Dispensing device, microchannel cartridge, nucleic acid analyzer and nucleic acid analysis method

Abstract

To dispense a fixed volume of solution filled into a main chamber without using an external sensor.SOLUTION: A dispensing device has a solution inlet section at a predetermined position in a direction of gravity and an outlet section at the lowest position in the direction of gravity. The solution supplied from a main chamber via the solution inlet section is filled from the lowest position of a dispensing chamber to a height of the solution inlet section.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a dispensing device that is applied to a microchannel device having a microchannel for passing a liquid sample, a microchannel cartridge including the dispensing device, a nucleic acid analysis device including the dispensing device or the microchannel cartridge, and a nucleic acid analysis method using the dispensing device or the microchannel cartridge. [Background technology]

[0002] A microchannel device is a device having microchannels that are fabricated using so-called microfabrication technology and enable specific chemical reactions and biological analyses. With the microchannel device, biochemical analyses and the like can be carried out easily and quickly using minute amounts of samples. Microchannel devices have attracted attention not only in biochemical research but also in various fields such as medicine, drug discovery, health care, the environment, and food.

[0003] The microfluidic device can be combined with a DNA chip (also called a DNA microarray), which is known as a system for detecting gene mutations. That is, by using a microfluidic device equipped with a DNA chip, it is possible to test for gene mutations and markers that are causative of various diseases, including cancer. In particular, gene mutations related to cancer prognosis prediction and gene mutations related to drug side effects have also been identified, and these gene mutations can be tested using a microfluidic device to predict cancer prognosis and determine the possibility of developing drug side effects. Note that gene mutations include, for example, single nucleotide variants (SNVs) and single nucleotide polymorphisms (SNPs).

[0004] Meanwhile, a DNA chip has a structure in which nucleic acid probes (a wild-type probe corresponding to the wild-type and a mutant probe corresponding to the mutant) corresponding to the gene mutation to be detected are fixed on a substrate. Using genomic DNA collected from a subject as a template, a nucleic acid amplification reaction is carried out using a pair of primer sets that sandwich a region containing the gene mutation to be detected, to obtain a nucleic acid fragment having a label. A solution containing the obtained nucleic acid fragment is brought into contact with the DNA chip under specified reaction conditions. If the solution contains a nucleic acid fragment having a gene mutation, the nucleic acid fragment and the mutant probe hybridize, and a signal based on the label can be detected from the mutant probe.

[0005] Based on the above-mentioned basic principle, the presence or absence of genetic mutations in the genomic DNA of a subject can be examined. Note that genetic mutations are not limited to SNVs and SNPs, and DNA chips having nucleic acid probes corresponding to sequences are also used in microsatellite, DNA methylation, and chimera gene testing.

[0006] Thus, testing for gene mutations using a DNA chip requires a series of processing steps using solutions, such as genomic DNA extraction, nucleic acid amplification reaction, hybridization reaction, etc., and a microfluidic device is designed to accommodate these steps. That is, the microfluidic device is designed to have reaction sections that perform each processing step, and flow channels between the reaction sections and for supplying liquid samples to the reaction sections. In addition, various liquid samples are supplied to the reaction sections in microfluidic devices designed for various reactions, and are not limited to microfluidic devices equipped with DNA chips.

[0007] In particular, it is preferable that the microchannel device is provided with a mechanism capable of weighing and delivering a liquid reagent, i.e., a dispensing device, for filling a reaction section with a predetermined volume of liquid sample. Patent Document 1 discloses an apparatus that includes a chamber having a structure deeper than a microchannel through which a liquid sample passes, and a connection section that connects the microchannel and the chamber, and quantitatively separates a liquid sample flowing through the microchannel into the chamber. Patent Document 2 discloses a configuration in which a liquid sample is filled into the test chamber through an opening formed on the side thereof, and an excess liquid sample is sucked from the opening, thereby separating the liquid sample by a volume from the bottom surface of the test chamber to the lower end of the opening. Patent Document 3 discloses an apparatus provided with a rotating table on which a microchannel is formed, and a device capable of separating a liquid sample equivalent to the volume of the microchannel formed in the rotating body. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2013-253938 A [Patent Document 2] Patent No. 6585119 [Patent Document 3] JP 2009-014439 A Summary of the Invention [Problem to be solved by the invention]

[0009] In conventional microchannel devices, there has been no known dispensing device that dispenses a fixed amount of solution from a main chamber filled with the solution and supplies the dispensed solution to another chamber. In general, to dispense a fixed amount of solution, a series of operations are performed, such as weight measurement, weighing using a scale, or measuring the dispensed solution using an external sensor such as infrared or heat detection, and then transferring the solution by an operator. However, in a microchannel device, the dispensing operation must be performed without the intervention of an operator.

[0010] Therefore, an object of the present invention is to provide a dispensing device that can be applied to a microchannel device and is capable of dispensing a fixed amount of a solution filled in a main chamber without the use of an external sensor, a microchannel cartridge equipped with the dispensing device, a nucleic acid analysis device equipped with the dispensing device or the microchannel cartridge, and a nucleic acid analysis method using the dispensing device or the microchannel cartridge. [Means for solving the problem]

[0011] In order to achieve the above-mentioned object, the inventors conducted intensive research and as a result developed a dispensing chamber connected to a main chamber which can dispense a fixed amount of solution with a simple operation, thereby completing the present invention.

[0012] The present invention encompasses the following: (1) A dispensing chamber having an inlet portion connected to a main chamber in which a solution is filled, and an outlet portion, the inlet portion being at a predetermined position in the direction of gravity, and the outlet portion being at the bottom in the direction of gravity; A dispensing device, characterized in that the solution supplied from the main chamber through the inlet portion fills the dispensing chamber from the lowest portion to the height of the inlet portion. (2) The dispensing device according to (1), wherein the dispensing chamber has a tapered shape toward the outlet portion. (3) The dispensing device according to (1), characterized in that the main chamber is connected to the inlet portion of the dispensing chamber and has a solution outlet portion for discharging the solution in the main chamber, and the inlet portion of the dispensing chamber is located at a higher position in the direction of gravity than the solution outlet portion of the main chamber. (4) The dispensing device according to (1), wherein the inlet of the dispensing chamber is provided at a position higher in the direction of gravity than the liquid level of the solution filled in the main chamber. (5) The dispensing device according to (1), wherein the dispensing chamber has an air vent located at a higher position in the direction of gravity than the inlet portion. (6) The dispensing device according to (5), wherein the flow resistance of the outlet portion of the dispensing chamber is greater than the flow resistance of the vent hole. (7) The dispensing device according to (1), wherein the main chamber has at least one inlet port, a vent port, and an outlet port connected to the dispensing chamber. (8) The dispensing device according to (1), wherein the outlet portion of the dispensing chamber is connected to a discharge port disposed at a high position in the direction of gravity. (9) The dispensing device according to (1), wherein the outlet portion of the dispensing chamber is connected to a discharge port disposed at a higher position in the direction of gravity than the inlet portion of the dispensing chamber. (10) The dispensing device according to (1), characterized in that it is a micro-channel cartridge comprising a substrate having a recess formed therein that serves as a channel connecting the main chamber, the dispensing chamber, and the main chamber and the dispensing chamber, and another substrate bonded together.

[0013] (11) A dispensing method using the dispensing device according to any one of (1) to (10) above, A dispensing method comprising the steps of: filling the dispensing chamber with solution from a main chamber filled with the solution up to the height position of an inlet of the dispensing chamber; and discharging the solution filled in the dispensing chamber from an outlet of the dispensing chamber. (12) The dispensing method according to (11), characterized in that in the step of filling the solution, the solution is filled to a height exceeding the inlet part of the dispensing chamber, and then, with the outlet part blocked, the solution that has exceeded the inlet part is returned from the inlet part to the main chamber, thereby filling the solution up to the height position of the inlet part of the dispensing chamber.

[0014] (13) A nucleic acid analyzer comprising the dispensing device according to any one of (1) to (10) above. (14) The nucleic acid analyzer according to (13), further comprising: a nucleic acid amplification section having the above-mentioned dispensing device and amplifying the nucleic acid contained in the solution supplied to the main chamber; a hybridization section connected to the dispensing chamber in the nucleic acid amplification section and performing hybridization between the nucleic acid contained in the solution supplied from the dispensing chamber and a nucleic acid probe; and a detection section for detecting hybridization between the nucleic acid probe and the nucleic acid in the hybridization section. (15) The nucleic acid analyzer according to (14), further comprising a nucleic acid extraction section for extracting nucleic acid from a sample, and supplying a solution containing nucleic acid from the nucleic acid extraction section to the main chamber of the nucleic acid amplification section. (16) The nucleic acid analyzer according to (14), wherein the hybridization section includes a DNA chip on which the nucleic acid probe is fixed. (17) The nucleic acid analyzer according to (14), characterized in that it quantitatively analyzes a target nucleic acid containing a base to be detected in a gene mutation.

[0015] (18) A method for analyzing nucleic acid using the nucleic acid analyzer according to any one of (14) to (17) above, comprising the steps of: amplifying a nucleic acid contained in a solution supplied to a main chamber of a nucleic acid amplification unit in the nucleic acid analyzer through a plurality of thermal cycles; and, in a predetermined thermal cycle in the step of amplifying the nucleic acid, supplying the solution filled in the main chamber to a dispensing chamber, supplying the solution filled in the dispensing chamber to a nucleic acid analysis unit in the nucleic acid analyzer, and analyzing the nucleic acid contained in the supplied solution in the nucleic acid analysis unit, wherein, in the step of analyzing the nucleic acid, a target nucleic acid containing a base to be detected in a gene mutation is quantitatively analyzed. (19) The method for analyzing nucleic acid according to (18), wherein the step of analyzing the nucleic acid is carried out multiple times using different thermal cycles. Effect of the Invention

[0016] The dispensing device and the micro-channel cartridge equipped with the dispensing device according to the present invention can dispense a fixed amount of solution with a simple configuration. Therefore, in a nucleic acid analysis device equipped with the dispensing device or the micro-channel cartridge, the device configuration involved in the dispensing operation can be simplified. Furthermore, in the nucleic acid analysis method according to the present invention, a fixed amount of solution containing nucleic acids can be dispensed with a simple operation, so that nucleic acid analysis can be performed easily. [Brief description of the drawings]

[0017] [Figure 1] 1 is a perspective view of a main portion showing an example of a dispensing device to which the present invention is applied. [Diagram 2] FIG. 2 is an exploded perspective view of the dispensing device shown in FIG. [Diagram 3] 2 is a plan view of a main part showing one procedure of a dispensing operation using the dispensing device shown in FIG. 1. [Figure 4] 2 is a plan view of a main part showing one procedure of a dispensing operation using the dispensing device shown in FIG. 1. [Diagram 5] 2 is a plan view of a main part showing one procedure of a dispensing operation using the dispensing device shown in FIG. 1. [Figure 6] 2 is a plan view of a main part showing one procedure of a dispensing operation using the dispensing device shown in FIG. 1. [Figure 7] 1A and 1B are a plan view and a perspective view of a main portion, respectively, showing another example of a dispensing device to which the present invention is applied. [Figure 8] FIG. 8 is an exploded perspective view of the dispensing device shown in FIG. 7. [Figure 9] 8 is a plan view of a main part of the dispensing device shown in FIG. 7, a sectional view taken along line AA and a sectional view taken along line BB of the plan view of the main part. [Figure 10] FIG. 11 is an exploded perspective view of a main part showing still another example of a dispensing device to which the present invention is applied. [Figure 11] FIG. 11 is a plan view of the main part of the dispensing device shown in FIG. [Figure 12] 11 is a plan view of a main part of the dispensing device shown in FIG. 10, a cross-sectional view taken along CC and a cross-sectional view taken along DD in the plan view of the main part. [Figure 13]1A and 1B are schematic diagrams for explaining the relationship between the bonding area of ​​the substrates and the area of ​​an opening. [Figure 14] FIG. 1 is a schematic diagram showing the configuration of a genetic mutation testing device equipped with a dispensing device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] A dispensing device, a micro-channel cartridge equipped with the dispensing device, a nucleic acid analysis device, and a nucleic acid analysis method according to the present invention will be described in detail with reference to the drawings.

[0019] [First embodiment] One embodiment of the dispensing device according to the present invention is shown in Figures 1 and 2. As shown in Figures 1 and 2, the dispensing device 1 according to this embodiment has a main chamber 2 filled with a solution such as a nucleic acid-containing solution, a dispensing chamber 3 connected to the main chamber 2, a connecting flow path 4 connecting the main chamber 2 and the dispensing chamber 3, and an outlet flow path 5 connected to the dispensing chamber 3. That is, the dispensing chamber 3 can receive a solution injected from an inlet portion 6 which is a connection portion of the connecting flow path 4, and can discharge the solution from an outlet portion 7 which is a connection portion of the outlet flow path 5.

[0020] In the dispensing device 1, the inlet section 6 is formed at a predetermined position in the direction of gravity in the dispensing chamber 3 (synonymous with the vertical direction, sometimes referred to as the height direction; direction H in Figs. 1 and 2). In other words, the inlet section 6 is formed upward in the height direction from the lowest part of the dispensing chamber 3 in the direction of gravity. Meanwhile, the outlet section 7 is formed at the lowest part of the dispensing chamber 3. Also, in the dispensing device 1, the outlet flow path 5 is formed so as to extend upward from the outlet section 7 in the direction of gravity. In particular, the outlet flow path 5 is formed so that a discharge port 5A, which is the end opposite to the outlet section 7, is located higher in the direction of gravity than the inlet section 6.

[0021] Here, the shape of the dispensing chamber 3 is not particularly limited, but it is preferable that the dispensing chamber 3 has a shape tapering toward the outlet 7, as shown in Figures 1 and 2. In other words, the dispensing chamber 3 preferably has a shape (which may also be called a funnel shape) in which the area of ​​its cross section (cross section perpendicular to the direction of gravity) decreases toward the outlet 7.

[0022] The dispensing chamber 3 has a first vent 8 formed at a position higher in the direction of gravity than the inlet 6, and is connected to a first air passage 9 at the first vent 8. Although not shown, the first air passage 9 is connected to a pump device such as a peristaltic pump, a vacuum pump, or a syringe pump. A pump device may also be connected to the outlet passage 5. In particular, in the dispensing chamber 3, the flow resistance of the inlet 6 and the flow resistance of the outlet 7 are greater than the flow resistance of the first vent 8. Here, the flow resistance of the inlet 6 means the flow resistance in the vicinity of the inlet 6 in the connecting passage 4, and acts on the flow of the solution supplied from the inlet 6 to the dispensing chamber 3. Similarly, the flow resistance of the outlet 7 means the flow resistance in the vicinity of the outlet 7 in the outlet passage 5, and acts on the flow of the solution discharged from the outlet 7. The flow resistance of the first vent 8 means the flow resistance in the vicinity of the first vent 8 in the first air passage 9, and acts on the flow of the solution flowing in the first air passage 9.

[0023] Here, the flow resistance of the inlet section 6, the flow resistance of the outlet section 7, and the flow resistance of the first vent port 8 can be adjusted by the diameters of the connecting flow path 4, the outlet flow path 5, and the first vent passage 9, respectively, and the pipe friction coefficient. For example, by making the diameters of the connecting flow path 4 and the outlet flow path 5 smaller than the diameter of the first vent port 8, the flow resistance of the inlet section 6 and the flow resistance of the outlet section 7 can be made larger than the flow resistance of the first vent port 8.

[0024] In addition, in the dispensing device 1, the main chamber 2 is formed with a first solution inlet section 10 and a second solution inlet section 11, which are connected to a first solution flow path 12 and a second solution flow path 13, respectively. Note that, although the dispensing device 1 shown in Figs. 1 and 2 is configured to be connected to the first solution flow path 12 and the second solution flow path 13, the dispensing device according to the present invention is not limited to such a configuration. The dispensing device 1 may be configured to have one flow path connected to the main chamber 2, or to have three or more flow paths connected to it. The number of solution flow paths connected to the main chamber 2 can be designed appropriately according to various reactions in the main chamber 2.

[0025] Similar to the dispensing chamber 3, the main chamber 2 is formed with a second vent port 14, which is connected to a second air passage 15. Although not shown, similar to the first air passage 9, the second air passage 15 is connected to a pump device such as a peristaltic pump, a vacuum pump, or a syringe pump.

[0026] Furthermore, the main chamber 2 has a solution outlet 16 formed at its bottom, which is connected to the connecting flow path 4. The shape of the main chamber 2 is not particularly limited, but it is preferable that the main chamber 2 has a shape that tapers toward the solution outlet 16, as shown in Figures 1 and 2. In other words, the main chamber 2 preferably has a shape (which can also be called a funnel shape) in which the area of ​​its cross section (a cross section perpendicular to the direction of gravity) decreases toward the solution outlet 16.

[0027] 1, the connecting flow path 4 is formed to extend upward in the direction of gravity from the solution outlet 16. In the dispensing device 1, the connection portion between the connecting flow path 4 and the dispensing chamber 3, i.e., the inlet 6 of the dispensing chamber 3, is formed to be at a higher position than the solution outlet 16 in the direction of gravity.

[0028] Also, the inlet 6 of the dispensing chamber 3 is formed so as to be at a position higher in the direction of gravity than the liquid level (not shown in FIGS. 1 and 2) of the solution filled in the main chamber 2. For example, the main chamber 2 and the dispensing chamber 3 having the inlet 6 can be designed so that the inlet 6 of the dispensing chamber 3 is at a position higher than the liquid level when the main chamber 2 is filled with the maximum amount of solution. Alternatively, the amount of solution filled in the main chamber 2 can be adjusted so as not to exceed the height of the inlet 6 of the dispensing chamber 3.

[0029] The dispensing device 1 shown in FIG. 1 can be produced by bonding a plurality of substrates together as shown in FIG. 2. In the example shown in FIG. 2, the dispensing device 1 is produced by bonding a first substrate 17 and a second substrate 18 together. The first substrate 17 can be produced by microfabrication technology using a polymeric material such as polyethylene terephthalate (PET), polycarbonate (PC), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), polypropylene (PP), polyethylene (PE), polystyrene (PS), ABS resin, cycloolefin polymer (COP), cycloolefin copolymer (COC), polydimethylsiloxane (PDMS), polyimide, polyether, silicone resin, or elastomer, or a flat plate of glass. That is, recesses 20 corresponding to the main chamber 2, the dispensing chamber 3, the connecting channel 4, the outlet channel 5, the first air passage 9, the first solution flow passage 12, the second solution flow passage 13, and the second air passage 15 are formed on one main surface 19 of the first substrate 17. The method for processing the recesses 20 is not particularly limited, and may be cutting of a flat plate, processing by a photolithography process, dry etching, or wet etching. In addition, for the outlet flow path 5, the first ventilation path 9, the first solution flow path 12, the second solution flow path 13, and the second ventilation path 15, through holes penetrating to the surface opposite to the one main surface 19 are formed at the ends.

[0030] The second substrate 18 is a flat plate formed in approximately the same shape as the outer shape of the first substrate 17, and is bonded to the first substrate 17 to cover the recess 20 and form the main chamber 2, the dispensing chamber 3, the connecting flow path 4, the outlet flow path 5, the first air passage 9, the first solution flow path 12, the second solution flow path 13, and the second air passage 15. In particular, the second substrate 18 is bonded to the first substrate 17 to form the dispensing chamber 3, and therefore may be made of a material selected from the same materials as those of the first substrate 17. The method of bonding the first substrate 17 and the second substrate 18 is not particularly limited, and examples of the method include a heat fusion method, a laser welding method, an ultrasonic welding method, a bonding method using a solvent, an adhesive, a pressure sensitive adhesive, or the like, and a method of activating the surface by plasma treatment or ultraviolet treatment and then bonding the substrates under pressure.

[0031] In the dispensing device 1 configured as above, a certain amount of the solution filled in the main chamber 2 can be transferred to the dispensing chamber 3, and can be discharged from the dispensing chamber 3 to the outside through the outlet 5A via the outlet flow path 5. The operation of discharging a certain amount of solution from the main chamber 2 to the outside in this manner is called dispensing. The procedure of dispensing by the dispensing device 1 will be described below. Note that the following description will not refer to the configuration of the dispensing destination, which is not particularly limited and may be a chamber connected to the outlet flow path 5, a reaction tank, a solution recovery tank, or the like.

[0032] First, solutions are injected into the main chamber 2 (FIG. 3). In this example, with the second air passage 15 of the main chamber 2 connected to the outside and with the first air passage 9 and the outlet passage 5 of the dispensing chamber 3 closed, different solutions are injected into the main chamber 2 from the first solution passage 12 and the second solution passage 13. The solutions injected from the first solution passage 12 and the second solution passage 13 are mixed in the main chamber 2. When the solutions are injected from the first solution passage 12 and the second solution passage 13, they may be injected simultaneously or sequentially.

[0033] Here, even when the first air passage 9 and the outlet flow passage 5 are blocked, a phenomenon may occur in which the solution in the main chamber 2 is self-transported in the direction of the connecting flow passage 4 due to Laplace pressure (capillary phenomenon). Therefore, in a state in which the solution remains in the solution outlet section 16 of the main chamber 2 and the main chamber 2 and the dispensing chamber 3 are separated by the solution, the inside of the dispensing chamber 3 is pressurized using a pump device connected to the first air passage 9 and / or the outlet flow passage 5. This allows the solution self-transported in the direction of the dispensing chamber 3 in the connecting flow passage 4 to be returned to the main chamber 2, and a state in which the solution is held back at the solution outlet section 16 of the main chamber 2 can be created. In this state, the internal pressure (P2) of the dispensing chamber 3 is higher than the internal pressure (P1) of the main chamber 2, and the Laplace pressure and the internal pressure (P2) of the dispensing chamber 3 are in balance (P2 = Laplace pressure > P1). At this time, in the dispensing device 1, the connecting flow path 4 is formed so as to extend upward in the direction of gravity from the solution outlet 16, and the inlet 6 of the dispensing chamber 3 is positioned higher in the direction of gravity than the solution outlet 16. For this reason, it is difficult for the solution in the main chamber 2 to flow out into the connecting flow path 4 or through the connecting flow path 4 to the dispensing chamber 3 due to capillary action.

[0034] In particular, as shown in Figure 3, when the inlet 6 of the dispensing chamber 3 is located at a position higher in the direction of gravity than the liquid level of the solution filled in the main chamber 2, it is possible to make it difficult for the solution in the main chamber 2 to flow into the dispensing chamber 3 through the connecting flow path 4 due to capillary action.

[0035] Next, a certain amount of the solution filled in the main chamber 2 is transferred to the dispensing chamber 3 (FIG. 4). Specifically, the first solution flow path 12 and the second solution flow path 13 are closed, and the inside of the main chamber 2 is pressurized using a pump device connected to the second air passage 15, so that the internal pressure (P1) of the main chamber 2 is made higher than the internal pressure (P2) of the dispensing chamber 3 (P1>P2). At this time, the first air passage 9 of the dispensing chamber 3 is opened and the outlet passage 5 is closed, or both the first air passage 9 and the outlet passage 5 are closed. As a result, the solution stored in the main chamber 2 is pressurized and sent to the dispensing chamber 3 through the connecting passage 4. The solution is injected from the inlet 6 of the dispensing chamber 3, and after filling the entire dispensing chamber 3 by maintaining P1>P2 as described above, it rises in the first air passage 9. In this process, the solution is injected to a height at least exceeding the inlet 6, but may be injected until it reaches the inside of the first air passage 9.

[0036] Here, even when the outlet flow path 5 is blocked, a phenomenon may occur in which the solution in the dispensing chamber 3 is self-transported from the outlet portion 7 toward the outlet flow path 5 due to Laplace pressure (capillary phenomenon). However, as described above, when the flow path resistance of the outlet portion 7 is made larger than the flow path resistance of the first ventilation port 8, this phenomenon can be made less likely to occur.

[0037] Next, a certain amount of solution is measured into the dispensing chamber 3 (FIG. 5). Specifically, by making the internal pressure (P2) of the dispensing chamber 3 greater than the internal pressure (P1) of the main chamber 2 (P2>P1), the portion of the solution injected into the dispensing chamber 3 that exceeds the inlet 6 is returned to the main chamber 2 via the connecting flow path 4. As a result, the dispensing chamber 3 is filled with solution up to the height position of the inlet 6. This operation makes it possible to measure out the solution in the dispensing chamber 3 up to the inlet 6.

[0038] Here, the following methods can be used to make P2>P1: blocking the outlet flow path 5 and pressurizing the inside of the main chamber 2 using a pump device connected to the first air passage 9; blocking the first air passage 9 and pressurizing the inside of the dispensing chamber 3 using a pump device connected to the outlet flow path 5; opening any one or more of the first solution flow path 12, the second solution flow path 13, and the second air passage 15 to reduce the internal pressure of the main chamber 2 that had been pressurized; reducing the pressure inside the main chamber 2; and a combination of these methods. Note that when the relationship between the internal pressure (P3) of the outlet flow path 5 and the internal pressure (P2) of the dispensing chamber 3 and the internal pressure (P1) of the main chamber 2 becomes P2>P1>P3, the solution in the dispensing chamber 3 will be sent from the outlet 7 to the outlet flow path 5. Therefore, by blocking the outlet flow path 5, pressurizing the outlet flow path 5, or lowering the internal pressure of the main chamber 2, it is possible to make P3≧P2>P1, and the portion of the solution injected into the dispensing chamber 3 that exceeds the inlet portion 6 can be returned to the main chamber 2 via the connecting flow path 4.

[0039] Next, the solution measured in the dispensing chamber 3 is discharged to the outside, completing the dispensing operation (FIG. 6). Specifically, the first solution flow path 12, the second solution flow path 13, and the second air passage 15 are closed, and the outlet flow path 5 is opened, and the inside of the dispensing chamber 3 is pressurized using a pump device connected to the first air passage 9. As a result, the internal pressure (P2) of the dispensing chamber 3 becomes greater than the internal pressure (P3) of the outlet flow path 5, and the solution stored in the dispensing chamber 3 can be discharged to the outside through the outlet flow path 5. At the start of this process, since P2>P3>P1, air is sent from the dispensing chamber 3 to the main chamber 2 through the connecting flow path 4, but after a while the internal pressures of the dispensing chamber 3 and the main chamber 2 become equal (P2≒P1), and the solution in the dispensing chamber 3 is sent to the outlet flow path 5.

[0040] As described above, the procedure of dispensing by the dispensing device 1 has been described. However, the opening and closing of the connecting flow path 4, the outlet flow path 5, the first air passage 9, the first solution flow path 12, the second solution flow path 13, and the second air passage 15 can be appropriately controlled by a valve using a diaphragm, a rotation method, or a shape memory element, although not shown. In addition, the internal pressure of the main chamber 2, the dispensing chamber 3, and the outlet flow path 5 can be controlled by internal pressure management using a pressure sensor, or by capturing the behavior of the fluid from the outside with an optical sensor or an image recognition camera. In addition, pressure can be controlled without using a sensor by pushing and pulling a predetermined amount of air, or by sending liquid for a predetermined time. Furthermore, the flow rate from the main chamber 2 to the dispensing chamber 3 can be controlled by, for example, flow rate management using a flow sensor, a method of capturing the behavior of the fluid in the same way as a pressure sensor, or flow rate management without using a sensor by pushing and pulling a predetermined amount of solution.

[0041] As described above, according to the dispensing device 1 of the present invention, a fixed amount of solution injected into the main chamber 2 can be measured out into the dispensing chamber 3, and can be discharged to the outside from the dispensing chamber 3. As described above, according to the dispensing device 1 of the present invention, dispensing can be performed with an extremely simple operation, without requiring a sensor or the like for measuring a fixed amount of solution.

[0042] Furthermore, in the dispensing device 1 according to the present invention, solution can be measured out from the bottom of the dispensing chamber 3 to the height position of the inlet section 6. In other words, in the dispensing device 1 according to the present invention, the amount of solution to be dispensed can be determined by the shape of the dispensing chamber 3 and the position of the inlet section 6. As described above, the dispensing chamber 3 and the connecting flow channel 4 (position of the inlet section 6) can be produced by microfabrication of the first substrate 17, so that the shape of the dispensing chamber 3 and the position of the inlet section 6 can be easily designed according to the amount of solution to be dispensed.

[0043] Second Embodiment Another embodiment of the dispensing device according to the present invention is shown in Figures 7 and 8. Dispensing device 25 shown in Figures 7 and 8 will be described below, but the same members and configurations as those of dispensing device 1 shown in Figures 1 and 2 will be given the same reference numerals and detailed description thereof will be omitted. In dispensing device 25 shown in Figures 7 and 8, a configuration in which valves are attached to various flow paths will be described.

[0044] The dispensing device 25 has valves 28A, 28B, 28C, and 28D disposed on the other main surface 27 opposite to the main surface 19 on which the recess 26 serving as the main chamber 2 of the first substrate 17 is formed. Here, the valves 28A, 28B, 28C, and 28D may be of any valve mechanism as long as they can control the opening and closing of each flow path. For example, the valves 28A, 28B, 28C, and 28D may be valves using a diaphragm, valves using a method such as rotation, or valves using a shape memory element. In the following example, the valves 28A, 28B, 28C, and 28D are valves using a diaphragm. A valve using a diaphragm generally means a valve having a structure in which a flow path is opened and closed by a flexible diaphragm (partition membrane) made of rubber or the like.

[0045] In dispensing device 25, main chamber 2 and dispensing chamber 3 are formed on different main surfaces of first substrate 17. That is, recess 26 which becomes main chamber 2 is formed on one main surface 19 of first substrate 17, and recess 29 which becomes dispensing chamber 3 is formed on the other main surface 27. In dispensing device 25, recess 26 and recess 29 which become main chamber 2 and dispensing chamber 3 are disposed downward in the height direction.

[0046] In the dispensing device 25, the outlet flow path 5, the first air passage 9, the first solution flow path 12, and the second air passage 15 are formed by grooves and through holes formed on both the one main surface 19 and the other main surface 27 of the first substrate 17, respectively. In the dispensing device 25, the outlet flow path 5, the first air passage 9, the first solution flow path 12, and the second air passage 15 are connected to the main chamber 2 or the dispensing chamber 3, respectively, but a predetermined portion (referred to as the upper portion of the flow path) toward the end opposite to the end connected to the main chamber 2 or the dispensing chamber 3 is located on the other main surface 27 side of the dispensing chamber 3 and above in the height direction. Here, "above" means above in the height direction relative to the "below" where the recesses 26 and the recesses 29 are formed.

[0047] The dispensing device 25 has a first substrate 17, a second substrate 30 bonded to one main surface 19 of the first substrate 17, a third substrate 31 bonded to a lower portion of the other main surface 27 of the first substrate 17, and a fourth substrate 32 bonded to an upper portion of the other main surface 27 of the first substrate 17. By bonding the first substrate 17, the second substrate 30, the third substrate 31, and the fourth substrate 32 together, a main chamber 2, a dispensing chamber 3, a connecting flow path 4, an outlet flow path 5, a first air passage 9, a first solution flow path 12, and a second air passage 15 are formed.

[0048] Here, valves 28A, 28B, 28C, and 28D use fourth substrate 32 as a diaphragm, and a drive unit (not shown) presses fourth substrate 32 at the upper portions of outlet flow path 5, first air vent path 9, first solution flow path 12, and second air vent path 15, thereby bringing fourth substrate 32 into pressure contact with first substrate 17, thereby controlling opening and closing of each flow path. Specifically, valves 28A, 28B, 28C, and 28D control opening and closing of outlet flow path 5, first air vent path 9, first solution flow path 12, and second air vent path 15, respectively.

[0049] Thus, since the fourth substrate 32 functions as a diaphragm, an elastic film is used as the fourth substrate 32. In contrast, the first substrate 17, the second substrate 30, and the third substrate 31 do not need to be elastic films, and a material with high rigidity can be appropriately used. In particular, since the first substrate 17, the second substrate 30, and the third substrate 31 form the main chamber 2 and the dispensing chamber 3, it is preferable to use a material with high rigidity and low deformation, such as glass, silicon, polydimethylsiloxane, and cyclic olefin polymer, from the viewpoint of preventing a change in shape due to injection of a solution.

[0050] 9, the first solution flow path 12 and the second air passage 15 communicating with the main chamber 2 are formed on the one main surface 19 side in the vicinity of communicating with the main chamber 2, and are formed so as to face the other main surface 27 via a through hole at a position facing the fourth substrate 32. The outlet flow path 5 and the first air passage 9 communicating with the dispensing chamber 3 are formed on the other main surface 27 side in the vicinity of communicating with the dispensing chamber 3, are formed on the one main surface 19 side via a through hole downstream thereof, and are formed so as to face the other main surface 27 again via a through hole at a position facing the fourth substrate 32 further downstream. By forming the outlet flow path 5, the first air passage 9, the first solution flow path 12, and the second air passage 15 on the one main surface 19 side and the other main surface 27 side in this manner, the outlet flow path 5, the first air passage 9, the first solution flow path 12, and the second air passage 15 can be formed on the one main surface 19 side at the boundary between the third substrate 31 and the fourth substrate 32. This makes it possible to reliably prevent liquid leakage and air leakage from the outlet flow path 5, the first air passage 9, the first solution flow path 12, and the second air passage 15.

[0051] In the dispensing device 25 configured as above, a fixed amount of solution from the lowermost part of the dispensing chamber 3 to the height position of the inlet part 6 can be dispensed to the outside via the outlet flow path 5, similarly to the dispensing device 1 described in the first embodiment. As described above, the dispensing device 25 according to the present invention can dispense with a very simple operation without requiring a sensor for measuring a fixed amount of solution. Also, in the dispensing device 25 according to the present invention, the amount of solution to be dispensed can be determined by the shape of the dispensing chamber 3 and the position of the inlet part 6.

[0052] In particular, in the dispensing device 25, the valves 28A, 28B, 28C and 28D are arranged on the other main surface 27 side of the first substrate 17, so that the drive system for driving the valves 28A, 28B, 28C and 28D can be simplified compared to the case where the valves 28A and 28B are arranged on one main surface 19 and the valves 28C and 28D are arranged on the other main surface 27.

[0053] Third embodiment Another embodiment of the dispensing device according to the present invention is shown in Figures 10 to 12. Dispensing device 35 shown in Figures 10 to 12 will be described below, but detailed description thereof will be omitted by assigning the same reference numerals to the same members and configurations as those of dispensing device 1 shown in Figures 1 and 2. Dispensing device 35 shown in Figures 10 to 12 is composed of a first substrate 36 in which recesses that become main chamber 2 and dispensing chamber 3 are formed, a second substrate 37, a third substrate 38, and a fourth substrate 39 bonded to one main surface 36A of first substrate 36, and a fifth substrate 40 bonded to the other main surface 36B of first substrate 36.

[0054] A recess 41 which becomes the dispensing chamber 3 is formed on one main surface 36A of the first substrate 36. That is, the dispensing chamber 3 is formed by bonding the first substrate 36 and the second substrate 37 together. In addition, a recess 42 which becomes the main chamber 2 and which partially penetrates to the one main surface 36A is formed on the other main surface 36B of the first substrate 36. That is, the main chamber 2 is formed by bonding the first substrate 36, the second substrate 37 and the fifth substrate 40 together.

[0055] 1 and 2 and dispensing device 25 shown in Figures 7 to 9, the opening areas of recess 42 constituting main chamber 2 and recess 41 constituting dispensing chamber 3 are small relative to the bonding area of ​​the substrates. In dispensing device 35 of the present example, in order to ensure that the volumes of main chamber 2 and dispensing chamber 3 are equivalent to those of dispensing device 1 shown in Figures 1 and 2 and dispensing device 25 shown in Figures 7 to 9, the thickness of first substrate 36 is made large, unlike dispensing device 1 shown in Figures 1 and 2 and dispensing device 25 shown in Figures 7 to 9.

[0056] A groove serving as the first solution flow path 12 and the second air passage 15 communicating with the main chamber 2, and a groove serving as the outlet flow path 5 communicating with the dispensing chamber 3 are formed on one main surface 36A and the other main surface 36B of the first substrate 36, and the grooves formed on the one main surface 36A and the other main surface 36B are connected by a through hole penetrating the first substrate 36 in the thickness direction. A groove serving as the first air passage 9 communicating with the dispensing chamber 3 is formed on the one main surface 36A of the first substrate 36, and an end of the groove faces the other main surface 36B through a through hole penetrating the first substrate 36 in the thickness direction. A groove serving as the connecting flow path 4 connecting the main chamber 2 and the dispensing chamber 3 is formed on the other main surface 36B of the first substrate 36, and the groove is connected to a recess 41 serving as the dispensing chamber 3 through a through hole penetrating the first substrate 36 in the thickness direction.

[0057] In the dispensing device 35 of this example, the first substrate 36, the second substrate 37, the third substrate 38, the fourth substrate 39 and the fifth substrate 40 are bonded together to form a connecting flow path 4, a first solution flow path 12, a second air passage 15, an outlet flow path 5 and a first air passage 9.

[0058] In the dispensing device 35 of this example, valves 43A and 43B are disposed in the grooves that become the outlet flow path 5 and the grooves that become the first solution flow path 12, respectively, on one main surface 36A of the first substrate 36. Here, the valves may be of any valve mechanism that can control the opening and closing of each flow path. For example, a valve using a diaphragm, a valve using a rotation method, or a valve using a shape memory element can be mentioned. In this example, the valve is a valve using a diaphragm. A valve using a diaphragm generally means a valve having a structure that opens and closes a flow path with a flexible diaphragm (septum) such as rubber.

[0059] In the dispensing device 35 of this example, the valve uses the third substrate 38 as a diaphragm, and a drive unit (not shown) presses the third substrate 38, thereby bringing the third substrate 38 and the first substrate 36 into pressure contact, thereby controlling the opening and closing of the outlet flow path 5. Also, in the dispensing device 35 of this example, the valve uses the fourth substrate 39 as a diaphragm, and a drive unit (not shown) presses the fourth substrate 39, thereby bringing the fourth substrate 39 and the first substrate 36 into pressure contact, thereby controlling the opening and closing of the first solution flow path 12.

[0060] In this way, since the third substrate 38 and the fourth substrate 39 function as diaphragms, elastic films are used. In contrast, the first substrate 36, the second substrate 37, and the fifth substrate 40 do not need to be elastic films, and a material with high rigidity can be used as appropriate. In particular, since the first substrate 36 forms the main chamber 2 and the dispensing chamber 3, it is preferable to use the aforementioned material that is highly rigid and difficult to deform from the viewpoint of preventing shape changes due to injection of the solution.

[0061] Moreover, the dispensing device 35 of this example is provided with a third air passage 44 in the middle of the outlet flow path 5. Although not shown, the third air passage 44 is connected to a pump device such as a peristaltic pump, a vacuum pump, or a syringe pump.

[0062] In the dispensing device 35 configured as above, a certain amount of the solution filled in the main chamber 2 can be transferred to the dispensing chamber 3 via the connecting flow path 4, and can be discharged from the dispensing chamber 3 to the outside via the outlet flow path 5. Specifically, in the dispensing device 35, first, the first air vent 8 is opened, the outlet flow path 5 is closed by the valve 43A, and the inside of the outlet flow path 5 is pressurized from the third air passage 44 by a pump (not shown), and in this state, the solution in the main chamber 2 is injected into the dispensing chamber 3 via the connecting flow path 4. To inject the solution in the main chamber 2 into the dispensing chamber 3 via the connecting flow path 4, the first solution flow path 12 communicating with the main chamber 2 is closed by the valve 43B, and the inside of the main chamber 2 is pressurized from the second air passage 15 by a pump (not shown).

[0063] At this time, the solution is injected into the dispensing chamber 3 until it exceeds the height of the inlet portion 6. In this step, the solution is injected to a height at least exceeding the height of the inlet portion 6, but may be injected until it reaches the first air passage 9.

[0064] Next, a certain amount of solution is measured into the dispensing chamber 3. Specifically, the inside of the dispensing chamber 3 is pressurized from the first air passage 9 by a pump (not shown), and the part of the solution injected into the dispensing chamber 3 that has passed through the inlet 6 is returned to the main chamber 2 via the connecting flow path 4. As a result, the inside of the dispensing chamber 3 is filled with the solution up to the height position of the inlet 6. This operation allows the solution to be accurately measured in the dispensing chamber 3 up to the inlet 6. At this time, the solution in the dispensing chamber 3 is prevented from flowing into the outlet flow path 5 by maintaining a pressurized state from the third air passage 44 by a pump (not shown).

[0065] Next, the solution measured in the dispensing chamber 3 is discharged to the outside, completing the dispensing operation. Note that before dispensing the solution to the outside, the solution may be stirred in the dispensing chamber 3, or even heated. For stirring the solution, for example, a method of stirring using magnetic beads or applying ultrasonic waves or vibrations can be adopted. To discharge the solution measured in the dispensing chamber 3 to the outside, the main chamber 2 connected to the connecting flow path 4 is made airtight, and with the outlet flow path 5 open, the inside of the dispensing chamber 3 is pressurized using a pump device connected to the first air passage 9. This allows the solution stored in the dispensing chamber 3 to be discharged to the outside via the outlet flow path 5.

[0066] As described above, in the dispensing device 35 of this embodiment, the inside of the main chamber 2 is pressurized to transfer the solution to the dispensing chamber 3, and the inside of the dispensing chamber 3 is pressurized to discharge the solution to the outside. In the dispensing device 35 of this embodiment, the thickness of the first substrate 36 is increased, so that the volumes of the main chamber 2 and the dispensing chamber 3 are secured, while the opening areas of the recesses 42 constituting the main chamber 2 and the recesses 41 constituting the dispensing chamber 3 can be made small relative to the bonding area of ​​the substrates. FIG. 13 shows the relationship between the bonding area of ​​the substrates and the opening area. In FIG. 13(a), the opening area is relatively large relative to the bonding area of ​​the substrates compared to (b). In (a) and (b), the same pressure is applied to the internal space formed by bonding the substrates. Then, it can be understood that in the case of (a), the load applied to the bonding surface of the substrates is large compared to the case of (b), and the substrates are easily peeled off.

[0067] In the dispensing device 35 of this example, which corresponds to the case of (b) in Figure 13, even when pressurizing the main chamber 2 or the dispensing chamber 3, peeling between the first substrate 36 and the second substrate 37, and peeling between the first substrate 36 and the fifth substrate 40 can be prevented.

[0068] [Fourth embodiment] The fourth embodiment describes a nucleic acid analyzer equipped with the dispensing device described in the first to third embodiments. The nucleic acid analyzer according to the present invention is not limited in any way as long as it dispenses a part of the solution in the main chamber using the above-mentioned dispensing device. In this embodiment, a device is described that performs a nucleic acid amplification reaction using the nucleic acid recovered by the nucleic acid extraction device as a template, and uses a part of the reaction solution to test for gene mutation based on hybridization between the amplified fragment contained in the reaction solution and the nucleic acid probe. In the nucleic acid analyzer shown in this embodiment, the above-mentioned dispensing device is applied as a device that dispenses a part of the reaction solution of the nucleic acid amplification reaction to the reaction vessel in which the hybridization is performed. Note that when the above-mentioned dispensing device according to the present invention is used in a nucleic acid analyzer, the dispensing device according to the present invention can be applied as a micro-channel cartridge that is detachable and replaceable from the nucleic acid analyzer.

[0069] As shown in Figure 14, a genetic mutation testing device 50 includes an extraction unit 51 equipped with a nucleic acid extraction device, and an analysis unit 53 connected to the extraction unit 51 via a flow path 52 and configured to analyze the nucleic acids contained in the solution recovered by the nucleic acid extraction device.

[0070] Specifically, the analysis unit 53 includes a nucleic acid amplification reaction unit 54 that performs a nucleic acid amplification reaction to amplify a region containing a genetic mutation to be tested, and a detection unit 55 that detects the target nucleic acid contained in the reaction solution to which a portion of the reaction solution is dispensed from the nucleic acid amplification reaction unit 54. The nucleic acid amplification reaction unit 54 includes a reaction vessel 56 that stores reagents and substances necessary for the nucleic acid amplification reaction, and a first temperature control unit 57 that controls the reaction solution in the reaction vessel 56 to a predetermined temperature.

[0071] The above-mentioned dispensing device is included in the nucleic acid amplification reaction unit 54, and a reaction vessel 56 is used as the main chamber 2, and a part of the reaction solution in the reaction vessel 56 is dispensed to the detection unit 55. This nucleic acid amplification reaction unit 54 constitutes the testing device 50, and can be a detachable and replaceable micro-channel cartridge. That is, the micro-channel cartridge includes the above-mentioned dispensing device and the reaction vessel 56 which is the main chamber, and is attached so as to be connected to the extraction unit 51 and the detection unit 55.

[0072] The detection unit 55 includes a DNA chip mounting unit 59 mounting a DNA chip 58 having a nucleic acid probe for detecting a target nucleic acid, a nucleic acid probe for detecting a non-target nucleic acid, a common probe, and the like (these various probes will be described later), a second temperature control unit 60 controlling the temperature of the hybridization reaction liquid supplied to the DNA chip 58 mounted on the DNA chip mounting unit 59 at a predetermined temperature, and an imaging unit 61 disposed at a position facing the surface of the DNA chip 58 on which the various probes are fixed. In the inspection device 50, the nucleic acid amplification reaction unit 54 and the detection unit 55 are connected to the outlet flow path 5 in the dispensing device and are connected via a flow path 62 through which a solution can pass. The first temperature control unit 57 and the second temperature control unit 60 include a metal block having high thermal conductivity and a temperature control device for heating / cooling the metal block.

[0073] The inspection device 50 also includes a hybridization buffer tank 64 connected to the DNA chip mounting section 59 via a flow path 63, and a cleaning liquid tank 66 connected to the DNA chip mounting section 59 via a flow path 65. The inspection device 50 also includes a waste liquid tank 68 connected to the DNA chip mounting section 59 via a flow path 67. The inspection device 50 also includes a pump device 70 connected to the waste liquid tank 68 via the flow path 69.

[0074] In the inspection device 50, liquid supply cutoff valves 71A, 71B, 71C, and 71D are provided in the flow paths 62, 63, 65, and 67. In addition, in the inspection device 50, intake valves 72A, 72B, 72C, and 72D are attached to the reaction chamber 56, the hybridization buffer tank 64, the washing liquid tank 66, and the waste liquid tank 68, respectively. These liquid supply cutoff valves 71A, 71B, 71C, and 71D can open and close the flow paths, and guide the pressure of the pump device 70 to the flow path to which the liquid is to be sent. That is, the inspection device 50 is provided with a liquid supply device including the pump device 70, the liquid supply cutoff valves 71A, 71B, 71C, and 71D, and the intake valves 72A, 72B, 72C, and 72D. This liquid delivery device can deliver various solutions to the detection unit 55 via the flow paths 62 , 63 and 65 , and can deliver waste liquid to a waste liquid tank 68 via a flow path 67 .

[0075] Furthermore, when the reagent in the reaction chamber 56 or the DNA chip mounting portion 59 temporarily expands or contracts due to temperature fluctuation, these liquid supply cutoff valves 71A, 71B, 71C, and 71D can be moved to another portion connected by a flow path. The liquid supply cutoff valve is not particularly limited, and any valve that has been subjected to hydrophobic or hydrophilic treatment, or a material that changes shape due to heat or the like can be used without any limitation, but a shape that can be physically opened and closed by external stress is preferable, and as an example, it is preferable to use a diaphragm valve that is pressed by an actuator, or a rotary valve that is opened and closed by rotation.

[0076] In the testing device 50 configured as above, the target nucleic acid and non-target nucleic acid contained in the nucleic acid amplification reaction solution are detected, and the target nucleic acid can be quantitatively analyzed based on the detection. To quantitatively analyze the target nucleic acid, first, a nucleic acid amplification reaction of a region containing a gene mutation to be tested is performed in the reaction vessel 56. At this time, the reaction vessel 56 is filled with a reaction solution consisting of reagents necessary for the nucleic acid amplification reaction. The reaction vessel 56 may be filled with the reaction solution by an operator, or the reaction vessel 56 may be filled with the reaction solution from a different flow path (not shown). In addition, by setting a thermal cycle in the nucleic acid amplification reaction in advance, the first temperature control unit 57 can control the temperature and processing time of the reaction solution in the reaction vessel 56 according to the set thermal cycle.

[0077] In the testing device 50, next, a part of the reaction liquid is sent to the DNA chip mounting portion 59 on which the DNA chip 58 is mounted, using the above-mentioned dispensing device according to the present invention. Specifically, the pump device 70 is operated while the intake valve 72A and the liquid supply cutoff valves 71A and 71D are opened and the other intake valves 72B, 72C, and 72D and the liquid supply cutoff valves 71B and 71C are closed, and a fixed amount of the reaction liquid in the reaction tank 56 can be sent to the DNA chip mounting portion 59 by the dispensing operation in the above-mentioned dispensing device.

[0078] At this time, the inspection device 50 can be set to temporarily halt the thermal cycle performed by the first temperature control unit 57 and wait until the liquid transfer is completed in one temperature range of the thermal cycle (for example, 95° C.).

[0079] Further, in addition to a part of the reaction solution, the hybridization buffer is supplied from the hybridization buffer tank 64 to the DNA chip mounting portion 59. To supply (send) the hybridization buffer to the DNA chip mounting portion 59, the pump device 70 is operated while the intake valve 72B and the liquid supply cutoff valves 71B and 71D are opened and the other intake valves 72A, 72C and 72D and the liquid supply cutoff valves 71A and 71C are closed. This allows the hybridization buffer in the hybridization buffer tank 64 to be sent to the DNA chip mounting portion 59. The hybridization buffer may have a composition containing a blocking nucleic acid, which will be described later.

[0080] As a result, hybridization of the target nucleic acid and non-target nucleic acid contained in the reaction solution with various probes of the DNA chip 58 proceeds in the DNA chip mounting section 59. At this time, the second temperature control section 60 adjusts the temperature of the solution supplied to the DNA chip mounting section 59 to a hybridization temperature set in advance. The second temperature control section 60 can also perform preheating before the reaction if necessary. In this example, the reaction solution and the hybridization buffer solution are separately sent through different flow paths, but the reaction solution and the hybridization buffer solution can also be mixed and then sent to the DNA chip mounting section 59 as a mixed solution.

[0081] Next, in the inspection device 50, the detection unit 55 detects the target nucleic acid and the non-target nucleic acid hybridized to the probe. Specifically, first, a cleaning liquid is sent to the DNA chip mounting unit 59 to clean the DNA chip 58. The cleaning liquid can be sent from the cleaning liquid tank 66. To supply (send) the cleaning liquid to the DNA chip mounting unit 59, the intake valve 72C and the liquid supply cutoff valves 71C and 71D are opened, and the other intake valves 72A, 72B, and 72D and the liquid supply cutoff valves 71A and 71B are closed, and the pump device 70 is operated. During this cleaning, the second temperature control unit 60 can also lower the cleaning temperature (for example, 25° C.) lower than the hybridization reaction temperature.

[0082] When washing the DNA chip 58, the mixture of the reaction solution and hybridization buffer solution may be discharged into the waste tank 68, and then the washing solution may be pumped as described above, or the mixture of the reaction solution and hybridization buffer solution may be discharged into the waste tank 68 by pumping the washing solution as described above.

[0083] The surface of the DNA chip 58 on which the nucleic acid probes for detecting target nucleic acids, the nucleic acid probes for detecting non-target nucleic acids, and the common probe are immobilized is imaged by a camera 61, and the signal intensity of each probe is measured by image analysis software in a computer (not shown). Based on the measured signal intensity, the target nucleic acid can be quantitatively analyzed by the same computer.

[0084] As described above, by using the inspection device 50 equipped with the dispensing device according to the present invention, it is possible to detect target nucleic acid and non-target nucleic acid based on the hybridization reaction with each probe using the DNA chip 58. In the above-mentioned example, the reaction vessel 56 and the DNA chip mounting section 59 are connected via the flow path 62, and a part of the nucleic acid amplification reaction liquid is sent to the detection section 55 by the dispensing device according to the present invention. In addition, the hybridization buffer solution and the washing solution are sent to the detection section 55 by the configuration of the pump device 70, the liquid sending cutoff valves 71B, 71C, and 71D, and the intake valves 72B, 72C, and 72D. However, the mechanism (liquid sending device) for sending the hybridization buffer solution and the washing solution to the detection section 55 is not limited to such a configuration, and the dispensing device according to the present invention or a pipette device arranged on the drive arm can be used in the same manner as the nucleic acid amplification reaction liquid.

[0085] 14, the above-mentioned liquid sending operation is performed by suction using the pump device 70. However, the testing device 50 can also perform the above-mentioned liquid sending operation by using a pressure pump connected upstream of the nucleic acid amplification reaction unit 54, the hybridization buffer tank 64, and the washing liquid tank 66.

[0086] In addition, in the testing device 50 shown in Figure 14, the hybridization buffer solution is sent directly to the DNA chip mounting section 59 from the hybridization buffer solution tank 64 connected to the DNA chip mounting section 59 via the flow path 63, but it may also be sent to the DNA chip mounting section 59 after first merging with the flow path 62.

[0087] 14 includes a DNA chip mounting section 59 on which a DNA chip 58 can be mounted, and the DNA chip 58 is detachably configured. However, the testing device 50 is not limited to such a configuration, and may have a configuration in which each probe is directly fixed to the detection section 55.

[0088] 14, the nucleic acid amplification reaction liquid is delivered from the nucleic acid amplification reaction unit 54 having one reaction chamber 56 by a dispensing device, but the testing device to which the present invention is applied is not limited to this configuration. That is, the testing device to which the present invention is applied may be configured to include a nucleic acid amplification reaction unit 54 having a plurality of reaction chambers 56 and a plurality of dispensing devices corresponding to the plurality of reaction chambers 56. In this case, different dispensing devices are connected to the plurality of reaction chambers 56, and the temperatures of the reaction chambers 56 may be collectively controlled by a single first temperature control unit 57, or the temperatures of the reaction chambers 56 may be individually controlled by a plurality of first temperature control units 57.

[0089] In this case, the inspection device 50 has a plurality of flow paths corresponding to the outlet flow paths 5 of the dispensing device connected to each of the plurality of reaction chambers 56, and a switching device disposed on the flow paths and switching the flow paths that supply the reaction liquid to the detection unit 55. In this case, a fixed amount of the nucleic acid amplification reaction liquid can be dispensed from one of the plurality of reaction chambers 56 to the detection unit 55 by the switching device.

[0090] The following describes gene mutations to be tested in the above-mentioned testing device 50 and various probes to be mounted on the DNA chip.

[0091] The genetic mutation to be tested is, for example, a single nucleotide variant (SNV) or a single nucleotide polymorphism (SNP), which means a difference in a base at a specific position of genomic DNA. That is, for a specific genetic mutation, multiple bases are present. In the following description, one of these bases is referred to as a detection target base, and bases other than the detection target base are referred to as non-detection target bases.

[0092] The detection target base means, for example, a specific nucleic acid residue at a specific position of genomic DNA, and is not particularly limited, but means a specific type of base in a base sequence such as a single nucleotide polymorphism (SNP). For example, if a specific single nucleotide polymorphism can take A (adenine) or C (cytosine), either one of the bases, that is, A (adenine) in the single nucleotide polymorphism, can be the detection target base. Here, the detection target base may be either a major allele or a minor allele in a gene polymorphism, and may or may not be a risk allele.

[0093] Here, the target nucleic acid means a nucleic acid molecule containing a base to be detected, that is, a nucleic acid fragment. The target nucleic acid may be a nucleic acid molecule made of DNA, a nucleic acid molecule made of RNA, a nucleic acid molecule containing DNA and RNA (DNA-RNA complex), or a fragmented one such as cell-free DNA. In addition, the nucleic acid includes adenine, cytosine, guanine, thymine, and uracil, as well as artificial nucleic acids such as peptide nucleic acid (PNA) and locked nucleic acid (LNA).

[0094] The target nucleic acid containing the detection target base can be prepared by amplifying a specific region on genomic DNA containing a gene mutation by a nucleic acid amplification method. The target nucleic acid may also be cDNA obtained by reverse transcription reaction from a transcription product collected from an individual organism, tissue, or cell. The base length of the target nucleic acid is not particularly limited, but may be, for example, 60 to 1000 bases, preferably 60 to 500 bases, and more preferably 60 to 200 bases.

[0095] In addition, a nucleic acid molecule (nucleic acid fragment) containing a non-target base corresponding to a target base to be detected is called a non-target nucleic acid. For example, when one base among a plurality of bases that can be taken at a predetermined position in genomic DNA is taken as a target base to be detected, bases other than the target base to be detected are taken as non-target bases to be detected. More specifically, when a single nucleotide polymorphism at a predetermined position can take A (adenine) or C (cytosine), if A (adenine) in the single nucleotide polymorphism is taken as a target base to be detected, C (cytosine) in the single nucleotide polymorphism is taken as a non-target base to be detected.

[0096] Furthermore, for example, when a mutant type (forward or reverse strand of a minor allele) in a specific polymorphism or variant is used as a base to be detected, a nucleic acid fragment containing the mutant type can be used as a target nucleic acid, and a nucleic acid fragment containing a wild type (forward or reverse strand of a major allele), which is a base not to be detected, can be used as a non-target nucleic acid.

[0097] When the non-target base exists on genomic DNA for a specific gene mutation, the non-target nucleic acid containing the non-target base is obtained at the same time as the target nucleic acid containing the target base is obtained as described above.For example, when the target nucleic acid is obtained by a nucleic acid amplification reaction such as polymerase chain reaction, if one allele is a non-target base, the non-target nucleic acid is amplified together with the target nucleic acid.

[0098] In the above-mentioned genetic mutation testing device, in order to detect a target nucleic acid containing a target base, a nucleic acid probe having a base sequence complementary to at least a region containing the target base in the target nucleic acid is used. The nucleic acid probe is not particularly limited, but may be, for example, 10 to 30 bases long, and preferably 15 to 25 bases long. In addition, when the bases constituting the nucleic acid probe are viewed as a character string, the base complementary to the target base is preferably located at the center of the character string. Note that the center of the character string includes the case where the nucleic acid probe is shifted by one base toward the 5' end or 3' end in the case of a nucleic acid probe consisting of an even number of bases.

[0099] In particular, the above-mentioned genetic mutation testing device can use a common probe having a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid. The common region is a region of a base sequence common to the target nucleic acid and the non-target nucleic acid, and does not contain a detection target base or a non-detection target base. For example, the common region can be a region that does not overlap with a region where a nucleic acid probe for detecting a target nucleic acid hybridizes and a region where a nucleic acid probe for detecting a non-target nucleic acid hybridizes.

[0100] The above-mentioned genetic mutation testing device can be applied to any system as long as it includes hybridization, which means complementary binding between nucleic acid molecules in a target nucleic acid and a nucleic acid probe. That is, the above-mentioned genetic mutation testing device can be based on Southern hybridization, Northern hybridization, or in situ hybridization. In particular, the above-mentioned genetic mutation testing device is preferably used in a system in which a nucleic acid probe is fixed to a carrier (including a substrate, hollow fiber, and microparticles) and the target nucleic acid is detected (including qualitative and quantitative) using the fixed nucleic acid probe. More specifically, the above-mentioned genetic mutation testing device uses a DNA chip (DNA microarray) in which a nucleic acid probe is fixed to a substrate.

[0101] Hereinafter, a system used for detecting a target nucleic acid using a DNA chip (DNA microarray) will be described. The above-mentioned nucleic acid probe for detecting a target nucleic acid (nucleic acid probe for detecting a target nucleic acid), the nucleic acid probe for detecting a non-target nucleic acid (nucleic acid probe for detecting a non-target nucleic acid), and the common probe are more preferably single-stranded DNA, and can be obtained by chemical synthesis using a nucleic acid synthesizer, for example. As the nucleic acid synthesizer, a device called a DNA synthesizer, a fully automatic nucleic acid synthesizer, an automatic nucleic acid synthesizer, etc. can be used.

[0102] In this example, the nucleic acid probe for detecting the target nucleic acid, the nucleic acid probe for detecting the non-target nucleic acid, and the common probe are preferably used in the form of a microarray by immobilizing their 5'-end or 3'-end on a carrier. The material of the carrier can be any known in the art and is not particularly limited. For example, conductive materials such as precious metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten, and compounds thereof, and carbon such as graphite and carbon fiber; silicon materials such as single crystal silicon, amorphous silicon, silicon carbide, silicon oxide, silicon nitride, and composite materials of these silicon materials such as SOI (silicon-on-insulator); inorganic materials such as glass, quartz glass, alumina, sapphire, ceramics, forsterite, and photosensitive glass; polyethylene, polypropylene, rings, etc. Examples of the organic materials include polyolefins, polyisobutylene, polyethylene terephthalate, unsaturated polyesters, fluorine-containing resins, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resins, polyacrylonitrile, polystyrene, acetal resins, polycarbonate, polyamide, phenolic resins, urea resins, epoxy resins, melamine resins, styrene-acrylonitrile copolymers, acrylonitrile-butadiene styrene copolymers, polyphenylene oxide, and polysulfone. The shape of the carrier is not particularly limited, but is preferably flat.

[0103] As the carrier, a carrier having a carbon layer such as diamond-like carbon (DLC) and a chemical modification group such as an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and an active ester group on the surface is preferably used. The carrier having a carbon layer and a chemical modification group on the surface includes a substrate having a carbon layer and a chemical modification group on the surface, and a substrate made of a carbon layer having a chemical modification group on the surface. The substrate material can be any known material in the art, and is not particularly limited, and the same materials as those listed as the above-mentioned carrier materials can be used.

[0104] The above-mentioned genetic mutation testing device can quantitatively analyze the target nucleic acid in the subject using the DNA chip thus prepared. Specifically, the method includes the steps of: first, extracting DNA from a sample derived from the subject; and amplifying a region containing the genetic mutation of the test subject by a nucleic acid amplification reaction using the extracted DNA as a template, and detecting at least the target nucleic acid using the DNA chip in the exponential amplification phase of the nucleic acid amplification reaction.

[0105] The subject is usually a human being, and is a subject to be examined for genetic mutations, and may be a patient suffering from a disease associated with a specific genetic mutation. The sample derived from the subject is not particularly limited. For example, blood-related samples (blood, serum, plasma, etc.), lymph, feces, cancer cells, tissue or organ fragments and extracts, etc. may be included.

[0106] First, DNA is extracted from a sample collected from a subject. Next, an amplification reaction is performed using the obtained DNA as a template to amplify a nucleic acid region containing a gene mutation to be tested, preferably DNA. As the amplification reaction, polymerase chain reaction (PCR), LAMP (Loop-Mediated Isothermal Amplification), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids), etc. can be applied. In the amplification reaction, it is desirable to add a label so that the region after amplification can be identified. At this time, the method of labeling the amplified nucleic acid is not particularly limited, but for example, a method of labeling the primer used in the amplification reaction in advance may be used, or a method of using a labeled nucleotide as a substrate in the amplification reaction may be used. As the labeling substance, it is not particularly limited, but a radioisotope, a fluorescent dye, or an organic compound such as digoxigenin (DIG) or biotin, etc. can be used.

[0107] This reaction system also contains a buffer necessary for nucleic acid amplification and labeling, a heat-stable DNA polymerase, a pair of primers specific to the region to be amplified, labeled nucleotide triphosphates (specifically, nucleotide triphosphates to which a fluorescent label or the like has been added), nucleotide triphosphates, and magnesium chloride.

[0108] Nucleic acid amplification reaction consists of three steps: denaturation, annealing, and extension. These three steps constitute one cycle, and by repeating multiple cycles, a nucleic acid fragment containing the genetic mutation to be tested can be synthesized. The procedure for repeating these three steps is called a thermal cycle, and the number of times the three steps are repeated is called the number of thermal cycles.

[0109] The nucleic acid amplification reaction may also be composed of two steps, a thermal denaturation step and an annealing and extension step, by performing annealing and extension at the same temperature, which is called 2-step PCR. In this case, by repeating these two steps as one cycle, a nucleic acid fragment containing the gene mutation to be tested can be synthesized. In this case, the procedure for repeating these two steps is called a thermal cycle, and the number of times the two steps are repeated is called the number of thermal cycles.

[0110] The thermal denaturation step is a step in which a reaction solution containing template DNA is heated to 90°C or higher to form single-stranded DNA. The first thermal denaturation step in the thermal cycle is preferably carried out for a relatively long time (1 to 5 minutes) when genomic DNA is used as a template. The second and subsequent thermal denaturation steps are preferably carried out for a relatively short time (1 to 90 seconds) since the purpose is to convert the amplified product obtained as a double strand into a single strand.

[0111] The annealing step is a step in which a reaction solution containing a single-stranded template DNA and a pair of primer sets is heated to a temperature usually in the range of 40 to 75°C, and these primers are specifically bound to the template DNA. The temperature of the annealing step is appropriately set depending on the sequence and length of the primers. The temperature of the annealing step is usually set within a range of ±5°C of the Tm value of the primer used.

[0112] The extension step is a step in which the temperature of the reaction solution is set to about 72° C. with the primer annealed to the template DNA, and a complementary strand is synthesized from the end of the primer by a heat-resistant polymerase (such as Taq DNA polymerase). In this reaction, the complementary strand of the template DNA is synthesized from a labeled primer, or the complementary strand of the template DNA is synthesized while incorporating a labeled free nucleotide. Thus, for the genetic mutation to be tested, the target nucleic acid containing the base to be detected and the non-target nucleic acid containing the base not to be detected are labeled. The time of the extension step is appropriately set depending on the length of the nucleic acid fragment to be amplified and the reactivity of the enzyme to be used.

[0113] In the above-mentioned genetic mutation testing device, a fixed amount of nucleic acid amplification reaction solution is dispensed, the target nucleic acid and non-target nucleic acid contained therein are detected, and the target nucleic acid can be quantitatively analyzed based on the detection. In particular, in the above-mentioned genetic mutation testing device, a fixed amount of nucleic acid amplification reaction solution is dispensed at a predetermined number of thermal cycles in the nucleic acid amplification reaction, and the target nucleic acid contained therein can be quantitatively analyzed. In addition, in the above-mentioned genetic mutation testing device, the operation of dispensing a fixed amount of nucleic acid amplification reaction solution is performed multiple times at different numbers of thermal cycles, and the target nucleic acid contained in the reaction solution dispensed each time can be quantitatively analyzed.

[0114] To quantitatively analyze a target nucleic acid, a hybridization reaction between a nucleic acid probe for detecting a target nucleic acid and the target nucleic acid is carried out, and the amount of the nucleic acid hybridized to the nucleic acid probe for detecting a target nucleic acid can be measured, for example, by detecting a label. For example, when a fluorescent label is used, the signal from the label can be detected as a fluorescent signal using a fluorescent scanner, and the signal intensity can be quantified by analyzing the signal using image analysis software.

[0115] The hybridization reaction is preferably carried out under stringent conditions. Stringent conditions refer to conditions under which specific hybrids are formed and non-specific hybrids are not formed, for example, conditions under which the hybridization reaction is carried out at 50°C for 16 hours, followed by washing at 2xSSC / 0.2% SDS at 25°C for 10 minutes and 2xSSC at 25°C for 5 minutes. That is, the hybridization buffer composition according to the present invention may contain salts necessary for the hybridization reaction, such as SSC, and known blocking agents, such as SDS.

[0116] Specifically, when a target nucleic acid is quantitatively analyzed based on signals from a nucleic acid probe for detecting a target nucleic acid and a nucleic acid probe for detecting a non-target nucleic acid, the signal intensities of the nucleic acid probe for detecting a target nucleic acid and the nucleic acid probe for detecting a non-target nucleic acid are measured, respectively, and a judgment value for evaluating the signal intensity from the nucleic acid probe for detecting a target nucleic acid is calculated. An example of the calculation of the judgment value is a method using the formula: [signal intensity from the nucleic acid probe for detecting a target nucleic acid] / ([signal intensity from the nucleic acid probe for detecting a target nucleic acid]+[signal intensity from the nucleic acid probe for detecting a non-target nucleic acid]).

[0117] Incidentally, the above-mentioned genetic mutation testing device is not limited to the above-mentioned configuration, and in order to prevent non-target nucleic acids in the nucleic acid amplification reaction solution from non-specifically hybridizing to the nucleic acid probe for detecting the target nucleic acid, a so-called blocking nucleic acid can also be used. The blocking nucleic acid can be mixed with a reaction solution containing a target nucleic acid and a non-target nucleic acid, and the resulting mixture can be brought into contact with a DNA chip to allow the hybridization reaction between the target nucleic acid and the nucleic acid probe for detecting the target nucleic acid to proceed. In addition, the reaction solution containing the target nucleic acid and the non-target nucleic acid and the solution containing the blocking nucleic acid can be mixed on the DNA chip to allow specific hybridization between the target nucleic acid and the nucleic acid probe to proceed simultaneously. In any of these cases, the blocking nucleic acid hybridizes to the non-target nucleic acid, thereby preventing the non-target nucleic acid from hybridizing to the probe for detecting the target nucleic acid.

[0118] The blocking nucleic acid has a base sequence complementary to a region containing a non-target base in the non-target nucleic acid, and can hybridize with the non-target nucleic acid containing the non-target base under conditions in which the target nucleic acid containing the target base can hybridize with the nucleic acid probe.

[0119] In the blocking nucleic acid, the position of the base corresponding to the non-detection target base is not particularly limited. The blocking nucleic acid is not particularly limited, but is preferably 60% or more in length relative to the base length of the nucleic acid probe. The blocking nucleic acid is preferably 140% or less in length relative to the base length of the nucleic acid probe. For example, if the length of the nucleic acid probe is 25 bases, the blocking nucleic acid is preferably 15 to 35 bases in length.

[0120] Furthermore, the blocking nucleic acid may contain mismatched bases (non-complementary bases) at positions corresponding to bases other than the non-detection target bases contained in the non-target nucleic acid. When the blocking nucleic acid is 15 bases long, the number of mismatched bases may be 1 to 3, preferably 1 to 2. When the blocking nucleic acid is 25 bases long, the number of mismatched bases may be 1 to 5, preferably 1 to 4.

[0121] Furthermore, the concentration of the blocking nucleic acid is not particularly limited, and can be appropriately set according to, for example, the concentration of the non-target nucleic acid and / or the concentration of the target nucleic acid, or according to the primer concentration. Specifically, the concentration of the blocking nucleic acid in the composition can be 0.01 to 2 μM, preferably 0.02 to 1.5 μM, and more preferably 0.05 to 1.5 μM.

[0122] In addition, when the blocking nucleic acid is used to prevent non-specific hybridization between the non-target nucleic acid and the nucleic acid probe for detecting the target nucleic acid, the specific hybridization between the non-target nucleic acid and the nucleic acid probe for detecting the non-target nucleic acid is also inhibited. In this case, the signal intensity from the nucleic acid probe for detecting the non-target nucleic acid is greatly reduced. In this case, the determination value according to the above formula: [signal intensity from the nucleic acid probe for detecting the target nucleic acid] / ([signal intensity from the nucleic acid probe for detecting the target nucleic acid]+[signal intensity from the nucleic acid probe for detecting the non-target nucleic acid]) may not be sufficient for quantitative analysis of the target nucleic acid. In this case, it is preferable to measure the total amount of the target nucleic acid and the non-target nucleic acid using a common probe.

[0123] As described above, the common probe has a sequence complementary to a common region common to the target nucleic acid and the non-target nucleic acid, and therefore can hybridize to the same non-target nucleic acid even if the blocking nucleic acid is hybridized to the same non-target nucleic acid.

[0124] When a common probe is used, the formula for calculating the above-mentioned judgment value can be [signal intensity from the nucleic acid probe for detecting the target nucleic acid] / [signal intensity from the common probe]. According to this formula, the influence of the blocking nucleic acid can be eliminated and the ratio of the target nucleic acid to the total of the target nucleic acid and non-target nucleic acid can be calculated.

[0125] In addition, the target nucleic acid can be absolutely quantified by preparing a calibration curve in advance. The calibration curve can be prepared by detecting the target nucleic acid and the non-target nucleic acid contained in the nucleic acid amplification reaction solution using various known mutation ratio samples (samples containing the target nucleic acid and the non-target nucleic acid at a known ratio), and calculating the determination value by the above formula. That is, the calibration curve shows the relationship between the determination value calculated as described above and the ratio of the target nucleic acid and the non-target nucleic acid contained in the nucleic acid amplification reaction solution, or the amount of the target nucleic acid.

[0126] As described above, the above-mentioned genetic mutation testing device can dispense a fixed amount of nucleic acid amplification reaction solution with a simple configuration, quantitatively analyze the target nucleic acid contained in the dispensed reaction solution, and quantitatively test whether or not there is a genetic mutation in the genomic DNA derived from the subject and the proportion of genetic mutations. [Explanation of symbols]

[0127] Reference Signs List 1... Dispensing device, 2... Main chamber, 3... Dispensing chamber, 4... Connecting flow path, 5... Outlet flow path, 6... Inlet portion, 7... Outlet portion, 8... First vent, 9... First vent passage

Claims

1. A dispensing chamber having an inlet portion connected to a main chamber filled with a solution and an outlet portion, the inlet portion being at a predetermined position in the direction of gravity, and the outlet portion being at the bottom in the direction of gravity; A dispensing device, characterized in that the solution supplied from the main chamber through the inlet portion fills the dispensing chamber from the lowest portion to the height of the inlet portion.

2. 2. The dispensing device of claim 1, wherein the dispensing chamber is tapered toward the outlet port.

3. the main chamber includes a solution outlet connected to the inlet of the dispensing chamber for discharging a solution in the main chamber; 2. The dispensing device according to claim 1, wherein the inlet of the dispensing chamber is provided at a position higher in the direction of gravity than the solution outlet of the main chamber.

4. 2. The dispensing device according to claim 1, wherein the inlet of the dispensing chamber is provided at a position higher in the direction of gravity than the liquid level of the solution filled in the main chamber.

5. 2. The dispensing device according to claim 1, wherein the dispensing chamber has an air vent located at a position higher than the inlet in the direction of gravity.

6. 6. The dispensing device according to claim 5, wherein the flow resistance of the outlet of the dispensing chamber is greater than the flow resistance of the vent hole.

7. 2. The dispensing device of claim 1, wherein the main chamber has at least one inlet, a vent, and an outlet connected to the dispensing chamber.

8. 2. The dispensing device according to claim 1, wherein the outlet of the dispensing chamber is in communication with a discharge port disposed at a high position in the direction of gravity.

9. 2. The dispensing device according to claim 1, wherein the outlet of the dispensing chamber is connected to a discharge port disposed at a higher position in the direction of gravity than the inlet of the dispensing chamber.

10. 2. The dispensing device according to claim 1, which is a micro-channel cartridge formed by bonding a substrate having a recess formed therein that serves as a channel connecting the main chamber, the dispensing chamber, and the main chamber and the dispensing chamber to another substrate.

11. A dispensing method using the dispensing device according to any one of claims 1 to 10, filling the dispensing chamber with the solution from a main chamber filled with the solution up to the height of an inlet of the dispensing chamber; Discharging the solution filled in the dispensing chamber from an outlet of the dispensing chamber; A dispensing method comprising:

12. The dispensing method according to claim 11, characterized in that in the step of filling the dispensing chamber with the solution, the solution is filled to a height exceeding the inlet portion of the dispensing chamber, and then, with the outlet portion blocked, the solution that has exceeded the inlet portion is returned from the inlet portion to the main chamber, thereby filling the dispensing chamber with the solution up to the height position of the inlet portion of the dispensing chamber.

13. A nucleic acid analyzer comprising the dispensing device according to any one of claims 1 to 10.

14. a nucleic acid amplification unit having the above-mentioned dispensing device and amplifying nucleic acid contained in the solution supplied to the main chamber; a hybridization unit connected to the dispensing chamber in the nucleic acid amplification unit and configured to perform hybridization between the nucleic acid contained in the solution supplied from the dispensing chamber and the nucleic acid probe; 14. The nucleic acid analyzer according to claim 13, further comprising a detection section for detecting hybridization between the nucleic acid probe and a nucleic acid in the hybridization section.

15. Further comprising a nucleic acid extraction unit for extracting nucleic acid from the sample, The nucleic acid analyzer according to claim 14, wherein a solution containing nucleic acid is supplied from the nucleic acid extraction unit to a main chamber of the nucleic acid amplification unit.

16. 15. The nucleic acid analyzer according to claim 14, wherein the hybridization section comprises a DNA chip on which the nucleic acid probe is fixed.

17. 15. The nucleic acid analyzer according to claim 14, wherein a target nucleic acid containing a base to be detected in a gene mutation is quantitatively analyzed.

18. A nucleic acid analysis method using the nucleic acid analysis device according to any one of claims 14 to 17, amplifying the nucleic acid contained in the solution supplied to the main chamber of the nucleic acid amplification unit of the nucleic acid analyzer through multiple thermal cycles; supplying the solution filled in the main chamber to a dispensing chamber in a predetermined thermal cycle in the step of amplifying the nucleic acid, supplying the solution filled in the dispensing chamber to a nucleic acid analysis unit in the nucleic acid analyzer, and analyzing the nucleic acid contained in the supplied solution in the nucleic acid analysis unit, The nucleic acid analysis method is characterized in that in the step of analyzing the nucleic acid, a target nucleic acid containing a base to be detected in a gene mutation is quantitatively analyzed.

19. 20. The method for analyzing nucleic acid according to claim 18, wherein the step of analyzing the nucleic acid is carried out multiple times using different thermal cycles.

Images