unevenly. If the indentation is small, the flow path becomes narrow, the resistance of the flow path increases, and the controllability of the liquid supply is thereby impaired. If, on the other hand, the indentation section is large, it is deformed to such an extent that it collapses, further narrowing the flow path.Simultaneously, it is likely that liquid residue will occur in the indentation section and that, due to the O-shaped cross-section of the ring, liquid will enter the space between the ring and the membrane storage section, leading to an increase in the amount of solution that cannot be collected. It is assumed that, in the configuration example shown in PTL 10, at least a few hundred µl of liquid will be conveyed, whereas it is necessary to allow a small amount of liquid, a few tens of µl, to pass through the membrane and then collect it for the application of a high-sensitivity analysis, as described above. Therefore, the configuration shown in PTL 10 does not assume the application of a high-sensitivity analysis, as described above, and it can be concluded that the expected effects cannot be obtained.

[0015] Although there are numerous methods in the field for sealing and fixing membranes, as described above, they are all limited to processes that assume a gas flow, a continuous liquid supply, or liquid collection, and do not assume the collection of a small quantity of liquid. In other words, the technical significance of all known membrane fixing methods lies in preventing the leakage of gas or liquid from flow paths or containers and its entry into parts where the gas or liquid should not naturally enter. On the other hand, for the application of highly sensitive analysis, it is necessary that a small quantity of liquid that has flowed in passes through a membrane without loss and can be collected at a later stage with high yield.

[0016] Another method for retaining a thin, film-like object in a predetermined storage section by contact without leakage of liquid or gas is a membrane fixation method based on a press fit using a hard ring, as exemplified in PTL 2. PTL 2 shows a configuration in which a circular porous element, a membrane, and a hollow resin element are pressed into a resin spin column in that order to fix the membrane. This is a general spin column configuration that allows a small amount of liquid to pass through the membrane without loss and enables high-yield liquid collection.However, the investigations of the present inventors have shown that the pressed-in ring or the flow path chip is damaged or deformed, and the configuration cannot be applied if an attempt is made to apply the configuration from PTL 2 to a highly sensitive analysis. It was found that this was due to the fact that the stiffness of the flow path chip, unlike that of the spin column, was high, and the stress during the pressing-in of the ring could not be absorbed by both the ring and the flow path chip.

[0017] Another problem that arises when the PTL 2 configuration is applied to a highly sensitive analysis is that some solution remains within the circular porous retaining element, which serves as the membrane holding stage, and that the yield decreases. It was found that this phenomenon occurs when using a low liquid feed pressure for the PTL 2 application, compared to solution delivery based on a centrifugal process as a prerequisite for implementation, in a case where no centrifugal process is performed.An additional problem with applying the PTL 2 configuration to high-sensitivity analysis is that the purification efficiency remains low, or a PCR reaction is inhibited at a later stage, even when the Boom method, a standard technology, is used as a highly efficient nucleic acid purification technique after addressing the aforementioned issues. This has been found to be caused by sample clogging and the retention of an inhibitor due to insufficient liquid supply pressure. In a scenario where a series of purification steps are performed by a chip to which a purification membrane is securely attached, a high liquid supply pressure (approximately > 200 kPa) is required to ensure that a sample solution passes through the purification membrane without loss.On the other hand, in the case of a general sample-to-response flow path chip, maintaining a sufficient liquid supply pressure is often difficult due to pressure loss in the flow path and the pressure resistance of a valve within the flow path. To achieve a liquid supply pressure similar to that obtained with centrifugal solution delivery by combining known technologies, the pressure resistance of the chip must be improved and the output power of a liquid supply pressure source increased. Therefore, it is not possible to achieve a fully automated and highly sensitive gene analysis system based on a device and a flow path chip with simple configurations at a low cost without increasing the cost of the chip and the system size.

[0018] In view of these circumstances, the present disclosure proposes a technology that enables a treatment for removing impurities from a sample containing a nucleic acid and an efficiently performed treatment for cleaning and collecting the nucleic acid by means of a device and the achievement of a flow path chip with a simple configuration at low cost. Solution to the problem

[0019] To solve the above problem, the present disclosure proposes a flow path device with a flow path formed on a substrate, wherein the flow path device comprises: a cleaning membrane capable of collecting nucleic acid, a membrane storage section which is a space formed on the substrate, connected to the flow path and which receives the cleaning membrane, and a resin ring which is pressed into the membrane storage section, wherein the membrane storage section has a base surface formed between an upper and a lower surface of the substrate, such that the cleaning membrane is installed thereon, wherein the membrane storage section is divided into a cylindrical upper part of the membrane storage section and a lower part of the membrane storage section which has a smaller diameter than the upper part of the membrane storage section.wherein the base surface in between is designed as a sandwich-like boundary, wherein the flow path comprises a first flow path, connected to at least one valve mechanism, formed on the upper surface of the substrate and connected to a side wall of the upper part of the membrane storage section, and a second flow path, connected to at least one valve mechanism, formed on the lower surface of the substrate and connected to the lower part of the membrane storage section, the resin ring having an outer diameter greater than or equal to the inner diameter of the upper part of the membrane storage section, the cleaning membrane being fixed between the base surface and the resin ring, the outer diameter of the pressed-in resin ring being smaller than the outer diameter of the resin ring in a state where the resin ring is not contained in the membrane storage section,and the inner diameter of the upper part of the membrane storage section is substantially the same as the inner diameter of the upper part of the membrane storage section in the state in which the resin ring is not pressed into the membrane storage section, and the inner diameter of the upper part of the membrane storage section and the outer diameter of the pressed-in resin ring are the same at least at a position where the side wall of the upper part of the membrane storage section and the pressed-in resin ring are closest to each other.

[0020] Further features relating to the present disclosure will become clear from the description in the patent specification and the accompanying drawings. Moreover, aspects of the present disclosure are achieved and realized through elements, combinations of various elements, and aspects of the following detailed description and the accompanying claims.

[0021] The description of the patent specification merely shows typical illustrative examples and is not intended to restrict the claims of the present disclosure or the application examples in any way. Advantageous effects of the invention

[0022] According to the technology of the present disclosure, a treatment for removing impurities from a sample containing a nucleic acid and a treatment for purifying and collecting the nucleic acid can be efficiently carried out by a device, and a flow path chip with simple configurations can be obtained cost-effectively. Brief description of the drawings

[0023] They show: Fig. 1. A diagram of a treatment process of a nucleic acid purification procedure based on the standard Boom procedure. Fig. 2A a diagram of the in Fig. 1 treatment process shown and the composition of a sample solution used in the analysis, Fig. 2B a diagram of a reagent and solution transfer procedure used in the Fig. The cleaning treatment process shown in section 1 is used. Fig. 3A A concept diagram of a configuration example of a sample pretreatment system 301, which describes the treatment process in Fig. 1 is carried out by a flow path device, Fig. 3B a diagram of a configuration along the cross-section AA in Fig. 3A in a state where the flow path device is installed, Fig. 4A a top view of a flow path device (Example 1) 101, on which a known membrane installation method is applied, Fig. 4B a diagram of an AA cross-sectional configuration in Fig. 4A, Fig. 5A a top view of a flow path device (Example 2) 101, on which a known membrane installation method is applied, Fig. 5B a diagram of an AA cross-sectional configuration in Fig. 5A, Fig. 6A a top view of a flow path device (Example 3) 101, on which a known membrane installation method is applied, Fig. 6B a diagram of an AA cross-sectional configuration in Fig. 6A, Fig. 7A a diagram of quantitative assessment results of nucleic acid purification efficiency and collected liquid volumes in a case where a spin column and flow path device with a known configuration are used, Fig. 7B a diagram of the amount of DNA found in a sample by quantifying the amount of DNA in a sample Fig. 7A collected eluent, purification efficiency calculated by real-time PCR (DNA collection efficiency: dark (left-hand) bar graph of each procedure) and DNA leakage rate calculated by quantifying the amount of DNA contained in a sample solution (hereinafter referred to as waste fluid) that has passed through a purification membrane (light (right-hand) bar graph of each procedure), Fig. 8 a view (photograph) of a phenomenon in which a liquid pool of an eluent 403 is formed in a flow path device 101 (an example) with a membrane attachment structure based on an O-ring after the collection of the eluent near a notch section (a liquid appears to be trapped at the notch), Fig. 9A a top view of a flow path device 101 according to the present embodiment, Fig. 9B a diagram of an example of a cross-sectional configuration along AA in Fig. 9A, Fig. 10A a diagram of a state in which a cleaning membrane 201 is installed from an upper part 110 of the membrane storage section onto a base surface 109, Fig. 10B a diagram of a state in which a resin ring 204 is pressed into the upper part 110 of the membrane storage section, Fig. 10C a diagram of a cleaning membrane 201, which is attached between the base surface 109 and the resin ring, Fig. 11A a diagram of results from performing a nucleic acid purification experiment using the in Fig. 3A sample pretreatment system and flow path device 101 shown Fig. 9 and the quantitative assessment of the amount of eluent collected, Fig. 11B a diagram of results from performing a nucleic acid purification experiment using the in Fig. 3A sample pretreatment system and flow path device 101 shown in Fig. 9 and the quantitative assessment of DNA purification efficiency, Fig. 12A a diagram of a configuration of the upper surface of a flow path device 101 to which a spin column configuration is applied, Fig. 12B a diagram of an AA cross-sectional configuration in Fig. 12A, Fig. 13A a diagram (an enlarged photograph) of an example of the resin ring 204 which could not be pressed in and fastened and was deformed on the membrane storage section 104, Fig. 13B a ​​diagram (a photograph) of an example of the resin ring 204 which could not be pressed in and fastened and was deformed on the membrane storage section 104, Fig. 13C a table summarizing examples of outer diameters and the success / failure of pressing in and fastening the resin ring 204, Fig. 14A a diagram of an example of the internal configuration of a fluid supply mechanism 302, Fig. 14B a diagram of an example of the internal configuration of the fluid supply mechanism 302, which has a multi-way valve mechanism (V0) that can open to the atmosphere, Fig. 15 a diagram of a configuration example to achieve a function corresponding to that of the multi-way valve mechanism 3006 by combining several on-off valve mechanisms 3005, Fig. 16 a diagram of an example of the internal configuration of a reagent holding section 303, Fig. 17 A flowchart to explain a nucleic acid purification treatment in the Fig. 16 nucleic acid purification systems shown, Fig. 18 a diagram of another example of the internal configuration (modification example) of the reagent holding section 303 in the nucleic acid purification system, Fig. 19 a flowchart to explain a nucleic acid purification process in the Fig. 18 nucleic acid purification systems shown, Fig. 20 a diagram of an example of the system configuration of a sample pretreatment system 301, wherein a flow path device (nucleic acid purification device) 101 integrated into a reagent tank is installed, Fig. 21A a diagram of an example of the configuration of the upper surface near a reagent tank 3105, Fig. 21B a diagram of an example of the cross-sectional configuration along AA in Fig. 21A, Fig. 22 A flowchart (example) to explain a process from the in Fig. 20 nucleic acid purification systems shown performed nucleic acid purification treatment, Fig. 23 a diagram of an example of the overall configuration of a sample-to-response DNA analysis system consisting of a flow path device 101 and a sample pretreatment system 301, which enables nucleic acid extraction, nucleic acid purification and nucleic acid amplification / detection to be performed automatically from a biological sample, Fig. 24 a diagram of a configuration example of a nucleic acid extraction section 1201 in the sample-to-response DNA analysis system, Fig. 25 a flowchart to explain a nucleic acid extraction treatment by the sample-to-response DNA analysis system, Fig. 26 a diagram of a configuration example of the periphery of a nucleic acid purification section 1101 in the sample-to-response DNA analysis system, Fig. 27A a diagram of a configuration example of the periphery of a nucleic acid amplification section 1301, Fig. 27B a diagram of an example of the cross-sectional configuration along AA in Fig. 27A, Fig. 28 a flowchart to explain a nucleic acid amplification treatment by the sample-to-response DNA analysis system, Fig. 29A a diagram of a configuration example of the periphery of a membrane storage section 104 from above in each modification example, Fig. 29B a diagram of an example of the cross-sectional configuration along AA in Fig. 29A in a first modification example, Fig. 29C a diagram of an example of the cross-sectional configuration along AA in Fig. 29A in a second modification example, Fig. 29D a diagram of an example of the cross-sectional configuration along AA in Fig. 29A in a third modification example, Fig. 29E a diagram of an example of the cross-sectional configuration along AA in Fig. 29A in a fourth modification example, Fig. 30A a diagram of a state in which a sample solution 401 is conveyed by a continuous conveying process, and Fig. Figure 30B shows a diagram of a condition in which a cleaning fluid 402 is conveyed by the continuous conveying process. Description of embodiments

[0024] One embodiment of the present disclosure proposes a technology that enables highly efficient nucleic acid purification by allowing a small amount of reaction solution to pass without leakage through a purification membrane in a flow path device (a flow path chip) and be collected with high efficiency. The following patent specification first describes problems that became apparent for the first time during the investigation according to the present disclosure, and then describes features of a sample pretreatment system (a sample pretreatment device) according to one embodiment. Next, the description proceeds to modifications of the present embodiment. In the drawings of the present patent specification, functionally identical elements may be designated by the same numbers.It should be noted that, although the accompanying drawings show specific embodiments and implementation examples in accordance with the principles of the present disclosure, these serve to aid understanding of the present disclosure and should not be used to interpret the present disclosure restrictively. While the description is sufficiently detailed to enable those skilled in the art to implement the present disclosure, it should also be understood that other implementations and modes are possible, and changes to configurations and structures can be made, and various elements can be exchanged, without deviating from the scope of protection and technical intent of the present disclosure. Therefore, the following description should not be interpreted as being limited to this. (1) Further discussion of problems in nucleic acid purification (detailed investigation)<Behandlungsprozess der Nukleinsäurereinigung>

[0025] Fig. Figure 1 is a diagram illustrating the treatment process of a standard nucleic acid purification procedure. First, a sample solution containing sample DNA is forced through a purification membrane to cause the DNA to bind to the membrane (Process I). If a nucleic acid purification procedure based on the Boom method is used, a chaotropic agent such as a guanidinium salt is added to a sample solution obtained by subjecting a biological sample to cell lysis. Next, a purification fluid is forced through the purification membrane to wash away impurities adhering to the membrane (Process II). Generally, an ethanol-containing solution is used as the purification fluid, and the purification can be performed multiple times.Furthermore, the purification membrane is dried to remove a purification fluid component (Process III). Finally, the DNA is bound by the aforementioned procedure, and a DNA eluent is passed through the purification membrane, from which the impurities and the purification fluid component have been removed, thus collecting the target DNA. (Process IV)

[0026] Fig. 2A is a diagram that shows the one in Fig. Figure 1 shows the treatment process and the composition of a sample solution used in the analysis. Furthermore, Fig. 2B a diagram showing a reagent and a solution delivery procedure used in the Fig. The purification process shown in Figure 1 is used. Here, a human genome sample (2800M) is used as the sample, and a QIAamp DNA Investigator Kit (Qiagen), a standard kit for laboratory procedures, is used as the lysis and purification reagent. In a spin column procedure performed in a laboratory, the conveyance of each solution is achieved through a centrifugal process. <Probenvorbehandlungssystem zur Nukleinsäurereinigung>

[0027] Fig. 3A is a concept diagram showing a configuration example of a sample pretreatment system 301, which is described in Fig. The treatment process shown in Figure 1 is carried out by a flow path device. The sample pretreatment system 301 comprises a liquid supply mechanism 302, a reagent holding section 303, a chip holding section 304 made of polyetheretherketone (PEEK), a PEEK cover 305 with a connector 306, and a pipeline 307. The liquid supply mechanism 302 consists of a liquid supply power source and a pressure control mechanism, which allows a solution delivery pressure to be applied at any given time, and the liquid supply mechanism 302 is connected to the reagent holding section 303 via the pipeline 307.The reagent holding section 303 has a receiving area for a solution and an area for collecting the solution after conveying, each connected to the flow path device 101 via the pipeline 307 and capable of conveying and collecting any solution in response to a pressure increase and decrease process controlled by the liquid supply mechanism 302. Specific examples of the internal system configuration of the liquid supply mechanism 302 and the reagent holding section 303 are described later.

[0028] Fig. 3B is a diagram showing a configuration along cross-section AA in Fig. Figure 3A shows the flow path device installed. The flow path device 101 is installed so that it can be held in a predetermined position between the chip holding section 304 and the cover 305. When the flow path device 101 is installed, a first vent 107 and a second vent 108 provided in the flow path device 101 are connected to the connector 306 provided in the cover 305.

[0029] The fluid supply power source of the fluid supply mechanism 302 can apply a pressure sufficient to deliver the solution without exceeding the pressure resistance of the chip. For example, a diaphragm pump or a syringe pump capable of controlling the applied pressure within a range of 0.1 kPa to 250 kPa can be used. Although an example of a fluid supply pressure source using atmospheric pressure is shown here, mechanical compression, centrifugal force, or the like can also be used.

[0030] The material of the chip holding section 304 and the cover 305 may be other than PEEK and is not particularly restricted as long as it has sufficient stiffness to support the flow path device 101. Besides PEEK, a material such as a resin material like polycarbonate (PC) or acrylic (PMMA), a metallic material like aluminum or stainless steel, or the like may be selected. Considering a case where a solution such as a surfactant or salt contained in the sample solution, or ethanol contained in the cleaning fluid, leaks, the material is preferably chemically resistant to these solutions. <Strömungswegvorrichtung zur Nukleinsäurereinigung>

[0031] Next, the flow path device 101 used here (for consideration) will be described. Fig. 4A, Fig. 5A and Fig. Figure 6A shows top views of the flow path device (Examples 1 to 3) 101, on which a known membrane installation method is applied. Fig. 4B, Fig. 5B and Fig. 6B are views that represent AA cross-sectional configurations of the Fig. 4A, Fig. Show 5A or 6A.

[0032] Each flow path device 101 typically comprises a substrate 102 with a flow path structure, sealing strips 103 made of polyolefin (PO) attached to the upper and lower surfaces of the substrate 102, and a cleaning membrane 201 installed in a membrane storage section 104. First, a membrane storage section 104 with an upper part measuring 6 mm in diameter and 1 mm in depth and a lower part measuring 4 mm in diameter and 2 mm in depth, which are coaxial and have different dimensions, a first flow path 105 and a second flow path 106 measuring 1 mm in width and 1 mm in depth, and a first vent 107 and a second vent 108 measuring 1 mm in diameter are formed by cutting on the PC substrate 102 measuring 20 mm vertically, 50 mm horizontally, and 4 mm thick.The first vent 107 is located at an end point of the first flow path 105, the second vent 108 is located at an end point of the second flow path 106, and the first vent 107 and the second vent 108 are formed as through-holes in the substrate 102, with their upper surfaces being opened. The first flow path 105 is formed on the upper surface of the substrate 102 and connected to a side wall of an upper part 110 of the membrane storage section. The second flow path 106 is formed on the lower surface of the substrate 102 and connected to a side wall of a lower part 111 of the membrane storage section.

[0033] The Fig. 4A and Fig. Figure 4B shows a configuration of the flow path device 101 without the membrane mounting structure, i.e., a configuration in which the cleaning membrane 201 rests on a base surface 109. Before the sealing tape 103 is applied to the upper surface of the substrate 102, the cleaning membrane 201 (silica membrane filter, GF / F, Whatman) with a diameter of 6 mm is installed by pressing it from the side of the upper part 110 of the membrane storage section towards the base surface 109. After confirming that the installation has been achieved without any outer edge section of the cleaning membrane 201 remaining unsupported, the sealing tape 103 is applied to the upper surface of the substrate 102, thus forming the flow path device 101 without the membrane mounting structure.

[0034] The Fig. 5A and Fig. Figure 5B shows a configuration of a flow path device 101 with a membrane fastening structure based on a notched O-ring. Before the sealing strip 103 is applied to the upper surface of the substrate 102, the cleaning membrane 201 (silica membrane filter, GF / F, Whatman) with a diameter of 6 mm is installed by pressing it from the side of the upper part 110 of the membrane storage section towards the base surface 109. A notched rubber O-ring 202 (outer diameter 6 mm x inner diameter 4 mm) is then inserted such that the notched section aligns with the first flow path 105, without obstructing the first flow path 105.After confirming that the cleaning membrane 201 and the O-ring 202 could be installed without floating, a sealing strip 103 is attached to the upper surface of the substrate 102, and a 10 mm square PMMA block is attached to the sealing strip 103 at the top of the membrane storage section 104 to compress the O-ring. As described above, the flow path device 101 is formed with a membrane mounting structure based on the O-ring.

[0035] The Fig. 6A and Fig. Figure 6B shows a configuration of a flow path device 101 with an adhesive-based membrane attachment structure. Before the sealing tape 103 is applied to the upper surface of the substrate 102, a double-sided tape (#9969, 3M) is applied to the base surface 109. The 6 mm diameter cleaning membrane 201 (silica membrane filter, GF / F, Whatman) is installed by pressing it from the side of the upper part 110 of the membrane storage section towards the base surface 109, thus bonding the cleaning membrane 201 to the base surface 109. After confirming that the cleaning membrane 201 has been installed without the outer edge section floating, the sealing tape 103 is applied to the upper surface of the substrate 102, forming the flow path device 101 with the adhesive-based membrane attachment structure. <Experimentelle Ergebnisse>

[0036] A nucleic acid purification experiment was performed using the [methods / methods] described in the Fig. 3A and Fig. 3B shown sample pretreatment systems and the one described in the Fig. 3A, Fig. 3B, Fig. 5A and Fig. Flow path devices 101 shown in 5B are executed. Fig. 7A and Fig. Figure 7B contains diagrams showing the results of the quantitative assessment of the amounts of eluent collected and the DNA purification efficiency of the nucleic acid purification experiment. As shown in Fig. As shown in Figure 7A, the average amount of eluent ultimately collected by the spin column method was 17 µl, while the average amount of eluent collected by the chip devices was at most 10 µl. In particular, the amount of liquid collected in the case of the flow path device 101 with the membrane mounting structure based on the notched O-ring was reduced to at most 1 / 10 of the amount of liquid introduced. Fig. Figure 8 is a view (photograph) showing a phenomenon in which a pool of liquid of eluent 403 is formed after the collection of the eluent near the notch section in the flow path device 101 (an example) with the membrane attachment structure based on the O-ring (a liquid appears to be trapped at the notch).

[0037] Fig. Figure 7B shows the cleaning efficiency (DNA collection efficiency) determined by quantifying the amount of DNA collected in the Fig. The amount of eluent collected in 7A was calculated by real-time PCR, using a dark (left) bar graph for each procedure. Additionally, it shows Fig. 7B The DNA leakage rate, calculated by quantifying the amount of DNA contained in the sample solution (hereinafter referred to as waste liquid) that passed through the purification membrane, is shown by a light (right) bar graph for each procedure. The DNA leakage rate was assessed by introducing the waste liquid into a new spin column, performing the purification treatment according to the present embodiment, and quantifying the DNA content by real-time PCR. As described in Fig. As shown in Figure 7B, it was found that for the spin-column method, the cleaning efficiency averaged approximately 50%, and DNA leakage was 0% in all six trials. For the chip device, the cleaning efficiency was at most half that of the spin-column method in all cases, and the DNA leakage rate was high. Additionally, with the flow-path device 101 and its adhesive-based membrane attachment structure, an instance was observed where the incorporation of a PCR inhibitor into the collected eluent was suspected.

[0038] The reason why the amount of liquid collected in the chip device was less than in the spin column method is presumably due to the different liquid supply methods and the liquid residues on the ring observed in the flow path device 101 with the membrane attachment structure based on the O-ring (which merely involves the insertion of an elastic body as opposed to the pressing-in process described later in the present disclosure). Because the cleaning membrane is made of a porous material and has a certain water retention capacity, the amount of eluent remaining in the membrane depends on the material and density of the membrane as well as the liquid supply pressure.The purification membrane 201 used in this study is a silica membrane that has essentially the same density in both the spin column method and the chip device. Therefore, it is assumed that the purification membrane 201 did not contribute to the differences in the collected liquid volumes observed in this study. On the other hand, the liquid feed pressure differed between the spin column method and the chip device because the solution delivery methods were different. In the spin column method, the solution introduced into the column was forced by centrifugal force through the purification membrane and conveyed to the collection tube. At this point, a force of 20,000 g (corresponding to a maximum delivery pressure of approximately 100 kPa to 150 kPa under the conditions of this study) was constantly applied to the solution.On the other hand, the chip apparatus used for this investigation employed a liquid supply via air pressure, with the supply pressure of the eluent being a maximum of only about 50 kPa. Therefore, it is assumed that in the scheme using the flow path device, the sample solution held in the membrane was probably not released into the flow path, and the amount of collected liquid decreased.

[0039] Regarding purification efficiency, the waste fluid in the case of the spin column method contained essentially no DNA. This indicates that the sample solution was able to pass through the purification membrane without leakage and that the binding efficiency between the purification membrane and the DNA was very high. On the other hand, in the case of flow path device 101 without a membrane attachment structure, there was no mechanism for securing the purification membrane. Therefore, it is assumed that membrane displacement or leakage from an outer circumferential section occurred. Since membrane displacement was suppressed in flow path device 101 with the membrane attachment structure based on the O-ring compared to flow path device 101 without a membrane attachment structure, it is assumed that the DNA leakage rate was reduced and there was a tendency toward improved purification efficiency.However, the purification efficiency was lower than with the spin column method, suggesting that the DNA leakage suppression force, i.e., the membrane attachment force, was insufficient.

[0040] A high DNA leakage rate and a remarkably low cleaning efficiency were also observed in the flow path device 101, which features the adhesive-based membrane attachment structure. This is attributed to insufficient adhesion, resulting in an unintended gap or clogging of the cleaning membrane by the adhesive. PCR inhibition did not occur except in the flow path devices using adhesive 203. Therefore, contamination by adhesive 203 was suspected as the cause of the PCR inhibition. It was confirmed that adhesive 203, used in this study, is chemically resistant to a cleaning reagent. However, adhesive 203 was not a product with guaranteed biocompatibility. Therefore, a negligible amount of eluted components, otherwise insignificant, could have acted as an inhibitory factor for the PCR analysis.Furthermore, it is assumed that because the assembly of the flow path device 101 in this study was performed manually, the adhesion could not be controlled with satisfactory reproducibility, the adhesive component likely protruded from the base or was unevenly attached to it, and a PCR-inhibiting substance could have entered it through contact with the solution. To solve this problem, the process for manufacturing the device must be more precisely controlled, and an adhesive component that does not contain a PCR-inhibiting substance must be used. However, considering the dimensions of the membrane storage section 104 and the base area 109 (a section that serves as an attachment edge) of the flow path device 101 according to this study, it is expected that it will be difficult to stably prototype this in a laboratory.At the same time, the fact that the cleaning reagent used in this study contains a highly concentrated solution of ethanol, an organic solvent, and that the selection of adhesives and double-sided tapes is considerably limited, is also an important point to consider. (2) Embodiment of the present disclosure

[0041] A sample pretreatment system 301 and a flow path device 101 according to the following embodiment, which are solutions to the problems discovered in the preceding considerations (investigations), are described. <System und Strömungswegvorrichtung>

[0042] The sample pretreatment system 301 is equipped with the in Fig. The system shown in 3A is identical. Fig. Figure 9A is a top view of the flow path device 101 according to the present embodiment. Furthermore, Fig. 9B a diagram showing a cross-sectional configuration along AA in Fig. Figure 9A shows the flow path device 101, which comprises a membrane storage section 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 strips (film layers) 103 attached to the upper and lower surfaces of the substrate 102, and a cleaning membrane 201 installed in the membrane storage section 104. The membrane storage section 104 consists of an upper part 110 and a lower part 111 with different inner diameters, with a base surface 109 arranged in a sandwich-like manner as a boundary, such that the base surface 109, on which the cleaning membrane 201 can be installed, is formed in the middle between the upper and lower surfaces of the substrate 102.The inner diameter of the upper part 110 of the membrane storage section is essentially equal to the diameter of the cleaning membrane 201, and the inner diameter of the lower part 111 of the membrane storage section is smaller than the diameters of the cleaning membrane 201 and the upper part 110 of the membrane storage section. The first vent 107 is located at one end point of the first flow path 105. The second vent 108 is located at one end point of the second flow path 106. Both the first vent 107 and the second vent 108 are, for example, formed as through-holes in the substrate 102, and their upper surfaces are open. The first flow path 105 is formed on the upper surface of the substrate 102 and connected to a side wall of an upper part 110 of the membrane storage section.The second flow path 106 is formed on the lower surface of the substrate 102 and is connected to a side wall of a lower part 111 of the membrane storage section.

[0043] In particular, the flow path device 101 according to the present embodiment is formed by cutting the membrane storage section 104 with an upper section having a diameter of 6 mm x a depth of 1 mm and a lower section having a diameter of 4 mm x a depth of 2 mm, which are coaxial and have different dimensions, a first flow path 105 and a second flow path 106 with a width of 1 mm x a depth of 1 mm and a first vent opening 107 and a second vent opening 108 with a diameter of 1 mm on the substrate 102 made of PC with a vertical length of 20 mm x a horizontal length of 50 mm and a thickness of 4 mm.

[0044] The materials of substrate 102 and sealing strips 103 are not particularly limited, as long as they are commonly used in technical applications. For example, polypropylene (PC), polypropylene (PP), a cyclic olefin polymer (COP), a cyclic olefin copolymer (COC), polyethylene terephthalate, and polyurethane can be used as materials with low DNA adsorption. Furthermore, the amount of adsorption can also be suppressed by modifying the surface so that it is negatively charged. Other examples of suitable materials include glass materials such as glass, fused silica, fused quartz, synthetic quartz, aluminum oxide, sapphire, ceramics, forsterite, and photosensitive glass; plastics such as polyester resins, polystyrene, polyethylene resins, dimethyl polysiloxane (PDMS), nylon, acrylic resins, fluorinated resins, polycarbonate resins, polyurethane resins, methylpentene resins, phenolic resins, melamine resins, epoxy resins, and vinyl chloride resins, as well as any combination thereof.The flow path device 101 can be formed by applying the sealing strips (film layer) 103 made of the material described above to the top and bottom of the substrate 102, wherein the first flow path 105, the second flow path 106 and the membrane storage section 104 are formed thereon (whereby the sealing strips 103 are adhered to it by an adhesive that does not inhibit PCR) and at least partially sealing the openings formed by the flow paths and the like (performing at least one sealing except for the parts corresponding to the first vent opening 107 and the second vent opening 108). <Verfahren zur Befestigung der Reinigungsmembran>

[0045] The Fig. Figures 10A to 10C are diagrams explaining a method for installing the cleaning membrane 201. Before the sealing tape 103 is attached to the upper surface of the substrate 102, the cleaning membrane 201, having a diameter P and a thickness Q, is installed on the base surface 109 of the upper part 110 of the membrane storage section, having a diameter R and a depth D. Fig. 10A). Furthermore, the resin ring 204 with an outer diameter a, an inner diameter b and a height c (where a > R) is pressed in to secure the cleaning membrane 201 between the resin ring 204 and the base surface 109 ( Fig. 10B and Fig. 10C). After confirming that the inner diameter of the upper part 110 of the membrane storage section and the outer diameter of the resin ring 204 are equal (R' = a': R' represents the diameter of the upper part 110 of the membrane storage section after pressing on the resin ring 204, and a' represents the outer diameter of the resin ring 204 after pressing onto the upper part 110 of the membrane storage section), and that the cleaning membrane 201 and the resin ring 204 could be installed without floating, the sealing strip 103 is attached to the upper surface of the substrate 102 to form the flow path device 101. It should be noted that the height of the resin ring 204 is preferably such that the first flow path 105 is not blocked in an assembled state ( Fig. 10C). In particular, it is desirable that the upper surface (an end surface on the side where the resin ring 204 is not in contact with the cleaning membrane 201) of the resin ring 204 lies on a side that is located at least near the connection section between the upper part 110 of the membrane storage section and the first flow path 105 below the bottom surface of the first flow path 105 (thickness T of the chip - (depth of the first flow path 105) ≥ Q + c). In other words, it is desirable that the upper surface of the resin ring 204 is located below the bottom surface section of the first flow path 105. However, it is also desirable that a side surface of the resin ring 204 is in contact with a side surface of the membrane storage section 104 over as large an area as possible.Therefore, it is only necessary to determine the height of the resin ring 204 such that the upper surface of the resin ring 204 corresponds to the height of the bottom surface of the first flow path 105. In this way, it can be prevented that the pumped solution remains in the space between the resin ring 204 and the membrane storage section 104, and the flow path resistance can also be reduced, allowing a small amount of the solution to be collected without loss.

[0046] In the technology according to the present disclosure, "pressing" means a fit condition that corresponds to a mechanical fit criterion from pressing to pressing. In other words, after completion of the pressing, the membrane storage section 104 and the resin ring 204 cannot be moved relative to each other and are in a state in which it is possible to remove them from each other if a strong force is applied, or it is not possible to take them apart without breaking parts. In the case of the membrane fastening structure based on the O-ring, as presented in PTL 10 or the preceding discussion (consideration of problems), the O-ring 202 is elastically deformed (its shape is restored if an external force is removed).Therefore, after being placed on the upper part 110 of the membrane storage section, the O-ring returns to its position away from the membrane, thus counteracting the external force of placement. Simultaneously, a frictional force acts between the outer circumference of the O-ring 202 and the side wall of the upper part 110 of the membrane storage section. Although the O-ring 202 does not pop out once placed, the force with which it presses down the cleaning membrane is therefore weak. Thus, in the technology of the present disclosure, this does not correspond to a "press-fit condition".

[0047] On the other hand, when the resin ring 204 is inserted (pressed into) the upper part 110 of the membrane storage section, it undergoes plastic deformation (the deformation remains even after the external force is removed, and the shape is not restored to its original state) and is held in place by a frictional force against the side wall of the upper part 110 of the membrane storage section. Therefore, a force acting in a direction that depresses the cleaning membrane 201 is maintained in the membrane storage section 104. At this time, the resin ring 204 can be moved from a depressed state to a pressed-in state and held in place by appropriately adjusting its dimensional range relative to the upper part 110 of the membrane storage section. In this way, a high membrane retention force can be achieved.Whether the membrane fastening structure was implemented by pressing the resin ring 204 into the flow path device 101 can be confirmed at least by the fact that, at the position where the side wall of the upper part 110 of the membrane storage section and the resin ring 204 are closest to each other in the assembled state, R' = a' holds, and more rigorously, at least by carefully observing the flow path device 101 in the assembled state and observing |R' - a'l < 1 µm (no more than the resolution of the optical microscope) at the position where the side wall of the upper part 110 of the membrane storage section and the resin ring 204 are closest to each other. The implementation of the technology of the present disclosure can also be confirmed by measuring the inner diameter of the upper part 110 of the membrane storage section and the outer diameter of the resin ring 204 in the unassembled state. Fig. 10B) and in the composite state ( Fig. 10C) and confirm that R / R' (≈ 1) > a' / a. In other words, the feature that the upper part 110 of the membrane storage section is hardly deformed in the unassembled and assembled states due to its high stiffness, and that the resin ring 204, with an outer diameter a larger than the inner diameter R of the upper part 110 of the membrane storage section, is installed in such a way that it contracts, is an important feature of the technology of the present disclosure. It should be noted that, although known examples of a membrane fastening method based on pressing in using a resin ring also include the spin column configuration exemplified in PTL 2, where the inner diameters of the spin column in the unassembled and assembled states are defined as S and S' respectively, and the outer diameters of the ring are defined as A and S' respectively, the technology of the present disclosure is not a significant feature of the technology of the present disclosure.A' is defined, S / S' < A' / A (≈ 1) is satisfied, and a relationship results that is opposite to that in the technology of the present disclosure. This is due to the fact that, in the case of the spin column configuration, the spin column is a thin container with low stiffness and can be deformed by pressing in the ring. For example, in the case of a spin column included in the QIAamp DNA Investigator Kit (Qiagen), P = 7 mm, S = approximately 6.4 mm to approximately 6.7 mm, A = approximately 7 mm to approximately 7.2 mm, and S' = A' ≈ A are satisfied.

[0048] In particular, according to the present embodiment, the cleaning membrane 201 (silica membrane filter, GF / F, Whatman) with a diameter of 6 mm x a thickness of 0.4 mm and the resin ring 204 made of PE with an outer diameter of 6.2 mm x an inner diameter of 4 mm x a height of 0.5 mm are installed in the membrane storage section 104 in the flow path device 101.

[0049] It is only required that the purification membrane 201 be a membrane capable of retaining particles of at least 100 µm. The film thickness is preferably at least 1 µm. Furthermore, because DNA can be collected more effectively with a smaller pore size, it is desirable that the purification membrane 201 be capable of retaining particles of at least 10 µm, preferably at least 1 µm, and even more preferably at least 0.1 µm. The membrane type may differ from a silica membrane, and a purification membrane made of a solid starting material may be used, containing as its main component cellulose, which can adsorb DNA, carboxylated particles, or an ion-exchange resin. If the volume of the purification membrane is too small, the number of biomolecules that can be adsorbed decreases.On the other hand, if the volume is too high, there are concerns that unintended molecular adsorption or a deterioration in the conveying efficiency of the solution is likely to occur during purification or a subsequent step, so the volume must be appropriately determined, although the size is not particularly limited.

[0050] The material of the resin ring 204 can be different from PE, and it is desirable to use a material that does not deform elastically, such as a polyester resin, polystyrene, polyethylene resin, nylon, acrylic resin, fluorocarbon resin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, or vinyl chloride resin. The resin ring 204 does not have to be made of a resin. Specific physical properties include that the Young's modulus of the material is greater than or equal to 5 MPa and less than or equal to 1 GPa, that the elastic modulus is greater than or equal to 100 kPa, and that the elastic yield strength is less than 100%.

[0051] The outer diameter a of the resin ring 204 can be arbitrary, as long as the resin ring 204 can be fitted into the upper part 110 of the membrane storage section and can be secured by press-fitting. In particular, the investigations of the present inventors have shown that the outer diameter a of the resin ring 204 is preferably larger than the inner diameter R of the upper part 110 of the membrane storage section and lies within a range that satisfies 0 ≤ a - R < 400 µm or (a - R) / R ≤ 0.2. Details of the outer diameter a of the resin ring 204 are described later.

[0052] Although it is only necessary that the inner diameter b of the resin ring 204 be such that the solution can pass through it, it is desirable that the inner diameter b be substantially equal to the inner diameter r of the lower part of the membrane storage section. This ensures that the solution is in contact with the cleaning membrane 201, allowing it to efficiently pass through the lower part 111 of the membrane storage section and maximizing the amount of solution that can be collected. Furthermore, it is desirable that the difference between the outer diameter a and the inner diameter b of the resin ring 204 be as small as possible. In particular, it is desirable that the difference be less than or equal to approximately 2.5 mm. This prevents the solution from being trapped and remaining on the upper surface of the resin ring 204, thus allowing for more efficient solution collection. <Experimentelle Ergebnisse>

[0053] The Fig. 11A and Fig. 11B are diagrams showing the results of performing a nucleic acid purification experiment using the [equation / method] described in [reference]. Fig. 3A sample preparation system and the one shown in Fig. Figure 9 shows the flow path device 101 and the quantitative assessment of the amount of eluent collected and the DNA purification efficiency. It should be noted that the process according to the present embodiment, as previously mentioned in “Further Consideration of the Problems of Nucleic Acid Purification”, is carried out using the sample solution and treatment process as described in Fig. 1 was shown, executed.

[0054] As in Fig. As shown in Figure 11A, the amount of eluent collected by the chip apparatus of the technology of the present disclosure averaged 13 µl. Although the amount of eluent collected was somewhat less than in the spin-column method, a satisfactory collection efficiency (greater than or equal to 50%) was obtained compared to the conventional chip apparatus. The dark (left-hand) bar graph of each scheme in Fig. 11B is a diagram that illustrates the amount of DNA present in the body by quantifying it. Fig. Figure 11A contains the collected eluent, and the purification efficiency (DNA collection efficiency) calculated by real-time PCR is shown. The light (right-hand) bar graph of each scheme in Fig. Figure 11B shows the DNA leakage rate calculated by quantifying the amount of DNA contained in the sample solution (hereinafter referred to as waste liquid) that passed through the purification membrane. As in Fig. As shown in Figure 11B, in a case where the chip device according to the present embodiment was used, the problem of a high DNA leakage rate, which occurred with the conventional chip device, was solved, and a purification efficiency was obtained that is comparable to that of the spin column method. It should be noted that in the purification treatment process according to the present embodiment, the required solution delivery pressure is a maximum of 65 kPa, which is at the same level as with the conventional chip device.As described above, according to the present embodiment, strong membrane fixation without sample leakage and a stable supply of a small amount of solution can be achieved by using the chip device and the small apparatus with simple configurations, thereby achieving high purification efficiency with a performance comparable to that of the spin column method. The aforementioned technical effects are similar in the embodiments and derivatives described below. (3) Consideration of the problems of the flow path device to which a spin column configuration is applied

[0055] As in Fig. As shown in Figure 11B, high cleaning efficiency can be achieved even when using the spin column configuration. Here, problems of the flow path device 101, to which the spin column configuration is applied—which is the only known example of achieving membrane fixation by injecting a resin ring—are investigated and considered, as well as advantages of the flow path device 101 (configuration without the use of the porous retaining element 205) according to the present disclosure, which is set forth in Figure 11B. Fig. Figure 9 will be further clarified.

[0056] Fig. Figure 12A is a diagram showing a configuration of the upper surface of the flow path device 101, to which the spin column configuration is applied. Fig. 12B is a diagram showing the AA cross-sectional configuration in Fig. Figure 12A shows the flow path device 101 used for consideration, which has a porous retaining element 205 with a diameter of 6.7 mm x a thickness of 2 mm, a cleaning membrane 201 with a diameter of 7.0 mm x a thickness of 0.4 mm and a resin ring 204 made of PE with an outer diameter a (seven types of a = 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 and 7.1) x an inner diameter of 5 mm x a height of 0.5 mm in the membrane storage section 104.The flow path device 101, to which the spin column configuration is applied, is formed by cutting a tapered membrane storage section 104, in which the diameter is continuously changed from 7.1 mm at the upper part to 5 mm at the lower part, the first flow path 105 and the second flow path 106 with a width of 1 mm x a depth of 1 mm and the first vent opening 107 and the second vent opening 108 with a diameter of 1 mm on the substrate 102 made of PC and a vertical length of 20 mm x a horizontal length of 50 mm x a thickness of 5 mm. The first vent 107 is located at one end point of the first flow path 105. The second vent 108 is located at one end point of the second flow path 106. Both the first vent 107 and the second vent 108 are formed as through-holes in the substrate 102, and their upper surfaces are open.The first flow path 105 is formed on the upper surface of the substrate 102 and connected to the side wall of the membrane storage section 104. The second flow path 106 is also formed on the lower surface of the substrate 102 and connected to the side wall of the membrane storage section 104.

[0057] The porous retaining element 205 serves to hold the cleaning membrane 201 in a specific position within the membrane storage section 104. It is deformed so that it collapses in the centripetal direction when pressed into the spin column and flow path device 101. First, the porous retaining element 205 was pressed into and secured within the membrane storage section 104 such that it was horizontal to the surface direction (Procedure 1). The cleaning membrane 201 was then placed onto the secured porous retaining element 205 (Procedure 2), and the resin ring 204 was pressed in and secured so that it was horizontal to the surface direction (Procedure 3). During the processes of Procedures 1 to 3 for securing the cleaning membrane, several problems were found to occur in practice, which are described below. (i) The first problem with the procedure for securing the cleaning membrane when using the flow path device 101 based on the spin-column configuration, which features the porous retaining element 205, is that it is difficult to control the securing position of the porous retaining element 205 in procedure 1. The porous retaining element 205 deforms easily in response to a load from the side wall of the membrane storage section 104 during pressing. Therefore, if the application of pressure from the upper part continues even after reaching the predetermined securing position, the porous retaining element 205 extends over the connecting section between the membrane storage section 104 and the second flow path 106, thereby increasing the flow path resistance. Eventually, the porous retaining element 205 reaches the bottom part of the membrane storage section 104 and completely blocks the second flow path 106.If the sample solution 401 is introduced from the first flow path 105 in this state, it is not possible to collect the sample solution 401 efficiently due to the flow path resistance. If, on the other hand, the application of pressure from the upper part is interrupted before reaching the predetermined attachment position, the resin ring 204 extends over the connection section between the membrane storage section 104 and the first flow path 105 when the cleaning membrane 201 and the resin ring 204 are attached, and the flow path resistance increases as a result. If the sample solution 401 is introduced from the first flow path 105 in this state, it may not be able to come into efficient contact with the cleaning membrane 201, or an unintended diversion of the sample solution 401 into an interstitial space may occur.

[0058] If the flow path is provided on the substrate 102 and the cleaning membrane 201 is installed in it, as with the flow path device 101 (see Fig. 9) According to the present disclosure, a further simplification, reduction in size, and cost reduction of the flow path device can generally be expected because the thickness of the substrate 102 is thinner, so it is preferable to provide the flow path in the surface direction of the cleaning membrane 201. On the other hand, in the case of the spin column method, there is no incentive to provide the flow path in the surface direction of the cleaning membrane, which is why the disadvantage referred to as the first problem does not occur. In other words, in the case of the spin column method, there are no disadvantages to using the porous retaining element 205.

[0059] (ii) The second problem with the procedure for securing the cleaning membrane when using the flow path device 101 with the spin-column configuration, which includes the porous retaining element 205, is that it is not possible to press and secure the resin ring 204 into the membrane storage section 104 by dimensional control at the same level as in the spin-column method in the preceding procedure 3. As described above for the configuration of the flow path device 101 according to the present embodiment ( Fig. As described in Figure 9, the resin ring 204 is deformed in response to a stress from the side wall of the membrane storage section 104 when it is pressed into the membrane storage section 104. Because the resin ring 204 is not made of an elastic material such as rubber, it either collapses without entering the membrane storage section 104, or cracks form on the substrate 102 near the membrane storage section 104, leading to a fracture if the resin ring 204 is too large in relation to the membrane storage section 104.

[0060] The Fig. 13A and Fig. 13B are views (photographs) showing an example of the resin ring 204 which could not be pressed in and fastened and was deformed on the membrane storage section 104. Fig. Table 13C summarizes examples of outer diameter dimensions and the success / failure of pressing in and securing the resin ring 204. A press-in failure as shown in Fig. As shown in Figure 13A, it was observed that when the inner diameter R of the membrane storage section 104 at the attachment point of the cleaning membrane 201 was approximately 6.5 mm and the outer diameter a of the resin ring 204 was greater than or equal to 6.9 mm, i.e., (a - R) was greater than or equal to at least 400 µm. With respect to this relationship, the same results were obtained in a case where the inner diameter b and the thickness (height) c of the resin ring 204 were changed, as well as in a case where the membrane storage section 104 ( Fig. 9) instead of the porous retaining element 205, a substructure section was also obtained. At the same time, at least in one case with 1 mm ≤ R ≤ 7 mm, an event was observed in which the pressing in and fastening failed within a region that met a - R ≥ 400 µm. The main reason for the failure of the pressing in and fastening is presumably that the stiffness of the substrate 102 is high compared to the spin-column method and the stress during the pressing in of the resin ring 204 cannot be absorbed by either the ring or the substrate 102.

[0061] In the case of the spin column process, the container holding the cleaning membrane 201 is thin and has low stiffness, which is why it is not necessary to consider a dimensional range as in the flow path device 101 according to the present disclosure. As described above, the flow path device 101 of the present disclosure can be manufactured stably at a practical level by designing the inner diameter R of the membrane storage section 104 and the resin ring 204 such that they satisfy, for example, 0 ≤ a - R < 400 µm. This dimensional range can be selected depending on physical properties such as materials, stiffness, and bending moment of the substrate 102 and the resin ring 204 and is preferably determined in a suitable manner.

[0062] (iii) Another problem with the use of the flow path device 101 based on the spin column configuration, which includes the porous retaining element 205, is that the eluent 403 is trapped by the porous retaining element 205, reducing the amount of liquid collected. The porous retaining element 205 is made of a porous material to collect the sample solution 401 that has passed through the cleaning membrane 201 while the cleaning membrane 201 is held in a specific position within the membrane storage section 104. The porous retaining element 205 has a water retention capacity that depends on the material, pore diameter, volume, and the like.In the case of the spin column method, because the eluent 403 introduced into the column is caused by a centrifugal force (for example, 20000 g) to pass through the purification membrane 201, most of the eluent 403 recovered within the porous holding element 205 can also be collected, and this problem does not occur.

[0063] On the other hand, if the liquid is supplied, for example, by a small general-purpose pneumatic pump, as in the technology of the present disclosure, without the use of centrifugal force, the eluent 403 captured by the porous retaining element 205 cannot be collected, and the amount of collected liquid decreases considerably. Furthermore, during the execution of the cleaning flow of the in the Fig. 1, Fig. 2A and Fig. In the Boom procedure shown as an example in Figure 2B, the sample solution 401 and the cleaning fluid 402 remain in the porous retaining element 205. This can lead to these components being mixed into the eluent 403 after collection, thus contaminating the eluent 403. Although this event (in the case of the Boom procedure) has been improved to some extent by using a hydrophobic porous retaining element 205, for example, made of PTFE, by increasing the pores, or by reducing the thickness and thus the volume of the porous retaining element 205, it has been found that such measures have not achieved a sufficient improvement in the case of the configuration according to the present disclosure. Therefore, it is preferable to choose a configuration that does not use the porous retaining element 205 when implementing the flow path device 101 according to the present disclosure.

[0064] (iv) As described above, it can be determined that the application of the spin column configuration to the flow path device 101 is not suitable and preferably the configuration of the flow path device 101 according to the present embodiment described above is used. (3) Details of the liquid supply mechanism and the reagent holding section

[0065] Detailed configurations of the liquid supply mechanism 302 and the reagent holding section 303 in the sample pretreatment system 301 according to the present embodiment are described here. <Konfiguration des Flüssigkeitszufuhrmechanismus 302>

[0066] Fig. Figure 14A is a diagram showing an example of the internal configuration of the fluid supply mechanism 302. Fig. Figure 14B is a diagram showing an example of the internal configuration of the fluid supply mechanism 302, which has a multi-way valve mechanism (V0) that is able to open to the atmosphere.

[0067] According to Fig. 14A The liquid supply mechanism 302 comprises a first connection port 3001 and a second connection port 3002 connected to the reagent holding section 303, a liquid supply power source 3003 connected to the first connection port 3001 to provide a liquid supply pressure, and a pressure control mechanism 3004 that adjusts the liquid supply pressure provided by the liquid supply power source 3003. For example, the liquid supply power source 3003 and the pressure control mechanism 3004 can be designed by combining a diaphragm pump or a DC fan with a specific output (e.g., 100 kPa) and a vacuum regulator, or can be integrated using a syringe pump or a DC fan that can control the discharge / intake rate.

[0068] According to Fig. 14B the fluid supply mechanism 302 includes a multi-way valve mechanism 3006 (V0), which in addition to the in Fig. The configuration shown in 14A between the pressure control mechanism 3004 and the first connection port 3001 to the atmosphere can be opened. In the case of the configuration from Fig. 14B The connection between the first connection port 3001 and the liquid supply power source 3003 can be immediately interrupted by actuating the multi-way valve mechanism 3006 at the time when the liquid supply process is to be interrupted, thus enabling a rapid response. Furthermore, because the liquid supply power source 3003 can be freely operated in the open-to-atmosphere state, an operation to initiate the liquid supply, after the outputs of the liquid supply power source 3003 and the pressure control mechanism 3004 have stabilized, and an operation to return a syringe piston to a predetermined position, if a syringe pump is used as the liquid supply power source 3003, can be performed, with the piping 307 connected to the reagent holding section 303.It is also possible to operate such that the timing at which pressure is applied to the first connection port 3001 is controlled by an operation of the multi-way valve mechanism 3006, while the fluid supply power source 3003 is constantly driven. The configuration consists of... Fig. 14B can be used in a case where it is desirable to minimize the switching frequency of the fluid supply power source 3003 and the pressure control mechanism 3004, for example, in a case where the fluid supply power source 3003 is used with high current consumption immediately after the power supply is applied. It should be noted that the multi-way valve mechanism 3006 can be combined by combining several on-off valve mechanisms 3005, as shown in Fig. As shown in 15, an equivalent function can be achieved.

[0069] The liquid supply power source 3003, the pressure control mechanism 3004, and the multi-way valve mechanism 3006 can be connected to the second connection port 3002. In this case, a corresponding operation can be achieved by performing pressure increase and pressure decrease operations in the opposite direction to those when connected to the first connection port 3001. It is also possible to use pairs of liquid supply power sources 3003, pressure control mechanisms 3004, and multi-way valve mechanisms 3006, connecting each pair to both the first connection port 3001 and the second connection port 3002. This allows access to the flow path device 101 from both connection ports via the reagent holding section 303 and enables the selection of a liquid supply path that minimizes the effects of a liquid supply pressure loss due to an increase in pipe length.Similarly, a multi-way valve mechanism with the function of a three-way valve, whereby a pair consisting of a fluid supply power source 3003 and the pressure control mechanism 3004 can be switched to the first connection port 3001, the second connection port 3002, or the atmospheric opening flow path 3007, can be used as a set consisting of the fluid supply power source 3003 and the pressure control mechanism 3004. In this way, the size of the device can be reduced while allowing pressure to be applied to both the first connection port 3001 and the second connection port 3002. <Konfiguration des Reagenzhalteabschnitts 303>

[0070] Fig. Figure 16 is a diagram showing an example of the internal configuration of the reagent holding section 303.The reagent holding section 303 comprises, for example, a first liquid supply port 3101 connected to the first connecting port 3001 of the liquid supply mechanism 302, a second liquid supply port 3102 connected to the second connecting port 3002, a first device connecting port 3103 connected to the first vent 107 of the flow path device 101, a second device connecting port 3104 connected to the second vent 108, at least one reagent tank 3105 (T1) which can be connected and disconnected between the first liquid supply port 3101 and the first device connecting port 3103 by means of the on / off valve mechanism 3005, and a waste liquid tank 3106 (solution collection tank 3107 (T2)) which is connected between the second liquid supply port 3102 and the second device connecting port 3103. 3104 can be attached and detached. It should be noted that tank T2 is in . Fig. 16 is designated as 3106 / 3107 because the waste liquid tank 3106 is installed first and then replaced by the solution collection tank 3107 after an intermediate work operation has been carried out.

[0071] The liquid supply mechanism 302 and the reagent holding section 303, as well as the reagent holding section 303 and the flow path device 101, can be connected via the pipeline 307 or directly via a pipe connection. The pressure of the first liquid supply port 3101 and the second liquid supply port 3102 is increased or decreased by the liquid supply mechanism 302, and the reagent stored in the reagent tank 3105 can then be conveyed to the flow path device 101 and collected in the waste liquid tank 3106 or in the solution collection tank 3107.

[0072] Fig. Figure 17 is a flowchart explaining a nucleic acid purification treatment in the Fig. 16 nucleic acid purification system shown. First, the reagent tank 3105 containing the sample solution 401 and the waste liquid tank 3106 are connected at position T1 and position T2, respectively. Fig. 16 connected (S1701). The connection can be made automatically by a robot (robot arm) in the system or by an operator. The liquid feed mechanism 302 introduces the sample solution 401 (sample) contained in the reagent tank 3105 into the first vent 107 of the flow path device 101 and collects in the waste liquid tank 3106 the sample solution 401 that has passed through the cleaning membrane 201, which is installed in the membrane storage section 104 of the flow path device 101, and is expelled from the second vent 108 (S1702). Next, the operator or the robot removes the empty tank from T1 in Fig. 16 and replaces it with the reagent tank 3105, which contains a first cleaning fluid 402 (S1703). The fluid supply mechanism 302 introduces the first cleaning fluid 402 (cleaning fluid 1) contained in the reagent tank 3105 into the first vent 107 of the flow path device 101 and collects in the waste fluid tank 3106 the first cleaning fluid 402 that has passed through the cleaning membrane 201, which is installed in the membrane storage section 104 of the flow path device 101, and is expelled from the second vent 108 (S1704). Next, the operator or robot removes the empty tank from T1 in Fig. 16 and replaces it with the reagent tank 3105, which contains a second cleaning fluid 402 (S1705). The fluid supply mechanism 302 introduces the second cleaning fluid 402 contained in the reagent tank 3105 (cleaning fluid 2) into the first vent 107 of the flow path device 101 and collects in the waste fluid tank 3106 the second cleaning fluid 402 that has passed through the cleaning membrane 201, which is installed in the membrane storage section 104 of the flow path device 101, and is expelled from the second vent 108 (S1706). Similarly, the operator or robot removes the empty tank from T1 in Fig. 16 and replaces the empty tank with reagent tank 3105, which contains a third cleaning fluid 402 (S1707). The liquid supply mechanism 302 introduces the third cleaning fluid 402 contained in reagent tank 3105 (cleaning fluid 3) into the first vent 107 of the flow path device 101 and collects in waste liquid tank 3106 the third cleaning fluid 402 that has passed through the cleaning membrane 201, which is installed in the membrane storage section 104 of the flow path device 101, and is expelled from the second vent 108 (S1708). After conveying the third cleaning fluid 402, the liquid supply mechanism 302 sends dry air to the flow path device 101 for three minutes (S1709). Finally, the operator or robot removes the empty tank from T1 in Fig. 16 and replaces it with the reagent tank 3105 containing the eluent 403. At the same time, the operator or robot removes the waste liquid tank 3106 from T2 and replaces it with the solution receiving tank 3107 (S1710). The liquid supply mechanism 302 introduces the eluent 403 contained in the reagent tank 3105 into the first vent 107 of the flow path device 101 and collects in the solution receiving tank 3107 the eluent 403 that has passed through the cleaning membrane 201, which is installed in the membrane storage section 104 of the flow path device 101, and is expelled from the second vent 108 (S1711).

[0073] It should be noted that the reagent tank 3105, the waste liquid tank 3106, and the solution collection tank 3107 are not particularly restricted as long as they are generally used in this technical field. Microtubes, conical tubes, blister packs, pouch packs, or the like can be used for all of them, provided they are chemically resistant to the reagent to be used and can be connected to the liquid supply mechanism 302 and the flow path device 101 with a suitable connector. For example, combinations of microfluidic containers (Elveflow) and a microtube can be used. Additionally, the reagent tank 3105 can be used for treatment in Fig. 17. Perform this by placing tanks storing reagents in advance, one after the other, at the position of T1 in Fig. 16 exchanged. Alternatively, reagents can be introduced sequentially into the empty reagent tank 3105 after each process in Fig. The 17th process was completed, and treatment can continue with the next process in Fig. 17. As described above, the process of exchanging the tank or introducing the reagents can be carried out manually or automatically using a robotic arm or the like.

[0074] Furthermore, the first fluid supply port 3101 can be connected to the second connection port 3002, and vice versa. Similarly, the first device connection port 3103 can be connected to the second vent port 108, and the second device connection port 3104 can be connected to the first vent port 107. <Modifikationsbeispiel des Reagenzhalteabschnitts 303>

[0075] A configuration example of a nucleic acid purification system (reagent holding section 303) that is better suited for automation is given with reference to the Fig. 18 and Fig. 19 described. Fig. Figure 18 is a diagram showing another example of the internal configuration (modification example) of the reagent holding section 303 in the nucleic acid purification system. Fig. Figure 19 is a flowchart explaining a nucleic acid purification treatment in the Fig. 18 nucleic acid purification systems shown.

[0076] In Fig. 18 The reagent holding section 303 comprises a first liquid supply port 3101 connected to the first connecting port 3001 of the liquid supply mechanism 302, the second liquid supply port 3102 connected to the second connecting port 3002, the first device connecting port 3103 connected to the first vent 107 of the flow path device 101, the second device connecting port 3104 connected to the second vent 108, at least one reagent tank 3105 connected between the first liquid supply port 3101 and the first device connecting port 3103 via the on / off valve mechanism 3005 and containing a reagent, and at least one waste liquid tank 3106 and at least one solution collection tank 3107.which are arranged between the second liquid supply port 3102 and the second device connection port 3104 via the multi-way valve mechanism 3006 and collect a solution ejected from the flow path device. The liquid supply mechanism 302 and the reagent holding section 303, as well as the reagent holding section 303 and the flow path device 101, can be connected via the pipe 307 or directly via a pipe connection. The pressure in the first liquid supply port 3101 and in the second liquid supply port 3102 is increased or decreased by the liquid supply mechanism 302, and the reagents can be conveyed sequentially to the flow path device 101 by switching the flow paths via the on / off valve mechanism 3005 and the multi-way valve mechanism 3006. Therefore, no operation is required to exchange the reagent tank 3105.of the waste liquid tank 3106 and the solution collection tank 3107 is required and can automate the nucleic acid purification treatment through a simpler system than in the configuration in , Fig. 16 will be reached.

[0077] In Fig. 19 The control device (not shown: for example, formed from a computer connected to the system) first actuates the on / off valve mechanism 3005 to open V1 and V6 and to close V2 to V5 and V7 to V10. Simultaneously, the control device actuates the multi-way valve mechanism 3006 (V11 and V12) and connects the second fluid supply port 3102 and the second device connection port 3104 to the waste fluid tank 3106 (S1901).The control device actuates the liquid supply mechanism 302, introduces the sample solution 401 (sample) contained in the first reagent tank 3105 into the first vent 107 of the flow path device 101, and collects the sample solution 401, which has passed through the cleaning membrane 201 installed in the membrane storage section 104 of the flow path device 101 and is expelled from the second vent 108, in the waste liquid tank 3106 (S1902). Next, the control device actuates the on / off valve mechanism 3005 to open V2 and V7 and close V1, V3 to V6, and V8 to V10 (S1903).The control device actuates the liquid supply mechanism 302, introduces the first cleaning fluid 402 (AW1) contained in the second reagent tank 3105 into the first vent 107 of the flow path device 101, and collects the first cleaning fluid 402, which passes through the cleaning membrane 201 installed in the membrane storage section 104 of the flow path device 101 and is expelled from the second vent 108, in the waste fluid tank 3106 (S1904). After the delivery of the first cleaning fluid 402 has ceased, the control device actuates the on / off valve mechanism 3005 to open V3 and V8 and close V1, V2, V4 to V7, V9, and V10 (S1905).The control device actuates the liquid supply mechanism 302, directs the second cleaning fluid 402 (AW2), contained in the third reagent tank 3105, to the first vent 107 of the flow path device 101, and collects the second cleaning fluid 402, which passes through the cleaning membrane 201 installed in the membrane storage section 104 of the flow path device 101 and is expelled from the second vent 108, in the waste fluid tank 3106 (S1906). The control device actuates the on / off valve mechanism 3005 to open V4 and V9 and to close V1 to V3, V5 to V8, and V10 (S1907).The control device activates the liquid supply mechanism 302, introduces the third cleaning fluid 402 (EtOH), contained in the fourth reagent tank 3105, into the first vent 107 of the flow path device 101, and collects the third cleaning fluid 402, which passes through the cleaning membrane 201, installed in the membrane storage section 104 of the flow path device 101 and is expelled from the second vent 108, in the waste fluid tank 3106 (S1908). After the third cleaning fluid 402 has been pumped, the control device activates the liquid supply mechanism 302 to send dry air to the flow path device 101 for three minutes (S1909).Finally, the control device actuates the on-off valve mechanism 3005 to put V5 and V10 into an open state and V1 to V4 and V6 to V9 into a closed state, and actuates the multi-way valve mechanism 3006 (V11 and V12) to connect the second fluid supply port 3102 and the second device connection port 3104 to the solution collection tank 3107 (S1910). The control device initiates the actuation of the liquid supply mechanism 302, introduces the eluent 403 (ATE) contained in the fifth reagent tank 3105 into the first vent opening 107 of the flow path device 101 and collects the eluent 403, which passes through the cleaning membrane 201, which is installed in the membrane storage section 104 of the flow path device 101 and is expelled from the second vent opening 108, in the solution collection tank 3107 (S1911).It should be noted that the solution collected in the waste liquid tank 3106 or the solution collection tank 3107 is preferably contained within the waste liquid tank 3106 and the solution collection tank 3107 without leaking from the second liquid supply port 3102 and returning to the flow path device 101. Therefore, it is only necessary that the waste liquid tank 3106 and the solution collection tank 3107 be designed to have sufficient capacity with respect to the total volume of liquid to be conveyed into them. Alternatively, this can also be achieved by providing a filter venting mechanism that allows gas to pass through but not liquid to pass through the waste liquid tank 3106 and the solution collection tank 3107, and by placing the connection to the second liquid supply port 3102 in a closed state in a system that meets the requirements of the waste liquid tank 3106 and the solution collection tank 3107. Fig. The nucleic acid purification process shown in section 17 is achieved by a pressure increase process from the first connection port 3001.

[0078] The on / off valve mechanism 3005 and the multi-way valve mechanism 3006 can be interchanged, as shown in Fig. 15 shown. If the multi-way valve mechanism 3006 (V11 and V12) in Fig. If the on / off valve mechanism 3005 replaces the valve mechanism 16, the same valve mechanisms can be aligned in parallel, which is advantageous with regard to controllability, expandability, and the like. If the on / off valve mechanism 3005 (V1 to V10) is in Fig. 16 On the other hand, if the multi-way valve mechanism 3006 is replaced, the valve mechanisms can be integrated, and a reduction in the system size and the division of the control mechanism can be easily achieved. Furthermore, the valve mechanism need not necessarily be provided at both ends of the reagent tank 3105, but can be located only on the upstream side (V1 to V5) or only on the downstream side (V6 to V10). As in Fig. As shown in Figure 16, if the valve mechanism is provided at both ends of the reagent tank 3105, the probability of unintentional reagent leakage due to capillary action, changes in the internal pressure of the flow path, or the like can be reduced, and a more stable liquid supply can be achieved. Conversely, if the valve mechanism is provided only on one side of the reagent tank, the number of valve mechanisms can be reduced, resulting in a simpler, smaller system.The on-off valve mechanism 3005 and the multi-way valve mechanism 3006 can be any mechanism as long as they are chemically resistant to the reagent used for nucleic acid purification treatment and can allow a fluid to pass through or stop and adjust the flow rate, such as diaphragm valves, needle valves, bellows valves or shut-off valves, and the operation can be performed manually or controlled by an electrical signal or the like.

[0079] (3) Nucleic acid purification system integrated into a reagent tank, to which the configuration of the flow path device 101 of the present disclosure is applied.

[0080] A sample pretreatment system and a nucleic acid purification process of a nucleic acid purification device integrated into a reagent tank are described with reference to the Fig. described in sections 20 to 22. <Konfigurationsbeispiel des in einen Reagenztank integrierten Nukleinsäure-Reinigungssystems>

[0081] Fig. Figure 20 is a diagram showing an example of the system configuration of a sample pretreatment system 301 in which a flow path device (nucleic acid purification device) 101 integrated into a reagent tank is installed. The flow path device (nucleic acid purification device) 101 comprises a membrane storage section 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 strips 103 attached to the upper and lower surfaces of the substrate 102, a purification membrane 201 attached to the membrane storage section 104 by pressing in a resin ring 204, at least one reagent tank 3105 installed between the first vent 107 and the first flow path 105 via an on / off valve mechanism 3005, and a waste liquid tank 3106.which is connected to the second flow path 106 via the on / off valve mechanism 3005 and has a vent filter 3109, and a solution collection flow path 3108, which is connected to the second flow path 106 and to the second vent opening 108 via the on / off valve mechanism 3005. The configuration near the membrane storage section 104 is similar to that in the , Fig. 9A, Fig. 9B, Fig. 14A and Fig. 14B. The second vent opening 108 is connected to the solution collection tank 3107 provided in the reagent holding section 303.

[0082] Fig. 21A is a diagram showing an example of the configuration of an upper surface near reagent tank 3105. Fig. 21B is a diagram showing an example of the cross-sectional configuration along AA in Fig. Figure 21A shows that in reagent tank 3105, the reagent solution is sealed in a hollow container whose internal volume is slightly larger than the amount of reagent liquid (for example, 800 µl of the sample solution, 1000 µl of the cleaning liquid, and 20 µl of the eluent). Reagent tank 3105 is attached to the substrate 102 at the upper section of the connecting flow path 1001, which is provided with a projecting section 1004. As soon as reagent tank 3105 is pressed and folded by the reagent tank actuator 3110, the projecting section 1004 comes into contact with a sealing element 1003 at the bottom section of reagent tank 3105 and ruptures the sealing element 1003. In this way, the reagent can flow into the connecting flow path 1001.In the nucleic acid purification device integrated into the reagent tank, the reagent tank 3105 is formed by bending a 50 µm thick aluminum sheet. The reagent filling port 1005 and the vent 1006 are provided within the tank, and a 10 µm thick aluminum sheet is welded to the bottom section as a sealing element 1003. A micropipette is then inserted through the reagent filling port 1005, the interior of the reagent tank 3105 is filled with the reagent, and the reagent filling port 1005 and the vent 1006 are sealed with a sealing tape. Finally, a double-sided tape is applied to an upper section of the connecting flow path 1001 provided on the substrate 102. A small linear actuator with a thrust force of 10 N, to which a tapered tip is connected, can be used as the reagent tank actuator 3110.

[0083] The on / off valve mechanism 3005 consists of a valve section 1002, which is provided on the flow path device 101, and a valve actuator 3111. The valve section 1002 has a shape that matches the tip of the valve actuator 3111. The connecting flow path 1001 can be sealed by pressing the valve actuator 3111 against the valve section 1002. The nucleic acid purification device integrated into the reagent tank features the dome-shaped valve section 1002 with a curvature of φ 1 mm in the connecting flow path 1001, which has a width of 1 mm and a depth of 1 mm. A small linear actuator with a thrust force of 5 N and a spherical stainless steel tip with a curvature of φ 1 mm is used as the valve actuator 3111.

[0084] The material, shape, and similar characteristics of the reagent tank 3105 are not particularly restricted, as long as they are generally used in the technical field, and it is possible to use blister packaging, bag packaging, or the like. Furthermore, the reagent tank 3105 can be any reagent tank as long as it is chemically resistant to the reagent to be used, can hold the reagent in a predetermined position in the flow path device 101, and can convey the reagent at any given time.Preferably, the flow path device 101 is designed such that the reagent can be held in the predetermined position even when the flow path device is not installed in the sample pretreatment system 301 and the reagent tank 3105 is opened by an external force exerted by the sample pretreatment system 301 after the flow path device 101 has been installed in the sample pretreatment system 301. However, the flow path device 101 may have a configuration in which the reagent cannot be held in the predetermined position when the flow path device 101 is not installed in the sample pretreatment system 301.In this case, it is only necessary to introduce the reagent from reagent filling port 1005 and seal reagent filling port 1005 and vent opening 1006 after installation in the sample pretreatment system 301, and to close all on-chip valves, for example. It should be noted that reagent tank 3105 does not necessarily have reagent filling port 1005 and vent opening 1006. The sealing element 1003 can be assembled after filling with the reagent. Also, the shape of reagent tank 3105 does not have to be hexagonal, but can be, for example, square, triangular, circular, or the like. However, a shape in which the liquid flow direction is diagonal can minimize the amount of liquid remaining inside reagent tank 3105.Furthermore, the external force exerted on the reagent tank 3105 by the sample pretreatment system 301 cannot be a mechanical pressure force that deforms the reagent tank 3105, such as that exerted by the reagent tank actuator 3110. For example, pressure within the flow path can be used to rupture the sealed section of the reagent tank 3105 via the flow path connected to the reagent tank 3105. In particular, in the configuration where the reagent tank 3105 is opened by pressure within the flow path, the liquid supply mechanism 302 and the piping flow path required to effect the nucleic acid purification flow can be shared, which is advantageous in terms of simplifying and miniaturizing the system mechanism.

[0085] The on / off valve mechanism 3005 is not particularly limited as long as it is generally used in the technical field, and it can be any on / off valve mechanism as long as it is chemically resistant to any reagent to be used, its pressure resistance is greater than or equal to the liquid supply pressure (for example, 100 kPa), and it can be installed on the flow path device 101 and can control the liquid supply direction at any given time. In particular, examples of the on / off valve mechanism 3005 are an on-chip diaphragm valve, a valve using magnetic beads, a heat-deforming valve, a piezo valve, and the like, in addition to the film-deforming valve, which achieves a closed state by deforming a flow path lid material attached to the surface of the substrate 102 by a mechanical pressure force.Alternatively, a rotary on-chip valve can be used to replace the multi-way valve mechanism 3006. One advantage of the film deformation valve is that it can be operated with a relatively simple structure and can operate stably at low cost. Therefore, the film deformation valve is a particularly promising option in a high-performance sample pretreatment system, which necessarily requires a large number of valve mechanisms due to a complex treatment sequence, and because it must be small and cost-effective. On the other hand, in the case of a technology using a rubber O-ring or a sealing ring, which is one of the conventional diaphragm mounting technologies, applying pressure in the mounting direction (surface direction of the diaphragm) is essential.Therefore, it is necessary that the material of the flow path cover is a material with high stiffness that does not deform, or, in a case where a readily deformable film layer is applied (see . Fig. 21B), requires the provision of a pressure application actuator at the upper section. Adding the pressure application actuator increases the size and cost of the device, while the rigid material of the flow path cover precludes the use of the film deformation valve, thus raising concerns about the increased size and cost of the system. The method for securing the cleaning membrane according to the present disclosure does not require the application of an external force from the upper surface after the resin ring 204 has been pressed in, so it can be easily combined with the film deformation valve, which is one of the effects achieved by the technology of the present disclosure.

[0086] The reagent tank actuator 3110 and the valve actuator 3111 need not be linear actuators; for example, solenoid locks, XYZ stages, stepper motors, DC motors, or the like can be used, or the reagent tank actuator 3110 and the valve actuator 3111 can be actuated by a single actuator of several integrated valve mechanisms. The external power source and the actuation section can be separated using an arm, a wire spool, or the like. In this way, it is possible to reduce the size of the mechanisms to be arranged on the flow path device 101 and thus reduce the size of the flow path device 101 itself. <Nukleinsäure-Reinigungsbehandlung durch das in den Reagenztank integrierte Nukleinsäure-Reinigungssystem>

[0087] Fig. 22 is a flowchart (example) to explain a process from in Fig. The nucleic acid purification treatment performed in the nucleic acid purification system shown in Figure 20 is as follows. First, the control device (not shown: for example, formed from a computer connected to the system) actuates the valve actuator to open V1 and close V2 through V10, and actuates the reagent tank actuator 3110 to break the seal of reagent tank 3105 (sample), which contains the sample solution 401 (S2201). Next, the control device actuates the valve actuator 3111 to open V1, V4, and V8 and close V2, V3, and V5 through V7.Furthermore, the control device actuates the liquid supply mechanism 302, introduces the sample solution 401 into the first flow path 105 of the flow path device 101, and collects the sample solution 401, which has passed through the cleaning membrane 201 installed in the membrane storage section 104 of the flow path device 101 and is expelled from the second flow path 106, in the waste liquid tank 3106 (S2202). Next, the control device actuates the valve actuator 3111 to open V2 and V8 and close V1 and V3 to V7, and further actuates the reagent tank actuator 3110 to break the seal of the reagent tank 3105 (wash buffer), which contains the cleaning liquid 402 (S2203).The control device then actuates the valve actuator 3111 to open V2, V5, and V8 and to close V1, V3 to V4, V6, and V8. It further initiates the operation of the liquid supply mechanism 302, introduces the cleaning fluid 402, contained in the second reagent tank 3105, into the first flow path 105 of the flow path device 101, and collects the sample solution 401, which has been caused to pass through the cleaning membrane 201 installed in the membrane storage section 104 of the flow path device 101 and is expelled from the second flow path 106, in the waste liquid tank 3106 (S2204). After the cleaning fluid 402 has been conveyed, the control device actuates the liquid supply mechanism 302 and sends dry air to the flow path device 101 (S2205) for three minutes.Next, the control device actuates the valve actuator 3111 to open V3 and close V1, V2 and V4 to V8, and further actuates 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 actuates the valve actuator 3111 to open V3, V6 and V7 and close V1, V2, V4, V5 and V8, furthermore initiates the operation of the liquid supply mechanism 302, introduces the eluent 403 into the first flow path 105 of the flow path device 101, introduces the sample solution 401, which has passed through the cleaning membrane 201 installed in the membrane storage section 104 of the flow path device 101 and has been expelled from the second flow path 106, to the solution collection flow path 3108 and collects the sample solution 401 in the solution collection tank 3107, which is connected to the second vent opening 108 (S2207).

[0088] It should be noted that, although Fig. Figure 22 shows the process of nucleic acid purification treatment using a type of cleaning fluid for the purification membrane 201, various treatment processes, including the one in Fig. The nucleic acid purification process shown in Figure 2 can be used by changing the arrangement and combination of the reagent tank 3105 and the on-off valve mechanism 3005. (4) Sample-to-response DNA analysis system to which the configuration of the flow path device 101 of the present disclosure is applied

[0089] A sample pretreatment system and a flow path device according to a sample-to-response DNA analysis system are described with reference to the Fig. 23 to 28 are described here. A configuration and the content of the system's processing are described using an example in which a person is identified by a short tandem repeat analysis (STR analysis) using a capillary electrophoresis device based on a swab sample with several µl of adhering human blood. <Konfigurationsbeispiel eines Probe-zu-Antwort-DNA-Analysesystems>

[0090] Fig. Figure 23 is a diagram showing an example of the overall configuration of a sample-to-response DNA analysis system, consisting of a flow path device 101 and a sample pretreatment system 301, which enables the automated performance of nucleic acid extraction, nucleic acid purification, and nucleic acid amplification / detection from a biological sample. The configuration of the sample pretreatment system 301 is essentially similar to the configuration of the system shown in Figure 23. Fig. The system shown in Figure 3A comprises a liquid supply mechanism 302, a reagent holding section 303, and a chip holding section 304. The first vent opening 107 and the second vent opening 108 of the flow path device 101 attached to the chip holding section 304 are connected to the reagent holding section 303 via the pipe 307.The flow path device 101 comprises a nucleic acid extraction section 1201, which has a sample introduction port 1202 and dissolves a sample to extract a nucleic acid contained therein; a nucleic acid purification section 1101, which is connected to the nucleic acid extraction section 1201 and removes impurities from the sample solution 401 that is ejected from the nucleic acid extraction section 1201 to collect a target nucleic acid; and a nucleic acid amplification section 1301, which is connected to the nucleic acid purification section 1101 and amplifies and detects the target nucleic acid contained in the eluent 403 that is ejected from the nucleic acid purification section 1101. <Nukleinsäure-Extraktionsbehandlung des Nukleinsäure-Extraktionsabschnitts 1201>

[0091] Fig. Figure 24 is a diagram showing a configuration example of the nucleic acid extraction section 1201 in the sample-to-response DNA analysis system. The nucleic acid extraction section 1201 comprises, on substrate 102, a sample introduction port 1202, a nucleic acid extraction chamber 1203 connected to the sample introduction port, at least one reagent tank 3105 containing reagents for performing a sample dissolution and nucleic acid purification reaction, at least one on / off valve mechanism 3005, a purification section connecting flow path 1204 linking the nucleic acid extraction chamber 1203 and the nucleic acid purification section 1101, and a liquid supply flow path 1205 connected to the first vent 107 or the second vent 108, and a liquid supply through the operation of the liquid supply mechanism. 302 makes it possible.

[0092] A sample to be analyzed (for example, a swab sample (sample 400): a swab sample obtained by wiping a sample (blood) with the tip of a swab) is introduced into the sample introduction port 1202. A polyurethane introduction port sealing element 1206 is then attached to the sample introduction port 1202. This prevents the sample and reagent from leaking out of the flow path device 101 while ensuring air permeability within the nucleic acid extraction chamber 1203.

[0093] Additionally, the nucleic acid extraction chamber 1203 is designed to have, for example, an internal volume of 4 ml. A temperature control mechanism 3202 can control a nucleic acid extraction section heat source 3201 and maintain the nucleic acid extraction chamber 1203, which is in contact with the nucleic acid extraction section heat source 3201 at a set temperature, at a predetermined temperature. It should be noted that the on / off valve mechanism 3005 and the reagent tank 3105 are similar mechanisms to those found in the Fig. 21A and Fig. 21B are shown, can be used.

[0094] The inlet port sealing element 1206 need not be made of polyurethane and can be designed using an element that allows air to pass through to a certain extent and prevents leakage of a sample and solution within the nucleic acid extraction chamber 1203, such as a vent filter, a porous sintered resin filter, or a perforated sealing strip. The internal pressure of the nucleic acid extraction chamber 1203 can be reduced during the nucleic acid extraction reaction by using a structure in which the nucleic acid extraction chamber 1203 is not tightly sealed. Therefore, it is also possible to use a flow path device 101 with low compressive strength without fracture, thus reducing the cost of the device.Furthermore, the inlet port sealing element 1206 can be sealed using a material that is not air-permeable (for example, a rubber element). In this case, similar effects to those of the inlet port sealing element 1206 can be obtained by providing a filter venting mechanism in the nucleic acid extraction chamber 1203. <Nukleinsäure-Extraktionsbehandlung>

[0095] Fig. Figure 25 is a diagram explaining the nucleic acid extraction treatment performed by the sample-to-response DNA analysis system.

[0096] First, an operator inserts a swab sample (sample 400) with blood adhering to its tip (e.g., 0.5 µl) from the sample introduction port 1202 into the nucleic acid extraction chamber 1203 and closes the sample introduction port 1202 with the introduction port sealing element 1206 (S2501). Next, the control device (not shown: for example, formed by a computer connected to the system), in response to an instruction from the operator to initiate the nucleic acid extraction treatment, actuates the valve actuator 3111 (in Fig. 23 (not shown) of the on / off valve mechanism 3005 to open V2 and close V1 and V3 to V5, and actuates the reagent tank actuator 3110 to break the seal of the reagent tank 3105 containing a nucleic acid extract (lysis buffer; for example, 300 µl). Simultaneously, the control device actuates the temperature control mechanism 3202 to set the surface temperature of the nucleic acid extraction section heat source 3201 to 56 °C (S2502). Once the control device has confirmed (detected) that the temperature of the nucleic acid extraction chamber 1203 has reached 56 °C, the control device actuates the valve actuator 3111 to open V1 and V2 and close V3 to V5, furthermore initiates the actuation of the fluid supply mechanism 302 and introduces the nucleic acid extract into the nucleic acid extraction chamber 1203 (S2503).After the nucleic acid extract has been pumped, the control device directs the system to introduce air into the nucleic acid extraction chamber 1203 and stirs the nucleic acid extract and sample 400 by means of air bubbles and heat convection for thirty minutes (S2504). In this way, the cell membranes of the blood cells contained in sample 400 are lysed, and the nucleic acid in the cells is released into the nucleic acid extract. Next, the control device actuates the valve actuator 3111 of the on / off valve mechanism 3005 to open V3 and close V1, V2, V4, and V5, and further actuates the reagent tank actuator 3110 to break the seal of the reagent tank 3105, which contains the cleaning reagent (sample buffer, for example, 500 µl).Simultaneously, the control device actuates the temperature control mechanism 3202 to set the surface temperature of the nucleic acid extraction section heat source 3201 to 72 °C (S2505). Once the control device has confirmed (detected) that the temperature of the nucleic acid extraction chamber 1203 has reached 72 °C, the control device actuates the valve actuator 3111 to open V3 and V4 and close V1, V2, and V5, furthermore actuates the liquid supply mechanism 302, and introduces the cleaning reagent into the nucleic acid extraction chamber 1203 (S2506). After the purification reagent is conveyed into S2506, the control device controls the system to introduce air into the nucleic acid extraction chamber 1203 and stirs the sample 400 and the solution (the nucleic acid extract and the purification reagent) by means of air bubbles and heat convection for ten minutes (S2507).The purification reagent is a solution containing a chaotropic salt and ethanol, enabling highly efficient DNA adsorption to a silica membrane. The blood cells remaining in S2504 are completely dissolved, and simultaneously, the preparation of sample solution 401 (a mixed solution of the human genomic DNA, the nucleic acid extract, and the purification reagent in the sample), which will be used in a later stage for the Boom procedure in nucleic acid purification section 1101, is completed by the operation in S2507. Finally, the control device V3 to V5 opens, closes V1 and V2, and conveys sample solution 401 from the purification section connecting flow path 1204 to nucleic acid purification section 1101 (S2508). <Nukleinsäure-Reinigungsbehandlung>

[0097] Fig. Figure 26 is a diagram showing a configuration example of the periphery of the nucleic acid purification section 1101 in the sample-to-response DNA analysis system. The nucleic acid purification section 1101 has a configuration similar to that of the flow path device (nucleic acid purification device) 101 integrated into the reagent tank (see Figure 26). Fig. 20) is essentially similar. The main differences compared to the flow path device (nucleic acid purification device) 101 integrated into the reagent tank are that the sample solution 401 is conveyed from the purification section connecting flow path 1204, which is connected to the nucleic acid extraction section 1201, and that the solution collection flow path 3108 is connected to the nucleic acid amplification section 1301. The nucleic acid purification section 1101 can also be connected by the in Fig. The nucleic acid purification treatment shown in section 22 performs a very efficient nucleic acid purification. <Konfigurationsbeispiel des Nukleinsäure-Amplifikationsabschnitts 1301>

[0098] Fig. Figure 27A is a diagram showing a configuration example of the periphery of nucleic acid amplification section 1301. Fig. 27B is a diagram showing an example of the cross-sectional configuration along AA in Fig. 27A is shown. The nucleic acid amplification section 1301 comprises, on the substrate 102, a nucleic acid amplification chamber 1302 connected to the solution collection flow path 3108 of the nucleic acid purification section 1101, at least one reagent tank 3105 containing the reagent for carrying out a nucleic acid amplification reaction, at least one on / off valve mechanism 3005, an amplification product collection flow path 1303 connected to the nucleic acid amplification chamber 1302 and the first vent 107 or the second vent 108, which discharges the sample solution after the nucleic acid amplification reaction, and a liquid supply flow path 1205 connected to at least one of the first vent 107 and the second vent 108, and which Fluid supply is enabled by actuating the fluid supply mechanism 302.

[0099] The nucleic acid amplification chamber 1302 can, for example, have a linear shape with a width of 5 mm x a length of 8 mm x a thickness of 0.5 mm. Additionally, the substrate 102 on the lower surface of the nucleic acid amplification chamber 1302 is processed to be thinner than the peripheral section (for example, with a thickness of 0.5 mm). In this way, 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 within the nucleic acid amplification chamber can be changed rapidly. It should be noted that a temperature control mechanism 3302 can be installed in each of the upper and lower nucleic acid amplification section heat sources 3301.In this way, feedback control of the temperature setting can be performed using temperature data from each of the upper and lower surfaces, thus enabling more precise temperature control. On the other hand, the temperature control mechanism 3302 can be shared with that (temperature control mechanism 3202) of the nucleic acid extraction section heat source 3201 (see ). Fig. 24). In this way, the size of the device can be reduced. It should be noted that, as an on-off valve mechanism 3005 and as a reagent tank 3105, similar mechanisms to those in Fig. 20 can be shown and used. <Nukleinsäure-Amplifikationsbehandlung>

[0100] Fig. Figure 28 is a flowchart explaining a nucleic acid amplification treatment performed by the sample-to-response DNA analysis system. The control device mentioned above (not shown) first actuates the valve actuator 3111 to open V1 and V2 and close V3 and V4, and directs a portion (for example, 10 µl) of the eluent 403 from the solution collection flow path 3108 into the nucleic acid amplification chamber (S2801).Next, the control device actuates the valve actuator 3111 to open V3 and close V1, V2 and V4, further actuates the reagent tank actuator 3110 to break the seal of the reagent tank 3105, which contains the nucleic acid amplification reagent (PCR buffer; for example, 10 µl of the Globalfiler PCR kit reagent), and introduces the nucleic acid amplification reagent into the nucleic acid amplification chamber 1302 (S2802). Furthermore, the control device actuates the valve actuator 3111, once sets V1 to V4 all to an open state to eliminate air bubbles within the nucleic acid amplification chamber 1302, sets V1 to V4 all to a closed state and stores 20 µl of the reaction solution (2 µl of the eluent and 18 µl of the nucleic acid amplification reagent) in the nucleic acid amplification chamber 1302 (S2803).Here, the control device activates the temperature control mechanism 3302 to set the surface temperature of the nucleic acid amplification section heat source 3301 to 95 °C and maintains the temperature for one minute when the nucleic acid extraction chamber 1203 reaches (detects) 95 °C (S2804). In this way, the polymerase contained in the nucleic acid amplification reagent is activated, and the nucleic acid amplification reaction can be initiated. Next, the control device activates the temperature control mechanism 3302 to change the surface temperature of the nucleic acid amplification section heat source 3301 to 94 °C, maintains the temperature for ten seconds when the nucleic acid extraction chamber 1203 reaches 94 °C, and then performs a DNA denaturation reaction (S2805).The control device then activates the temperature control mechanism 3302 to change the surface temperature of the nucleic acid amplification section heat source 3301 to 59 °C, maintains the temperature for ninety seconds when the nucleic acid extraction chamber 1203 reaches (detects) 59 °C, and then performs an anneal / DNA extension reaction (S2806). The control device repeats the treatments in S2805 and S2806 for thirty cycles and amplifies the target nucleic acid (STR) to achieve a product concentration that allows detection by a capillary electrophoresis device. The control device performs the treatments in S2805 and S2806 for thirty cycles, then maintains the temperature at 59 °C for ten minutes and performs adenylation (S2807).Finally, the control device actuates the temperature control mechanism 3302 to change the surface temperature of the nucleic acid amplification section heat source 3301 to 4 °C and interrupts the nucleic acid amplification reaction (S2808). Simultaneously, the control device actuates the valve actuator 3111 to open V1, V2, and V4 and close V3, further actuates the liquid supply mechanism 302, and conveys the reaction solution from the nucleic acid amplification chamber 1302 to the amplification product collection flow path 1303 (S2809). <STR-Analysebehandlung durch Elektrophorese>

[0101] The reaction solution fed to the amplification product collection flow path 1303 is discharged through the second vent 108 and mixed with an electrophoresis reagent (e.g., a formamide solution) at a specific ratio (e.g., 200 µl of the electrophoresis reagent to 20 µl of the reaction solution). This mixed solution can be used as a sample solution in an electrophoresis apparatus and installed to perform STR analysis.

[0102] It should be noted that it is also possible to store the electrophoresis reagent in the reagent tank 3105, install it on the substrate 102, and mix the reaction solution and the electrophoresis reagent through the flow path device 101 in a similar manner to the steps from the nucleic acid extraction section 1201 to the nucleic acid amplification section 1301. Furthermore, the electrophoresis section can be integrated into the flow path device 101, and the introduction of the sample into the electrophoresis analysis can be fully automated. (5) Modification examples of the membrane storage section 104 and the resin ring 204

[0103] The Fig. Figures 29A to 29E are diagrams illustrating modification examples of the membrane storage section 104 and the resin ring 204 according to the present embodiment. Fig. Figure 29A is a diagram showing a configuration example of the periphery of the membrane storage section 104 in each modification example from above. Fig. 29B to 29E are diagrams showing examples of the cross-sectional configuration along AA in Fig. 29A shows the first to fourth modification examples.

[0104] As in Fig. As shown in Figure 29A, the resin ring 204, when viewed from the upper surface, preferably has an annular, seamless shape in each modification example. In this way, a condition is achieved in which the outer circumferential section of the cleaning membrane 201 is attached to the base surface 109 without any gaps over its entire periphery, and leakage of the solution can be minimized. Although the base surface 109 has an annular, seamless shape for a similar reason, it has been shown by experiment that the effects of the technology of the present disclosure can be sufficiently maintained even with a C-shaped base surface 109 having a notch corresponding to at most about 1 / 5 of the circumference.

[0105] In the Fig. In the modification examples shown in Figures 29B to 29E, a resin ring 204 with a circular cross-sectional shape in the vertical direction of the ring (an annular cross-sectional shape in the horizontal direction) can be used in the manner of a general O-ring. In this case, however, contact between the resin ring 204 and the cleaning membrane 201 and between the resin ring 204 and the side wall of the upper part 110 of the membrane storage section is preferably achieved over surfaces that are as large as possible. This further suppresses floating of the cleaning membrane 201 because the contact area between the resin ring 204 and the cleaning membrane 201 increases and because the frictional force acting between the resin ring 204 and the upper part 110 of the membrane storage section increases proportionally to the contact area.

[0106] As in Fig. As shown in Figure 29B, a preferred modification example is a resin ring 204, wherein a cross-section is inclined inwards in the vertical direction of the ring. In the Fig. In the form of the resin ring 204 shown in 29B, the contact surfaces between the resin ring 204 and the cleaning membrane 201 and between the resin ring and the upper part 110 of the membrane storage section are similar to those of the resin ring 204 ( Fig. 9) according to the embodiment mentioned above. Therefore, a strong cleaning membrane adhesion effect can be obtained, the amount of liquid remaining on the upper surface of the resin ring 204 is minimized, and a higher yield is thereby obtained.

[0107] Fig. Figure 29C shows a modification example using a resin ring 204 with tapered shapes on the top and bottom surfaces. As in Fig. As shown in Figure 29C, an upper tapered section 2001 is provided, which is inclined inwards in the central direction of the cleaning membrane 201 in the resin ring 204. In this way, similar to the modification in Fig. 29B, a reduction in liquid residues and an improvement in yield are expected. A lower tapered portion 2002 is provided on the outer edge of the cleaning membrane 201. This allows the resin ring 204 to be easily inserted into the upper part 110 of the membrane storage section during assembly. The resin ring 204 (according to the embodiment and each modification example) according to the present disclosure is characterized in that its inner diameter is greater than or equal to that of the upper part 110 of the membrane storage section. Therefore, it can be difficult to position and fit the resin ring 204 during assembly.Because the outer diameter of the base surface of the resin ring 204 decreases due to the provision of the lower tapered part 2002, it may be easy to place the resin ring 204 at the beginning of the fitting in the upper part 110 of the membrane storage section, thereby facilitating more precise pressing and fastening.

[0108] Another one in Fig. The modification example shown in Figure 29D relates to a resin ring 204 with a notched section 2003, which is provided at a connection section between the first flow path 105 and the upper part 110 of the membrane storage section. The notched section 2003 is preferably designed and arranged such that the upper surface of the notched section 2003 is located below the bottom surface of the first flow path 105 and the side wall of the notched section 2003 is located on the side that is further outward than the side wall surface of the first flow path 105, so that the first flow path 105 is not blocked. Through the resin ring 204 with an upper surface C (a predetermined portion of the upper part of the resin ring 204; a portion connected to the first flow path 105 is provided with a notched section) / a bottom surface O (a predetermined portion of the lower part of the resin ring 204) as shown in Fig. 29D allows the effective volume of the upper part 110 of the membrane storage section to be reduced. Therefore, the amount of air acting as a damper during liquid supply can be minimized, and the liquid supply pressure can be increased more effectively. In this way, the size of the liquid supply mechanism 302 can be reduced by lowering the required pressure, the controllability of the liquid supply can be improved, and a more stable nucleic acid purification treatment can be achieved automatically.

[0109] Another one in Fig. The modification example shown in Figure 29E relates to the membrane storage section 104, in which the upper part 110 and the lower part 111 of the membrane storage section have tapered shapes. In this case, the resin ring 204 preferably also has a tapered shape with which it can be pressed into the upper part 110 of the membrane storage section. The tapered shapes of the upper part 110 of the membrane storage section and of the resin ring 204 cause the resin ring 204 to be pressed into place during assembly, similar to the process described in Figure 29E. Fig. The lower tapered section 2002 shown in Figure 29C can be stably fitted. At the same time, the contact area between the resin ring 204 and the upper part 110 of the membrane storage section increases compared to a case where the side wall of the upper part 110 of the membrane storage section is nearly vertical. Therefore, a higher press-fit effect can be achieved. On the other hand, if the lower part 111 of the membrane storage section is provided with a tapered shape, the amount of solution remaining in the lower part 111 of the membrane storage section can be reduced, while ensuring the effective area of ​​the cleaning membrane 201, the solution passing through the cleaning membrane is efficiently conveyed to the second flow path 106, and the collection efficiency is further increased.

[0110] It should be noted that the characteristic configurations of the in the Fig. The modification examples shown in Figures 29B to 29E can be combined. It is possible to achieve a high force for securing the cleaning membrane and a high collection efficiency through suitable combinations. For example, it is preferred to determine a suitable shape taking into account the materials and processing loads of the substrate 102 and the resin ring 204, the material and size of the cleaning membrane 201, and the like. Furthermore, the membrane fastening method can also be used in combination with a conventional membrane fastening method. For example, a small gap between the pressed-in resin ring 204 and the side wall of the upper part 110 of the membrane storage section can be filled with an adhesive.In this way, errors resulting from processing errors and surface roughness caused by material processing or the like can be minimized, and highly effective nucleic acid purification can be achieved at a practical level according to the applications.

[0111] (6) Application of the continuous conveying method in the system to which the configuration of the flow path device 101 of the present disclosure is applied

[0112] (i) Herein we describe a nucleic acid purification treatment and a sample pretreatment system to which the continuous conveying method is applied. The composition of the sample solution 401 used in the continuous conveying method is as described in Fig. 2A, and pure ethanol is used as the cleaning fluid 402. Additionally, the configurations of the sample pretreatment system 301 and the flow path device 101 are essentially the same as those described in Fig. 20 are shown. Fig. Figure 30A is a diagram showing a state in which the sample solution 401 is conveyed by the continuous conveying process. Fig. Figure 30B is a diagram showing a condition in which the cleaning fluid 402 is conveyed by the continuous conveying process.

[0113] During the execution of the continuous conveying process, the rear end of the sample solution 401 stops at a liquid supply interruption point 1104, which is located in the middle of the sample solution flow path 1102, without the sample solution 401 being completely conveyed to the second flow path 106, as shown in the Fig. 30A and Fig. 30B shown. This fluid supply control differs from the one in Fig. 20. It should be noted that it is only necessary for the liquid supply interruption point 1104 to be located in a place where it can be detected that the rear end of the sample solution 401 has reached a predetermined position, and for example a liquid surface detection sensor or a camera can be arranged on the upper surface of the liquid supply interruption point 1104.

[0114] Next, the nucleic acid purification treatment based on the continuous conveying process will be briefly described with reference to the Fig. 30A and Fig. 30B described. First, the control device (not shown) conveys the sample solution 401 through the cleaning membrane 201, which is installed in the membrane storage section 104, from the first reagent tank 3105 to the second flow path 106 and interrupts the liquid supply at the time when the rear end of the sample solution 401 reaches the liquid supply interruption point 1104 ( Fig. 30A). Next, the control device ejects the cleaning fluid 402 from the second reagent tank 3105, causes the sample solution 401 to pass continuously through the cleaning membrane 201, and conveys the sample solution 401 to the second flow path 106 ( Fig. 30B). The subsequent nucleic acid purification treatment is similar to that used in Fig. 22 described process.

[0115] (ii) If the method for fixing the cleaning membrane using the resin ring 204 according to the present disclosure is applied, the space between the cleaning membrane 201 and the membrane storage section 104 becomes quite small, and the escape of liquid and gas is suppressed. Therefore, it was found by experiment that the liquid supply pressure required for pumping the solution in the technology of the present disclosure increases compared to a case in which the technology of the present disclosure is not used. When applying the method described in the Fig. 4A and Fig. In the configuration shown in 4B, the maximum fluid supply pressure is 20 kPa, and the maximum fluid supply pressure when applying the values ​​shown in the Fig. 9A and Fig. In the configuration shown in Figure 9B, for example, the pressure is approximately 120 kPa. The highest liquid supply pressure is required during the start-up process to initiate the flow of the cleaning fluid 402 after the sample solution 401 has been fully circulated. This is because, when air reaches the cleaning membrane 201 in a state where the membrane is moistened with the sample solution 401, it cannot pass through the membrane until the Laplace pressure is exceeded. Therefore, a high pressure is required until the cleaning fluid 402 reaches the membrane 201.

[0116] To prevent such a high liquid supply pressure, the technology of the present disclosure can employ a continuous conveying method in which the liquid supply pressure is reduced by continuously conveying the two solutions, which meet specific conditions, without trapping air. Experiments have shown that, because the main component of sample solution 401 is water and the main component of cleaning solution 402 is ethanol, the reduction of the liquid supply pressure can be easily achieved by applying the continuous conveying method. Furthermore, experiments have shown that it is advantageous if "the evaporation rate of the second liquid (cleaning solution 402) is higher 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."As the nucleic acid purification treatment, which is used in the . Fig. 30A and Fig. As shown in Figure 30B, and as actually carried out, it was confirmed that the maximum liquid supply pressure was approximately 40 kPa and that high cleaning efficiency could be achieved even with the flow path device 101 of the present disclosure. As described above, it is possible to achieve low-pressure liquid supply by the continuous conveying method and to realize very efficient nucleic acid purification with a small and cost-effective mechanism, while nucleic acid purification with high yield and high robustness is achieved by the membrane attachment method using the resin ring 204 according to the present disclosure. (7) Summary (i) The flow path device 101 according to the present embodiment comprises the cleaning membrane 201, which is formed on the substrate 102 and can collect a nucleic acid, the membrane storage section 104, which is a space formed on the substrate 102 and is connected to the first flow path 105 and the second flow path 106, which are formed on the substrate and accommodate the cleaning membrane 201, and the annular resin ring 204, which is pressed into the membrane storage section 104.Furthermore, the membrane storage section 104 comprises a base surface 109 formed between the upper and lower surfaces of the substrate 102, so that the cleaning membrane 201 can be installed on it, and it is divided into a cylindrical upper part 110 of the membrane storage section and the lower part 111 of the membrane storage section, which has a smaller diameter than the upper part 110 of the membrane storage section, with the base surface 109 sandwiched between them as a boundary.The first flow path 105 and the second flow path 106 comprise the first flow path 105, which is connected to at least one valve mechanism, is formed on the upper surface of the substrate 102 and is connected to the side wall of the upper part 110 of the membrane storage section, and the second flow path 106, which is connected to at least one valve mechanism, is formed on the lower surface of the substrate 102 and is connected to the lower part 111 of the membrane storage section. The outer diameter of the resin ring 204 is greater than or equal to the inner diameter of the upper part 110 of the membrane storage section. Furthermore, the cleaning membrane 201 is fixed between the base 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 in a state where the resin ring 204 is not housed in the membrane storage section 104, and the inner diameter of the upper part 110 of the membrane storage section is substantially equal to the inner diameter of the upper part 110 of the membrane storage section in a state where the resin ring 204 is not pressed into the membrane storage section. Moreover, the inner diameter of the upper part 110 of the membrane storage section and the outer diameter of the pressed-in resin ring 204 are equal at least at a position where the side wall of the upper part 110 of the membrane storage section and the pressed-in resin ring 204 are closest to each other.When the resin ring 204 is pressed into the membrane storage section 104, it is plastically deformed and held at the insertion point by the frictional force with the side wall of the upper part 110 of the membrane storage section. In other words, when pressed in, the resin ring 204 is not elastically deformed in the manner of a known O-ring. The resin ring 204 is made of a material with a Young's modulus between 5 MPa and 1 GPa. It should be noted that the Young's modulus of the material forming the resin ring 204 can be smaller than the Young's modulus of the material forming the substrate 102. Moreover, the elastic modulus of the resin ring is at least 100 kPa. On the other hand, the elastic yield strength of the resin ring 204 is less than 100%.In particular, if the inner diameter of the upper part 110 of the membrane storage section is defined as R' in a state in which the flow path device 101 has been mounted, and the outer diameter of the pressed-in resin ring 204 is defined as a' in the state in which the flow path device 101 has been mounted, |R' - a'| < 1 µm is satisfied at the position where the side wall of the upper part 110 of the membrane storage section and the pressed-in resin ring 204 are closest to each other. If, on the other hand, the inner diameter of the upper part 110 of the membrane storage section is defined as R in a state in which the flow path device 101 has not been mounted (state before pressing in the resin ring) and the outer diameter of the resin ring (before pressing in) 204 is defined as a in the state in which the flow path device 101 has not been mounted, then 0 ≤ a - R < 400 µm is satisfied.The flow path device (flow path chip) 101 can be manufactured cost-effectively by designing it in this way. Furthermore, highly efficient purification, in accordance with laboratory procedures, can be achieved fully automatically, and highly sensitive genetic analysis can be performed using the flow path device 101.

[0117] The inner diameter of the base surface 109 can be essentially the same as the inner diameter of the resin ring 204. In this way, it is possible to cause the solution in contact with the cleaning membrane 201 to efficiently pass through the lower part 111 of the membrane storage section and to maximize the amount of solution that can be collected.

[0118] The difference between the inner diameter of the upper part 110 of the membrane storage section and the inner diameter of the lower part 111 of the membrane storage section, and / or the difference between the outer and inner diameters of the resin ring 204, is preferably kept as small as possible, and it is desirable that the difference be no more than 2.5 mm. This prevents the solution from being trapped and remaining on the upper surface of the resin ring 204, and allows for more efficient solution collection. It should be noted that the difference between the outer and inner diameters of the resin ring 204 can be no more than 1 mm.

[0119] Furthermore, the pressed-in resin ring 204 and the side wall of the upper part 110 of the membrane storage section, as well as the pressed-in resin ring 204 and the cleaning membrane 201, are in surface contact. These are preferably in contact over the largest possible areas. In this way, the floating of the cleaning membrane 201 can be further suppressed by increasing the contact area between the resin ring 204 and the cleaning membrane 201, and a higher pressing-in effect can be achieved.

[0120] Furthermore, the pressed-in resin ring 204 is designed such that an end face on the side opposite the surface on which the pressed-in resin ring 204 is in contact with the cleaning membrane 201 lies on the side below the connecting section between the first flow path 105 and the side wall of the upper part 110 of the membrane storage section. This prevents the solution to be pumped from remaining in the space between the resin ring 204 and the membrane storage section 104. Moreover, the flow path resistance can be reduced, and a small quantity of solution can be collected without loss.

[0121] (ii) The present embodiment also proposes a nucleic acid purification method for generating a nucleic acid using the flow path device 101. The method features, as shown, for example, in Fig. Figure 17 shows the following: a process for connecting the sample tank (reagent tank 3105), which receives the sample solution, to the first flow path 105 of the flow path device 101; a process for connecting the second flow path 106 to the waste liquid tank 3106; a process for conveying the sample solution from the sample tank to the waste liquid tank 3106 via the first flow path 105, the cleaning membrane 201 and the second flow path 106 by actuating the liquid supply mechanism 302; a process for connecting the cleaning liquid tank, which receives the cleaning liquid for cleaning the cleaning membrane 201 (causing the reagent tank 3105 to contain the cleaning liquid 402), to the first flow path 105; a process for conveying the cleaning liquid 402 from the cleaning liquid tank to the waste liquid tank 3106 via the first flow path 105.The cleaning membrane 201 and the second flow path 106 are activated by actuating the valve mechanism. A process is initiated to send dry air from the first flow path 105 to the second flow path 106 via the cleaning membrane 201 by actuating the liquid supply mechanism 302. A process is initiated to connect the first flow path 105 to the eluent tank, which receives the eluent to elute the nucleic acid (causing the reagent tank 3105 to contain the eluent), via the valve mechanism. A process is initiated to connect the second flow path 106 to the solution collection tank 3107. Finally, a process is initiated to convey the eluent from the eluent tank to the solution collection tank 3107 via the first flow path 105, the cleaning membrane 201, and the second flow path 106 by actuating the liquid supply mechanism 302. In this way, nucleic acids can be efficiently purified, and genetic analysis can be performed with high sensitivity. become.,

[0122] (iii) The present embodiment also proposes another nucleic acid purification method. As in the Fig. 30A, Fig. 30B and Fig. As shown in Figure 22, the method comprises at least the following: a process for preparing a first liquid tank (the reagent tank 3105) which receives the first liquid (the sample solution 401), and a second liquid tank (the cleaning liquid tank) which receives the second liquid (cleaning liquid 402); a process for connecting the first flow path 105 of the flow path device 101 to the first liquid transfer flow path (sample solution flow path 1102) which is connected to the first liquid tank; a process for connecting the first flow path 105 of the flow path device 101 to the second liquid transfer flow path (cleaning liquid flow path) which is connected to the second liquid tank; and a process for connecting the second flow path 106 of the flow path device 101 to the waste liquid tank 3106.a process for conveying the first liquid from the first liquid supply tank to the waste liquid tank 3106 via the first flow path 105, the cleaning membrane 201 and the second flow path 106 by actuating the liquid supply mechanism 302, a process for interrupting the conveying of the first liquid before the rear end of the first liquid reaches the meeting point between the first liquid transfer flow path and the first flow path (at the liquid supply interruption point 1104) in order to achieve a state in which the first liquid is contained in the first transfer flow path,by actuating the liquid supply mechanism 302 and a process for initiating the transfer of the second liquid from the second liquid tank to the second liquid transfer flow path and for conveying the second liquid to the waste liquid tank 3106 via the meeting point between the second liquid transfer flow path and the first flow path, the first flow path 105, the cleaning membrane 201 and the second flow path 106 by actuating the liquid supply mechanism 302 in the state in which the first liquid is contained in the first transfer flow path, wherein the flow into the membrane storage section 104 is carried out continuously without air being trapped between the first and the second liquid.

[0123] Furthermore, the preparation process in this method comprises the following: a process for preparing a third liquid tank (reagent tank (elution buffer) 3105) which holds the eluent for eluting a nucleic acid; and furthermore, the following: a process for transferring dry air from the first flow path 105 to the second flow path 106 via the cleaning membrane 201 by actuating the liquid supply mechanism 302; and a process for conveying the eluent from the eluent tank to the solution collection tank 3107 via the first flow path 105, the cleaning membrane 201, and the second flow path 106 by actuating the liquid supply mechanism 302. In this way, the liquid supply pressure can be reduced. Therefore, no rupture of the flow path device 101 occurs due to the conveying of the solution.

[0124] (iv) The present embodiment proposes the sample pretreatment system 301, which comprises the flow path device 101 according to the present embodiment, the nucleic acid extraction section 1201, which is connected to the first flow path 105 of the flow path device 101 and extracts a nucleic acid from a biological sample, and the nucleic acid amplification section 1301, which is connected to the second flow path 106 of the flow path device 101 and amplifies the nucleic acid (see Fig. 23 to 27A and 27B).

[0125] (v) The technology according to the present disclosure can also form different functions by suitably combining several components disclosed in the present embodiment. For example, some or all of the components described in the embodiment can be removed, and the components can be suitably combined in various specific examples.

[0126] Although the present disclosure has been described above with specific examples, this is not intended to limit it in all aspects, but rather to explain it (to facilitate understanding of the technology of the present disclosure). Furthermore, those skilled in the art in the field can understand other implementations of the present disclosure by considering the present embodiment. It should be noted that the description and specific examples are merely typical examples and that the scope and concept of the technology of the present disclosure are specified by the following claims. Reference symbol list 101 Flow path device 102 Substrat 103 Sealing tape 104 Membrane storage section 105 first flow path 106 second flow path 107 first vent 108 second vent 109 Base area 110 upper part of the membrane storage section (diameter R, depth D) 111 lower part of the membrane storage section (diameter r, depth d) 201 Cleaning membrane (diameter P, thickness Q) 202 O-ring 203 Adhesive 204 Resin ring (outer diameter a, inner diameter b, height c) 205 porous retaining element 301 Sample Pretreatment System 302 Fluid supply mechanism 303 Reagent holding section 304 Chip retention section 305 Cover 306 Connector 307 Pipeline 400 samples 401 Sample solution 402 Cleaning fluid 403 Eluents 1001 Connecting flow path 1002 Valve section 1003 Sealing element 1004 preceding section 1005 Reagent filling ports 1006 Vent opening 1101 Nucleic acid purification section 1102 Sample solution flow path 1103 Cleaning fluid flow path 1104 Fluid supply interruption point 1105 Nucleic acid purification and amplification section 1201 Nucleic acid extraction section 1202 Sample Admission 1203 Nucleic acid extraction chamber 1204 Cleaning section collection flow path 1205 Fluid supply flow path 1206 Inlet sealing element 1301 Nucleic acid amplification section 1302 Nucleic acid amplification chamber 1303 Amplification product collection flow path 2001 upper tapered part 2002 lower tapered part 2003 Notch section 3001 first connection port 3002 second connection port 3003 Fluid intake-energy source 3004 Pressure control mechanism 3005 On / Off Valve Mechanism 3006 Multi-way valve mechanism 3007 Atmosphere Opening Flow Path 3101 First fluid supply port 3102 Second fluid supply port 3103 first device connection port 3104 second device connection port 3105 Reagent tank 3106 Waste liquid tank 3107 Solution collection tank 3108 Solution collection flow path 3109 Vent filter 3110 Reagent tank actuator 3111 Valve actuator 3201 Nucleic acid extraction section heat source 3202, 3302 Temperature control mechanism 3301 Nucleic acid amplification section heat source QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] JP 5815572

[0005] JP 2014-030397 A

[0005] JP 2002-512688 T

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[0005] JP 2010-257691 A

[0005] JP 2002-39984 A

[0005] WO 2022 / 030605 A

[0005] Cited non-patent literature

[0000] Microfluidic Devices for Nucleic Acid (NA) Isolation, Isothermal NA Amplification, and Real-Time Detection", Methods Mol. Biol. 1256, 15-40(2015

[0005] Magnetic particles for integrated nucleic acid purification, amplification and detection without pipetting“, Trends Analyt. Chem. 127:115912(2020

[0005] Purification of Nucleic Acids in Microfluidic Devices“, Anal. Chem. 80, 6472-6479(2008

[0005]

Claims

[1] Flow path device comprising a flow path formed on a substrate, comprising: a purification membrane that can collect nucleic acid, a membrane storage section, which is a space formed on the substrate, is connected to the flow path and accommodates the cleaning membrane, and a resin ring that is pressed into the membrane storage section, wherein the membrane storage section has a base surface formed between an upper and a lower surface of the substrate, so that the cleaning membrane can be installed on it, wherein the membrane storage section is divided into a cylindrical upper part of the membrane storage section and a lower part of the membrane storage section which has a smaller diameter than the upper part of the membrane storage section, wherein the base surface in between is formed in a sandwich-like manner as a boundary, wherein the flow path comprises a first flow path, connected to at least one valve mechanism, formed on the upper surface of the substrate and connected to a side wall of the upper part of the membrane storage section, and a second flow path, connected to at least one valve mechanism, formed on the lower surface of the substrate and connected to the lower part of the membrane storage section, the resin ring has an outer diameter that is greater than or equal to the inner diameter of the upper part of the membrane storage section, the cleaning membrane is attached between the base surface and the resin ring, the outer diameter of the pressed-in resin ring is smaller than the outer diameter of the resin ring in a state where the resin ring is not included in the membrane storage section, and the inner diameter of the upper part of the membrane storage section is substantially the same as the inner diameter of the upper part of the membrane storage section in the state where the resin ring is not pressed into the membrane storage section, and the inner diameter of the upper part of the membrane storage section and the outer diameter of the pressed-in resin ring are the same at least at one position where the side wall of the upper part of the membrane storage section and the pressed-in resin ring are closest to each other. [2] Flow path device according to claim 1, wherein the resin ring is formed from a material having a Young modulus of at least 5 MPa. [3] Flow path device according to claim 2, wherein the resin ring is formed from a material with a Young modulus of at most 1 GPa. [4] Flow path device according to claim 1, wherein the elastic modulus of the resin ring is at least 100 kPa. [5] Flow path device according to claim 1, wherein the elastic yield strength of the resin ring is less than 100%. [6] Flow path device according to claim 1, wherein if the inner diameter of the upper part of the membrane storage section in a state in which the flow path device has been mounted is defined as R' and the outer diameter of the pressed-in resin ring in the state in which the flow path device has been mounted is defined as a', |R' - a'| < 1 µm is satisfied at the position where the side wall of the upper part of the membrane storage section and the pressed-in resin ring are closest to each other. [7] Flow path device according to claim 1, wherein if the inner diameter of the upper part of the membrane storage section is defined as R in a state in which the flow path device has not been mounted and the outer diameter of the resin ring is defined as a in the state in which the flow path device has not been mounted, 0 ≤ a - R < 400 µm is satisfied. [8] Flow path device according to claim 1, wherein the inner diameter of the base surface is essentially the same as the inner diameter of the resin ring. [9] Flow path device according to claim 1, wherein the difference between the inner diameter of the upper part of the membrane storage section and the inner diameter of the lower part of the membrane storage section is at most 2.5 mm. [10] Flow path device according to claim 9, wherein the difference between the outer diameter and the inner diameter of the resin ring is at most 1 mm. [11] Flow path device according to claim 1, wherein the pressed-in resin ring and the side wall of the upper part of the membrane storage section as well as the resin ring and the cleaning membrane are in surface contact. [12] Flow path device according to claim 1, wherein an end surface of the pressed-in resin ring is located on a side opposite a surface on which the pressed-in resin ring and the cleaning membrane are in contact with each other, on a side which lies below a connecting section between the first flow path and the side wall of the upper part of the membrane storage section. [13] Flow path device according to claim 1, wherein the Young modulus of a material forming the resin ring is smaller than the Young modulus of a material forming the substrate. [14] Flow path device according to claim 1, wherein film layers are attached to the upper and lower surfaces of the substrate, such that the first flow path, the second flow path and at least part of an opening in the upper surface or the lower surface of the substrate, which the membrane storage section has, are blocked. [15] Flow path device according to claim 1, wherein the pressed-in resin ring is attached to the base surface of the membrane storage section by an adhesive. [16] Flow path device comprising a flow path formed on a substrate, comprising: a purification membrane that can collect nucleic acid, a membrane storage section, which is a space formed on the substrate, is connected to the flow path and accommodates the cleaning membrane, and a resin ring that is pressed into the membrane storage section, wherein the membrane storage section has a base surface formed between an upper and a lower surface of the substrate, so that the cleaning membrane can be installed on it, wherein the membrane storage section is divided into a cylindrical upper part of the membrane storage section and a lower part of the membrane storage section which has a smaller diameter than the upper part of the membrane storage section, wherein the base surface is arranged sandwich-like between as a boundary, the flow path comprises a first flow path formed on the upper surface of the substrate and connected to a side wall of the upper part of the membrane storage section, and a second flow path formed on the lower surface of the substrate and connected to the lower part of the membrane storage section, the resin ring has an outer diameter that is greater than or equal to the inner diameter of the upper part of the membrane storage section, the cleaning membrane is attached between the base surface and the resin ring, the outer diameter of the pressed-in resin ring is smaller than the outer diameter of the resin ring in a state where the resin ring is not included in the membrane storage section, and the inner diameter of the upper part of the membrane storage section is substantially the same as the inner diameter of the upper part of the membrane storage section in the state where the resin ring is not pressed into the membrane storage section, and the inner diameter of the upper part of the membrane storage section and the outer diameter of the pressed-in resin ring are the same at least at one position where the side wall of the upper part of the membrane storage section and the pressed-in resin ring are closest to each other. [17] Nucleic acid purification method for producing a nucleic acid using the flow path device according to claim 1, wherein the method comprises: Connecting the first flow path to a sample tank that holds a sample solution, Connecting the second flow path to a waste liquid tank, Transporting the sample solution from the sample tank to the waste liquid tank via the first flow path, the cleaning membrane and the second flow path by actuating a liquid supply mechanism, Connecting the first flow path to a cleaning fluid tank, which holds a cleaning fluid for cleaning the cleaning membrane, via the fluid supply mechanism, Transporting the cleaning fluid from the cleaning fluid tank to the waste fluid tank via the first flow path, the cleaning membrane and the second flow path by actuating the fluid supply mechanism, Sending dry air from the first to the second flow path across the cleaning membrane by activating the liquid supply mechanism, Connecting an eluent tank, which holds an eluent for eluting the nucleic acid, to the first flow path, Connecting the second flow path to a collection tank and Transport of the eluent from the eluent tank to the collection tank via the first flow path, the cleaning membrane and the second flow path by actuating the liquid supply mechanism. [18] Nucleic acid purification method for producing a nucleic acid using the flow path device according to claim 1, wherein the method comprises: Prepare at least one first liquid tank that holds a first liquid, and one second liquid tank that holds a second liquid, Connecting the first flow path of the flow path device to a first liquid transfer flow path that is connected to the first liquid tank, Connecting the first flow path of the flow path device to a second liquid transfer flow path that is connected to the second liquid tank, Connecting the second flow path of the flow path device to a waste liquid tank, Transferring the first liquid from the first liquid tank to the waste liquid tank via the first flow path, the cleaning membrane and the second flow path by actuating a liquid supply mechanism, Interrupting the transport of the first fluid before the rear end of the first fluid reaches a meeting point between the first fluid transfer flow path and the first flow path, by actuating the fluid supply mechanism to achieve a state in which the first fluid is contained in the first fluid transfer flow path, and Initiating the transport of the second liquid from the second liquid tank to the second liquid transfer flow path by actuating the liquid supply mechanism in the state where the first liquid is contained in the first liquid transfer flow path, in order to transport the second liquid via a meeting point between the second liquid transfer flow path and the first flow path, the first flow path, the cleaning membrane and the second flow path to the waste liquid tank, the flow into the membrane storage section is continuous, without air being sandwiched between the first and second liquids. [19] Nucleic acid purification method according to claim 18, wherein the first liquid is a sample solution the second liquid is a cleaning liquid and In the preparation of at least the first and second liquid tanks, a third liquid tank is further prepared which contains an eluent for eluting the nucleic acid, wherein The procedure further exhibits the following: Sending dry air from the first to the second flow path across the cleaning membrane by actuating the liquid supply mechanism, connecting the second flow path of the flow path device to a collection tank for collecting the eluent and Transport of the eluent from the third liquid tank to the collection tank via the first flow path, the cleaning membrane and the second flow path by actuating the liquid supply mechanism. [20] Sample pretreatment system comprising: the flow path device according to claim 1, a nucleic acid extraction section connected to the first flow path of the flow path device and extracting a nucleic acid from a biological sample, and a nucleic acid amplification section that is connected to the second flow path of the flow path device and amplifies the nucleic acid.

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