Microchannel dispensing device and microchannel device

Abstract

The present invention provides a flow path device that can be inexpensively manufactured to easily and quickly perform absolute quantitative testing of target genes using the LAMP method, and a dispensing device for use with the flow path device. [Solution] The dispensing device comprises a main channel 1, a branch channel 2, and a chamber 3. The main channel comprises a first in-channel resistance section 4 downstream of the branch position, which reduces the channel cross-sectional area and temporarily obstructs the downstream flow of fluid. A secondary channel 5 is provided between the main channel and the chamber. The secondary channel has a channel cross-sectional area smaller than the channel cross-sectional area of ​​the first in-channel resistance section, and is connected in a range from near the downstream end of the chamber to the downstream side near the first in-channel resistance section provided in the main channel. The microchannel device has multiple chambers, to which fluid is dispensed by the dispensing device, intermittently installed on one or both sides of the main channel.

Description

[Technical Field]

[0001] The present invention relates to a dispensing device for a microchannel and a microchannel device configured using the dispensing device. [Background technology]

[0002] Generally, when performing multiple virus tests (gene amplification reactions), a number of markers corresponding to the number of tests is required, and because the reactions are performed for the number of test subjects, samples and reagents must be prepared each time. This requires specialized knowledge and skill. Gene amplification reactions include PCR (Polymerase Chain Reaction) and LAMP (Loop-Mediated Isothermal Amplification). PCR uses three temperature settings to amplify DNA using a synthetic enzyme and primers, while LAMP amplifies the target base sequence in DNA using primers and strand-displacing synthetic enzymes. When using either of these gene amplification reactions, multiple tests require multiple reaction vessels.

[0003] The above-mentioned multiple virus tests are sometimes used, for example, to detect arthropod-borne viral infections (such as dengue fever and Zika fever). They involve immobilizing primers for amplifying genes associated with the infection in a reaction vessel, then adding a mixture of a genetic sample collected from an arthropod and a gene amplification reagent. Similar virus tests are also being conducted using PCR in the recent COVID-19 pandemic. Furthermore, the method is not limited to virus testing; it is also used for genetic testing of food allergens (such as wheat, buckwheat, and peanuts) contained in trace amounts in processed foods and illegal drugs (such as cannabis).

[0004] Meanwhile, in biological analysis, testing devices using microchips, such as μ-TAS (Micro Total Analysis Systems), are used, and these devices can perform reactions using small amounts of sample. Furthermore, such testing devices are configured to form microchannels and supply a predetermined test liquid to a reaction vessel. To simultaneously supply the same test liquid to multiple reaction vessels, a device has been developed that includes a main channel and branch channels, and the test liquid is supplied to multiple reaction vessels via the main channel (see Patent Document 1).

[0005] In the above technology, multiple branch channels are formed on a main channel, and storage units (reaction vessels) are formed on the branch channels, and an exhaust unit is connected to the storage unit to ensure smooth liquid supply to the storage unit. This exhaust unit configuration allows smooth liquid supply to the storage unit, but also smooths the flow of liquid passing through the main channel and branch channels, which can make the storage state of the liquid supplied to each storage unit (reaction vessel) unstable and raises concerns about mutual contamination.

[0006] Therefore, the inventors of the present application developed a dispensing device that provides resistance parts within the flow paths at appropriate positions in the main flow path and the branch flow paths and makes these parts behave like valves, thereby supplying an appropriate amount of liquid to reaction vessels provided in each branch flow path without causing backflow (see Non-Patent Document 1), and then developed a dispensing device that is an improvement over the above (see Patent Document 2).This technology configures branch flow paths according to the difference in resistance pressure (breakdown pressure) caused by the resistance parts within the flow path, but when many branch flow paths are configured, it is not possible to provide a sufficient pressure difference due to the resistance parts within the flow path, and there is a limit to the number of branch flow paths that can be installed (the number of branches that can be dispensed).

[0007] Therefore, the inventors of the present application have made further improvements and developed a dispensing device that dispenses using multiple branched channels and is not limited by the number of branched channels even when obtaining a wide variety of reaction results (see Patent Document 3). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-284769 [Patent Document 2] Japanese Patent Publication No. 2022-080026 [Patent Document 3] International Publication No. 2023-120648 [Non-patent literature]

[0009] [Non-Patent Document 1] Proceedings of the 2018 Autumn Meeting of the Japan Society for Precision Engineering, G02-12, pp. 772-773 Summary of the Invention [Problem to be solved by the invention]

[0010] The invention disclosed in the aforementioned Patent Document 3 arranges a main flow path and a discharge path in parallel, places a chamber between them, and connects a branch flow path branching off from the main flow path to the upstream side of the chamber, with the downstream side of the chamber connected to the discharge path.The discharge path is formed with an internal resistance section that inhibits the flow downward, and as the fluid that passes through the chambers reaches the discharge path one after another, the flow downward is inhibited by the internal resistance section.At the same time, by interposing internal air between the discharge paths of adjacent chambers, the inhibition of the flow downward in the discharge path is stabilized by balancing with the pressure that tends to flow back in the discharge path.

[0011] To eliminate the effects of viral infections, food allergens, and food poisoning bacteria on the human body, quantitative testing of the target genes that cause these diseases is necessary, and genetic diagnostic devices are essential for this purpose. These devices must be simple, fast, and low-cost. Recently, digital PCR has been developed, which estimates the absolute number of target genes in a sample based on statistical methods without the need for a calibration curve. However, no device has yet been developed that can easily and quickly perform absolute quantitative testing of target genes using the LAMP method, a type of isothermal gene amplification method.

[0012] When using a dispensing device with the above configuration, it is possible to dispense a fluid (specimen sample, etc.) into a large number of chambers, but it is not possible to dispense it quickly into the extremely large number of chambers used for quantitative testing of target genes. In other words, the fluid (specimen sample) that should flow down along the main flow path cannot flow down to the next chamber only after the dispensed fluid (specimen sample) has flowed into the chamber and reached the discharge path, and it takes time from dispensing into one chamber to dispensing into the next chamber. In addition, the discharge path connected to the downstream side of the chamber is required to precisely configure the resistance portion within the flow path for each connection, so it is difficult to dispense a large number of chambers (100 in units) for quantitative testing of target genes. n Equipment for forming such a chamber can be expensive.

[0013] The present invention has been made in consideration of the above points, and its purpose is to provide a flow path device that can be inexpensively manufactured to easily and quickly perform absolute quantitative testing of target genes using the LAMP method or the like, and a dispensing device for use with the flow path device. [Means for solving the problem]

[0014] Therefore, a first invention relating to a microchannel dispensing device includes a main channel that allows a fluid to flow down in one direction, a branch channel branching from the main channel, and a chamber connected to the branch channel, and is used to dispense a fluid into the chamber. The main channel includes a first internal resistance portion downstream of a branch position where the main channel branches off to the branch channel, the first internal resistance portion reducing a cross-sectional area of ​​the channel and temporarily obstructing the flow of fluid downstream. A secondary channel is provided between the main channel and the chamber, separate from the branch channel. The secondary channel has a cross-sectional area smaller than that of the first internal resistance portion and is connected in a range from near the downstream end of the chamber to the downstream side near the first internal resistance portion provided in the main channel. The connection portion between the secondary channel and the chamber constitutes a second internal resistance portion that obstructs the inflow of fluid into the chamber. The connection portion between the secondary channel and the main channel constitutes a third internal resistance portion that obstructs the inflow of fluid from the main channel to the secondary channel.

[0015] According to the above configuration, the first flow path resistance portion is located downstream of the branch point where the main flow path branches into the branch flow path. The first flow path resistance portion temporarily blocks the flow of fluid down the main flow path, causing the fluid to flow into the chamber via the branch flow path. After the fluid enters the chamber and fills the chamber, the fluid begins to flow into the secondary flow path near the downstream end. However, because the secondary flow path has a smaller cross-sectional area, it acts as a second flow path resistance, blocking the inflow of the fluid. At this time, the cross-sectional area of ​​the secondary flow path is configured smaller than the cross-sectional area of ​​the first flow path resistance portion. Therefore, the first flow path resistance portion breaks down without breaking down the second flow path resistance portion. At this stage, the fluid resumes flowing down the main flow path. As the fluid flows down the main flow path, it reaches the connection with the secondary flow path, but because the cross-sectional area of ​​the secondary flow path is small, the fluid cannot flow into the secondary flow path (backflow). As a result, the secondary flow path becomes filled with internal air, the pressure inside the flow path at each connection part at both ends is balanced, and the flow of fluid inside the chamber is maintained in a stopped state. Therefore, the fluid flowing down the main flow path continues to flow toward the next chamber and the first flow path resistance part nearby without flowing into the secondary flow path (backflow), and the flow into the chambers in sequence and the state is maintained continuously.

[0016] A second invention relating to a dispensing device for a microchannel is the first invention, wherein the secondary channel is formed by a uniform channel cross-sectional area and is connected to the chamber at any position of the secondary channel, and the second breakthrough pressure for releasing the temporary obstruction to inflow caused by the second channel resistance and the third breakthrough pressure for releasing the temporary obstruction to inflow caused by the third channel resistance are sufficiently larger than the first breakthrough pressure for releasing the temporary obstruction to downstream flow caused by the first channel resistance.

[0017] With the above configuration, the secondary flow path is formed with a small cross-sectional area overall, so that the breakthrough pressure of the second flow path resistance portion can be increased in relation to the flow path cross-sectional area regardless of the state of the opening portion at the connection portion near the downstream end of the chamber. Note that the breakthrough pressure of each flow path resistance portion is determined by the flow path cross-sectional area of ​​the respective flow path, but the breakthrough pressures of the second and third flow path resistance portions are adjusted to have a flow path cross-sectional area that is sufficiently larger than the breakthrough pressure of the first flow path resistance portion.

[0018] A third invention relating to a dispensing device for a microchannel is the first invention, wherein the secondary channel has a smaller channel cross-sectional area by at least lowering the channel height compared to the main channel.

[0019] According to the above configuration, the size of the flow path cross-sectional area is determined by the flow path width and flow path height in the case of a typical rectangular cross-section flow path. However, if the flow path cross-sectional area is reduced by the flow path width alone, the aspect ratio of the flow path cross-sectional shape (flow path height / flow path width) becomes large, and there are limitations due to manufacturing precision and other factors, which inevitably limits the amount of flow path height that can be increased. In contrast, reducing the flow path cross-sectional area by lowering the flow path height simply requires adjusting the relative height compared to the main flow path (the flow path in which the first flow path resistance portion is formed). This means that the height of the main flow path can be adjusted based on the flow path height of the secondary flow path, making manufacturing easier. Furthermore, adjusting the flow path width simultaneously with the flow path height further facilitates adjusting the flow path cross-sectional area compared to the main flow path (especially the first flow path resistance portion), which may also facilitate flow path design. In addition, it is known that in a microchannel with a rectangular cross section, the magnitude of the flow channel resistance is proportional to the channel width, but is also proportional to the cube of the channel height, and from this perspective, reducing the channel height is expected to be more effective in increasing the flow channel resistance. In this case, if the first intra-channel resistance section is configured to reduce only the channel width in the main channel and the chamber is configured to have the same height as the main channel, the channel height in the region where the fluid actually flows down (into) will be constant, and the flow state of the fluid will be stable.

[0020] A fourth invention relating to a dispensing device for a microchannel is the third invention, wherein the main channel, the branch channels, and the chamber all have a uniform channel height, and the secondary channels are configured at a predetermined channel height from the same bottom or ceiling as the main channel.

[0021] According to the above configuration, the system is configured with flow paths having two different flow path heights, and all flow paths other than the secondary flow path are provided with the same flow path height, and the entire flow path including the secondary flow path is formed based on the same bottom surface, so that the flow path height of the secondary flow path can be easily manufactured to a flow path height that is a predetermined ratio compared to the other flow path heights.

[0022] On the other hand, a first invention relating to a microchannel device is a microchannel device using any of the first to fourth inventions relating to the dispensing apparatus, comprising an inlet portion for supplying a fluid, the main channel connected to the inlet portion, an outlet portion connected to an end of the main channel, a plurality of branch channels provided at appropriate intervals from the main channel, chambers connected to each of the plurality of branch channels, and secondary channels connecting each of the chambers to the main channel, wherein the main channel is provided with a first in-channel resistance portion downstream of each branch position where the main channel branches off from the plurality of branch channels in the vicinity of the branch position, the first in-channel resistance portion temporarily obstructing the flow of fluid downstream, a second in-channel resistance portion formed at a connection portion between the secondary channel and the chamber, the second in-channel resistance portion obstructing the inflow of fluid into the chamber, and a third in-channel resistance portion formed at a connection portion between the secondary channel and the main channel, the third in-channel resistance portion obstructing the inflow of fluid from the main channel to the secondary channel.

[0023] According to the above configuration, the internal air in the chamber is exhausted to the main channel via a secondary channel provided in the range from the chamber to the main channel, so that the entire internal air can be moved to the exhaust section, eliminating the need to provide an exhaust channel alongside the main channel as in conventional microchannel devices. Furthermore, since only the internal air remaining in the secondary channel is stable, sealed between the chamber and the connecting section with the main channel, the fluid in the chamber can be stable once it has flowed into the chamber and fluid is present at the junctions at both ends of the secondary channel. Therefore, when the fluid flowing down the main channel is supplied to the downstream chamber, the fluid in the already filled chamber does not move, and the above stable state is maintained until the supply of fluid to all of the multiple chambers is completed. This allows for the same supply of fluid to all of the chambers, even if the number of chambers is increased. This allows for a large number of chambers, for example, 100 (or even 10 n Even if multiple chambers (number of chambers: 1 to 2) are connected to branch channels branching off from the main flow channel, fluid can be dispensed into all chambers, making it possible to use the system for absolute quantitative testing of target genes using methods such as the LAMP method.

[0024] A second invention relating to a microchannel device is the first invention, wherein the plurality of branch channels are provided only on one side wall of the main channel, and the connection portion of the secondary channel connected to the chamber on the upstream side of the main channel is located upstream of the next branch channel provided adjacent to the downstream side of the main channel.

[0025] In the above configuration, the chambers are installed on only one side of the main channel, and multiple chambers are arranged in a row parallel to the main channel. This allows for observation of the state of the chambers arranged in a row when used for absolute quantitative testing of target genes using the LAMP method or the like.

[0026] Furthermore, a third invention relating to a microchannel device is the first invention, wherein the branch channels are alternately provided on both side walls of the main channel, and the connection portion of the secondary channel connected to the chamber on the upstream side of the main channel is located upstream of the next branch channel provided on the opposite side wall on the downstream side of the main channel.

[0027] In the above configuration, the chambers are arranged alternately on both sides of the main channel, and multiple chambers are arranged in two rows parallel to the main channel. This allows the distance between adjacent chambers to be shorter than when multiple chambers are arranged in a single row, and the main channel can be correspondingly shorter. Furthermore, the number of chambers per unit area can be increased, resulting in a compact design of the microchannel device.

[0028] A fourth invention relating to a microchannel device is the first invention, wherein the main channel is composed of a plurality of supply channels to which fluid is supplied from a single inlet portion and which branch off, and the outlet portions are individually provided at the ends of the supply channels.

[0029] According to the above configuration, the supply channel, which is supplied from a single inlet and branches off, functions as a main channel, allowing fluid to be supplied to multiple main channels and the chambers surrounding them. Therefore, when this microchannel device is used for absolute quantitative testing of target genes using the LAMP method or other methods, if 100 chambers are connected to one supply channel (main channel), for example, by branching the supply channel (main channel) into 10 channels, reaction results from 1,000 chambers can be obtained. Furthermore, by increasing the number of chambers connected to one supply channel (main channel) or by increasing the number of branched supply channels (main channels), even more chambers can be installed. [Effects of the Invention]

[0030] According to the present invention, a microchannel dispensing device supplies a fluid to a main channel. When the first channel resistance temporarily blocks the flow of the fluid, the fluid flows into the chamber from the branch channel, allowing the air inside the chamber to be discharged from the secondary channel to the main channel. When the first channel resistance breaks and the flow of the fluid resumes in the main channel, the fluid reaches the secondary channel, trapping the air inside the secondary channel. As a result, the fluid flowing down the main channel, while temporarily blocked, flows toward the next chamber without flowing back into the secondary channel, enabling dispensing without the need for a separate discharge channel. This allows for a dispensing device with a simple configuration that can be manufactured inexpensively.

[0031] Furthermore, according to the present invention, after a fluid flows into an upstream chamber, the state of the fluid in the chamber that has already flowed therein stabilizes when the fluid flowing down the main channel reaches the junction with the secondary channel due to the breakdown of the first channel resistance portion. This allows for a channel device with multiple chambers in the main channel configuration without affecting the fluid in the chambers. In this case, the main channel can also function as a discharge channel, eliminating the need for a separate discharge channel, thereby providing an inexpensive channel device. Furthermore, the breakdown of the first channel resistance portion simultaneously initiates the supply of fluid to the next-order chamber (the downstream branch channel). This allows for only the supply of fluid to the main channel, thereby shortening the fluid supply time. Therefore, the device can be used for absolute quantitative testing of target genes using techniques such as the LAMP method, and the testing can be performed simply and quickly. Furthermore, because the dispensing device is constructed with a simple mechanism, the entire channel device can be manufactured inexpensively.

[0032] In particular, with the flow channel device of the present invention, the fluid in the chamber after dispensing is in a stable state, so that even if other fluids are flowed down the main channel after dispensing, the state of the fluid previously filled in the chamber is not affected. Therefore, for example, a sample (e.g., a solution of the substance to be tested) is dispensed and filled into the chamber in the first step, and then a solution serving as a separation phase (e.g., an oil phase) is flowed down the main channel in the second step. This prevents the separation phase from flowing into the chamber filled with the sample, allowing the sample to be isolated in the chamber. Therefore, adding a fluorescent reagent or the like to the sample to determine whether gene amplification is possible, dispensing the sample into the chamber, isolating it, and then subjecting it to gene amplification (LAMP) followed by fluorescence observation allows the absolute concentration of nucleic acids to be estimated by counting the number of chambers in which gene amplification is confirmed. [Brief explanation of the drawings]

[0033] [Figure 1]1 is an explanatory diagram showing an outline of an embodiment of a dispensing device. FIG. [Figure 2] 1A and 1B are explanatory views showing a dispensing mode in an embodiment of the dispensing device. [Figure 3] 1A and 1B are explanatory views showing a dispensing mode in an embodiment of the dispensing device. [Figure 4] FIG. 1 is an explanatory diagram showing a first embodiment of a microchannel device. [Figure 5] FIG. 10 is an explanatory diagram showing a second embodiment of the microchannel device. [Figure 6] FIG. 10 is an explanatory diagram showing a third embodiment of the microchannel device. DETAILED DESCRIPTION OF THE INVENTION

[0034] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. First, an embodiment relating to a flow dividing device will be described, and then an embodiment relating to a microchannel device will be described.

[0035] <Embodiment of Dispensing Device> An outline of an embodiment of the dispensing device of the present invention is shown in Figure 1. Note that Figure 1(a) shows only the flow path in three dimensions, and Figure 1(b) shows a cross section taken along line IB-IB in Figure 1(a).

[0036] As shown in this figure, this embodiment is generally configured to include a main channel 1, a branch channel 2 branching from the main channel 1, and a chamber 3 connected to the branch channel 2, and a fluid is dispensed from the main channel 1 to the chamber 3 via the branch channel 2. The main channel 1 is provided with an in-channel resistance section 4, which is formed by reducing the cross section of the main channel 1, near a branch position 11 with the branch channel 2 and downstream of this branch position 11, thereby temporarily impeding the flow of fluid down the main channel 1 and forcing the fluid to flow down the branch channel 2. In addition to the above configuration, a secondary channel 5 is connected between the main channel 1 and the downstream end 32 of the chamber 3 (the position farthest from the branch channel 2) and the main channel 1.

[0037] The main flow path 1 has a basically rectangular cross section, and the cross-sectional area of ​​the flow path is determined by the flow path width W1 and the flow path height H1. The first flow path resistance portion 4 has a flow path width W2 that is smaller than the main flow path 1 configured as described above, and is formed by a roughly rectangular protrusion protruding from one wall surface of the main flow path 1. The flow path cross-sectional area is reduced by making the flow path height H1 the same as the main flow path 1 and making the flow path width W2 smaller than the flow path width W1 of the main flow path 1. For example, by making the flow path width W2 approximately half the flow path width W1 of the main flow path 1, it is possible to temporarily stop the fluid flowing down the main flow path 1. Note that the flow path shapes of the main flow path 1 and the first flow path resistance portion 4 are shown as representative examples and are not limited to the above shapes. They can be changed to any appropriate shape from a manufacturing perspective.

[0038] The branch flow path 2 is provided upstream of the first internal resistance portion 4, and is basically configured with the same flow path height H1 and flow path width W1 as the main flow path 1. When the main flow path 1 branches into the branch flow path 2, the first internal resistance portion 4 temporarily stops the flow along the main flow path 1, allowing the branch to be smoothly branched, and enabling the fluid to be supplied to the chamber 3.

[0039] Chamber 3 is provided continuous with branch flow path 2, and its internal volume is adjusted to a predetermined amount by increasing the flow path width while maintaining the same flow path height H1 as branch flow path 2 (the same height as main flow path 1). By increasing the flow path width of chamber 3, flow flows from a small cross-section to a large cross-section flow path at its inlet (terminal end of branch flow path 2) 31, and flow path resistance due to surface tension acts. However, the flow path resistance that occurs at this time is adjusted to be much smaller than the flow path resistance due to first flow path resistance portion 4, so that first flow path resistance portion 4 will not break down when flowing into chamber 3.

[0040] The secondary flow path 5 is provided between an appropriate position of the downstream end 32 of the chamber 3 and the main flow path 1, and its flow path cross-sectional area is configured to be much smaller than the flow path cross-sectional area of ​​the main flow path 1, and further configured to be sufficiently smaller than the flow path cross-sectional area of ​​the first flow path resistance section 4. For example, it is preferably 1 / 5 or less of the flow path cross-sectional area of ​​the main flow path 1 (1 / 2 or less of the flow path cross-sectional area of ​​the first flow path resistance section 4), and more preferably 1 / 30 or less (1 / 15 or less of the first flow path resistance section 4).

[0041] In this embodiment, the channel width is 1 / 3 of that of the main channel 1, while the channel height H2 is 1 / 10, and the channel cross-sectional area is assumed to be 1 / 30 (1 / 15 of the first channel resistance portion 4). Specifically, if the channel width W1 of the main channel 1 is 60 μm and the channel height H1 is 100 μm, and the channel width of the secondary channel 5 is 20 μm and the channel height H2 is 10 μm, the channel cross-sectional area ratio can be 1 / 30. Furthermore, if the channel width W2 of the first channel resistance portion 4 is 30 μm, the channel cross-sectional area ratio can be 1 / 15. Incidentally, the channel height of the chamber 3 is 100 μm, the same as that of the main channel 1, and the shape is appropriately adjusted so that the volume is 1.2 nL.

[0042] The position of connection between the downstream end 32 of the chamber 3 and the secondary flow path 5 is not particularly limited, as long as it is located near the end (downstream end) in the flow direction when the chamber 3 is filled with fluid, and ultimately it is sufficient for the fluid to reach the connection part 51 of the secondary flow path 5 (opening near the tip of the secondary flow path 5) from the chamber 3. Basically, it is assumed that the wall surface 32 farthest from the inlet portion 31 connected to the branch flow path 2 is the downstream end, and therefore the connection part 51 of the secondary flow path 5 is provided at an appropriate location on this wall surface (downstream end) 32. Note that the connection part 51 of the secondary flow path 5 may be configured so that the tip of the secondary flow path 5 opens in the chamber 3, but is not limited to the tip, and it is sufficient if a part of the secondary flow path 5 is appropriately connected near the tip and opens in the chamber 3.

[0043] The other connection part 52 of the secondary flow path 5 (the connection part with the main flow path 1) is opened at a position where it is connected to the main flow path 1. This opening does not have to be at the tip either, but when the secondary flow path 5 is made to have a substantially L-shape as shown in the drawing for convenience of flow path design, it is preferable to use the tip of the secondary flow path 5 as the other connection part 52. The other connection part 52 of the secondary flow path 5 is connected downstream of the first intra-flow path resistance part 4 provided in the main flow path 1.

[0044] As described above, one connection part 51 of the secondary flow path 5 is connected to the chamber 3, and the other connection part 52 is connected to the main flow path 1, thereby enabling the movement of a fluid. Therefore, when a fluid (e.g., a solution such as a specimen) flows from the branch flow path 2 into the chamber 3, the internal air (fluid) present therein flows into the secondary flow path 5 and can move from the other connection part 52 to the main flow path 1. In this state, the fluid to be supplied (e.g., a solution such as a specimen) has not reached the downstream side of the first intra-flow path resistance part 4 of the main flow path 1, so the internal air can move freely into the main flow path 1.

[0045] However, when the fluid to be supplied (e.g., a solution such as a specimen) reaches the connection 51 of the secondary flow path 5, a large flow path resistance acts upon the inflow because the cross-sectional area of ​​the secondary flow path 5 is configured to be extremely small as described above. That is, the connection 51 acts as a second in-flow path resistance portion. The flow path resistance (breakdown pressure) at this time is larger (the flow path cross-sectional area is small) than the flow path resistance (breakdown pressure) of the first in-flow path resistance portion 4. Therefore, the fluid (solution such as a specimen) that has flowed into the chamber 3 breaks the first in-flow path resistance portion 4 (passes through the first in-flow path resistance portion 4) before flowing into the secondary flow path 5, and resumes flowing down along the main flow path 1. When the breakdown pressure was calculated for the flow path configuration assumed in this embodiment, the specific dimensions of which were exemplified above, the breakdown pressure at the first in-flow path resistance portion 4 was 3.56 kPa, while the breakdown pressure at both ends of the secondary flow path 5 was 8.55 kPa.

[0046] The other connection part 52 of the secondary flow path 5 is connected to the main flow path 1, and therefore functions to exhaust the air inside the chamber 3 while the flow of the fluid is obstructed by the first in-flow path resistance part 4. When the first in-flow path resistance part 4 breaks and the fluid reaches the connection part 52, the secondary flow path 5 branches off from the main flow path 1, and pressure acts to flow into the secondary flow path 5 from the connection part (opening) 52. However, because the flow path cross-sectional area of ​​the secondary flow path 5 is configured to be much smaller than the flow path cross-sectional area of ​​the main flow path 1, it is not possible to flow the fluid (solution such as a sample) into this secondary flow path 5. In other words, the connection part 52 acts as a third in-flow path resistance part. Furthermore, there is internal air inside the secondary flow path 5, and since the connection part 51 that opens on the chamber 3 side already has a fluid (a solution such as a specimen) present, the internal air cannot be moved. As a result, this internal air becomes sealed inside the secondary flow path 5, and the fluid pressure is balanced at both connection parts 51, 52, making it impossible for the fluid (a solution such as a specimen) to flow in either the downward or reverse direction.

[0047] With this configuration, the fluid (a solution such as a specimen) can be supplied only to the main channel 1, the branch channel 2, and the interior of the chamber 3, and the fluid (a solution such as a specimen) that has flowed into the chamber 3 does not flow back, and is maintained in a stably sealed state. In this way, even when a plurality of chambers 3 are installed, the fluid (a solution such as a specimen) can be dispensed sequentially into the chambers 3. Furthermore, with this dispensing device, the internal air generated when the fluid (a solution such as a specimen) is supplied to the chamber 3 is discharged from the main channel 1 via the secondary channel 5, eliminating the need for a discharge channel.

[0048] <Flow behavior in dispensing device> Next, we will explain how a fluid (a solution such as a specimen) flows down when dispensed into the chamber 3 by the dispensing device according to this embodiment. Figures 2 and 3 show how the dispensing device of this embodiment dispenses fluid. Both figures show the entire flow path in a plan view, and the height of the flow path differs between the secondary flow path 5 and the rest, as described above. Although not shown in detail in the figures, the connecting portions 51 and 52 at both ends of the secondary flow path 5 open into the main flow path 1 and the chamber 3, respectively.

[0049] First, as shown in FIG. 2(a), a predetermined pressure is applied to a main channel 1 to forcibly flow a fluid (such as a solution of a sample) from upstream to downstream, and the fluid basically flows down along the main channel 1. When the fluid reaches the first channel resistance section 4, a channel resistance occurs due to a reduction in the channel cross-sectional area, temporarily impeding the flow. Then, as the flow is impeded, the fluid flows down through the branch channel 2 toward the chamber 3. At this time, the channel width of the chamber 3 is expanded to ensure a predetermined storage volume (amount to be held). When the fluid flows from the branch channel 2 into the chamber 3, a channel resistance due to surface tension occurs. However, the large channel resistance due to the first channel resistance section 4 allows the fluid to easily flow into the chamber 3.

[0050] As shown in Figure 2(b), the fluid that has started to flow into the chamber 3 gradually flows from the inlet 31 of the chamber 3 toward the downstream end 32. Although the flow velocity of the fluid is slightly slower because the flow path width is expanded, the fluid continues to flow sequentially from the inlet 31 toward the downstream end 32. At this time, the internal air that was present inside the chamber 3 is exhausted via the secondary flow path 5 to the main flow path 1 downstream of the first intra-flow path resistance portion 4 (a region where the fluid is not flowing down).

[0051] As the flow into chamber 3 progresses, as shown in Figure 3(a), the fluid that has flowed into chamber 3 flows down to the vicinity of downstream end 32, and the flow of the fluid into chamber 3 is completed. At this point, the internal pressure of the fluid acts on secondary flow path 5 in the direction of flow from connecting portion (open end) 51. However, because the flow path cross-sectional area of ​​secondary flow path 5 is extremely small, flow path resistance acts significantly, and first flow path resistance portion 4 breaks before the fluid flows into secondary flow path 5. The fluid passes over this first flow path resistance portion 4 and flows down along main flow path 1. In other words, the flow down in main flow path 1 is resumed.

[0052] As the fluid resumes flowing down the main flow path 1, it reaches the connection 52 that branches off from the main flow path 1, but because fluid cannot flow in through this connection, the secondary flow path 5 remains in a state where the connection parts 51 and 52 at both ends are sealed with fluid, with internal air sealed in. In this state, the internal pressure of the fluid is uniform, so fluid cannot flow in through either of the connection parts 51 and 52 at both ends, and the secondary flow path 5 becomes stable with internal air sealed in, as shown in Figure 3(b).

[0053] As a result of the above, the dispensing of the fluid flowing down the main channel 1 into the chamber 3 is completed, and the fluid flowing down the main channel 1 is now in a state where it can be further dispensed into the chamber 3 via the next connected branch channel 2.

[0054] <Embodiments of Microfluidic Devices> A first embodiment of a microchannel device is shown in Figure 4. Microchannel device A of this embodiment is configured such that a main channel 1 is provided between an inlet 6 and an outlet 7, and multiple chambers 3a, 3b,..., 3m..., 3n are arranged on one side (the right side in the figure) of the main channel 1. This microchannel device is used to dispense fluids into these chambers 3a-3n. The dispensing apparatus described above is used to dispense fluids into each of the chambers 3a-3n. Each of the chambers 3a-3n is connected to the main channel 1 via branch channels 2a-2n (only 2m is shown as a representative example). First intra-channel resistance sections 4a-4n (only 4m is shown as a representative example) are provided downstream near each of the branch channels 2a-2n, respectively, so as to temporarily obstruct the flow of fluid down the main channel 1 when the fluid flows into each of the chambers 3a-3n. Further, secondary flow paths 5a to 5n (only 5m is shown as a representative example) are connected between the vicinity of the downstream end of each of the chambers 3a to 3n and the downstream side of the first intra-flow path resistance portion 4a to 4n of the main flow path 1. As described above, the connection positions of the secondary flow paths 5a to 5n with the main flow path 1 are downstream of the first intra-flow path resistance portion 4m, but are also upstream of the branch flow path (2m+1) for dispensing to the next chamber (3m+1).

[0055] With the above configuration, the fluid supplied from the introduction section 6 is dispensed sequentially from the upstream chamber 3m toward the downstream chamber. When the upstream chamber 3m is filled with fluid, the fluid flows downstream over the first in-flow path resistance section 4m. When the fluid flows downstream past the connection point of the secondary flow path 5m with the main flow path 1, dispensing of the upstream chamber is completed. Almost simultaneously with the completion of this dispensing, dispensing into the next chamber 3m+1 becomes possible. In other words, the downstream flow of the fluid in the main flow path 1 is temporarily blocked by the next-order first in-flow path resistance section 4m+1, and dispensing into chamber 3m+1 begins via branch flow path 2m+1.

[0056] By repeating this process up to the terminal chamber 3n, dispensing into all chambers 3a to 3n is completed, and excess fluid is discharged from the discharge section 7. The state inside each chamber 3a to 3n into which fluid has been dispensed is the same as in the dispensing device described above, with the flow paths closed by the main flow path 1 and secondary flow paths 5a to 5n (see FIG. 3(b)), and the sealed state becomes stable.

[0057] 5 shows a second embodiment of the microchannel device. The microchannel device B of this embodiment is basically the same as the first embodiment A, but has a configuration in which chambers 3a to 3n to which liquid is to be dispensed from the main channel 1 are arranged alternately on both sides of the main channel 1 (on both the left and right sides in the figure).

[0058] In this embodiment, each chamber 3a to 3n is connected to the main flow path 1 via a branch flow path 2a to 2n, respectively, and the first in-flow path resistance sections 4a to 4n provided in the main flow path 1 are arranged downstream of the branch flow paths 2a to 2n, and the secondary flow paths 5a to 5n are connected between the vicinity of the downstream end of the chambers 3a to 3n and the main flow path 1.

[0059] In this way, by arranging the multiple chambers 3a to 3n alternately on both sides of the main channel 1, the distance between the chambers 3a to 3n in the longitudinal direction of the main channel 1 can be shortened. Therefore, when dispensing into multiple chambers 3a to 3n using a linear main channel 1, the main channel 1 can be made relatively short. As a result, the number of chambers per unit area can be increased, making it possible to design the entire microchannel device compact. The dispensing state into the chambers 3a to 3n is the same as in the first embodiment.

[0060] 6 shows a third embodiment of the microchannel device. The microchannel device C of this embodiment has a configuration in which a plurality of microchannel devices B1, B2, and B3 configured similarly to the second embodiment are arranged in parallel, and three main channels 1a, 1b, and 1c are branched from an introduction section 6.

[0061] Even when the fluid is branched into multiple main channels 1a-1c and flows down as in this embodiment, the individual flow channel devices B1-B3 have the same configuration as in the second embodiment B, making it possible to dispense the fluid into all chambers. Of the main channels 1a-1c branched from the inlet 6, the central main channel 1b has a serpentine channel 8 on its upstream side. This is to synchronize the timing of fluid supply to the three flow channel devices B1-B3. Therefore, if fluid is supplied at different times, this serpentine channel 8 may be omitted. Furthermore, individual outlets 7a-7c are provided at the ends of the main channels 1a-1c in each of the flow channel devices B1-B3, respectively, to individually discharge internal air and excess fluid. When using a single common outlet 7a-7c, there is a risk of backflow into the other channels if one of the flow channel devices B1-B3 finishes flowing down early. Therefore, if all flow down is completed at the same time, a single common outlet may be used.

[0062] <How to use as an absolute quantitative test for target genes> The microchannel device according to the embodiment has the above-described configuration, and can therefore be used for absolute quantitative testing of target genes by the LAMP method. When in use, a solution of a sample or the like is first supplied from the inlet 6 to the main channel 1 (or 1a to 1c), and the solution of the sample or the like is then dispensed into each of the chambers 3a to 3n. Subsequently, a solution serving as a separation phase (oil phase, etc.) is similarly supplied from the inlet 6 to the main channel 1 (or 1a to 1c).

[0063] The secondarily supplied solution as a separation phase (oil phase, etc.) flows down the main channel 1 (or 1a to 1c), guiding the sample solution, etc., supplied to this main channel 1 (or 1a to 1c) downstream. During the flow, the flow channel resistance increases due to the reduction in the flow channel cross-sectional area caused by the first intra-channel resistance portions 4a to 4n, hindering smooth flow. However, unlike the initial sample solution, the flow does not stop temporarily. On the other hand, because chambers 3a to 3c are already filled with the sample solution, etc., that was primarily supplied, it cannot flow into chambers 3a to 3n. Therefore, even if the sample solution in branch channels 2a to 2n is replaced, it will not replace the sample solution filled in chambers 3a to 3n.

[0064] Although the solution serving as the separation phase (oil phase, etc.) is prevented from smoothly flowing down, it passes through the first intra-flow path resistance portions 4a-4n without flowing into the chambers 4a-4n, and flows down the main flow path 1 (or 1a-1c) in sequence, causing all of the solution of the specimen, etc. that was supplied to the main flow path 1 (or 1a-1c) to flow down, and instead the main flow path 1 (or 1a-1c) is left in a state where the solution serving as the separation phase (oil phase, etc.) is present. This allows the specimen, etc. to be isolated in the individual chambers 3a-3n.

[0065] By using the entire isolated flow channel device A (or B, C) in this way to carry out a gene amplification reaction (for example, by immersing it in 60°C water for 60 minutes), it becomes possible to perform processing for absolute quantitative testing of target genes using the LAMP method. After that, by counting the number of chambers in which gene amplification has occurred and estimating the absolute amount of nucleic acid concentration using the Poisson distribution equation (the following equation), absolute quantitative testing becomes possible.

[0066]

number

[0067] As described above, in the embodiment of the microchannel device, as in the embodiment of the dispensing apparatus, there is no need to provide an exhaust channel for discharging internal air using the main channel 1, and therefore, it can be manufactured inexpensively. Furthermore, since the fluid to be supplied to the main channel 1 (or 1a to 1c) can be supplied from the inlet 6, dispensing into all chambers can be performed extremely simply and quickly. Furthermore, after first dispensing a sample or the like (e.g., a solution of the test object) and filling the chambers, secondly, a solution serving as a separation phase (e.g., an oil phase) is allowed to flow down the main channel. This prevents the separation phase from flowing into the chambers filled with the sample or the like, allowing the sample or the like to be isolated by the chambers. This allows for simple and rapid absolute quantitative testing of target genes using the LAMP method.

[0068] <Experimental Example> Experiments were conducted to verify the feasibility of dispensing and isolation as described above. The microchannel device used in the experiments had chambers 3a to 3n provided on one side of a main channel 1, as shown in Figure 4, with a total of 100 chambers 3a to 3n. Specific dimensions were: main channel 1 width 60 μm, channel height 100 μm, first intra-channel resistance section 4 channel width 30 μm. Secondary channel 5 channel width 20 μm, channel height 10 μm. Chamber 3 channel height was 100 μm, and the shape was such that the volume was 1.2 nL.

[0069] The experimental device was fabricated using soft lithography, using a silicone resin (PDMS). Specifically, a thick negative photoresist (SU-8) mold was formed on a single-crystal silicon wafer (100 mm diameter) using two-step photolithography, and the flow channel pattern was transferred to the PDMS. The device was then attached to a glass substrate with double-sided tape (Nitto Denko No. 5303W) to seal each flow channel.

[0070] In the experiment, pure water colored with food coloring (red solution: 9 v / v%) was used instead of the sample solution, and silicone oil (Sigma-Aldrich 378348 (kinematic viscosity 20 cSt)) was used as the separation phase (oil phase, etc.). A syringe pump (YMC YSP-201) was used to introduce each liquid. The same syringe pump was first filled with silicone oil (0.7 mL) and then with the red solution (3 μL), so that the red solution and silicone oil could be supplied continuously during introduction.

[0071] As a representative example of the experiment, the introduction flow rate was set to 100 μL / min, and the dispensing process was observed by photographing it with an inverted microscope and a high-speed camera. As a result, it was found that the red colored liquid was dispensed extremely quickly, taking approximately 0.34 seconds for 100 pieces. Furthermore, both ends (connectors) 51, 52 of the secondary flow path 5 did not collapse, and the secondary flow path 5 maintained a state in which internal air was sealed inside. After the introduction of the silicone oil, it was confirmed that the red colored liquid was isolated in all of the chambers 3a to 3n.

[0072] <Summary> As is clear from the experimental results, a microfluidic device using the dispensing apparatus of this embodiment enables high-speed dispensing to multiple chambers. Therefore, although a post-process for gene amplification is required, this enables rapid testing in absolute quantitative testing of target genes.

[0073] Although the embodiment of the present invention is as described above, the present invention is not limited to the above embodiment, and each element of the above embodiment may be changed or other elements may be added. Therefore, the above embodiment can be modified as appropriate.

[0074] For example, the chamber 3 is used as a reaction vessel, and its shape may be changed as needed to provide a suitable capacity, and the channel width may also be changed accordingly. In this case, the channel heights of the main channel 1 and the branch channel 2 may be changed to increase or decrease the capacity. Also, although the first channel resistance portion 4 has only been exemplified as having a rectangular protrusion protruding into the channel, its specific shape may be arbitrary as long as it is capable of reducing the channel cross-sectional area and temporarily inhibiting downward flow.

[0075] Furthermore, in explaining the method of use for absolute quantitative testing of target genes, the LAMP method has been exemplified, but the gene amplification method performed with the microfluidic device of the present invention is not limited to the LAMP method, and PCR and other isothermal gene amplification methods (e.g., RPA (Recombinase Polymerase Amplification) method, etc.) can also be used. [Explanation of symbols]

[0076] 1,1a,1b,1c Main flow path 2, 2a to 2n Branch flow path 3, 3a to 3n Chambers 4, 4a to 4n: First flow path resistance portion 5, 5a~5n Secondary flow path 6 Introduction 7 Discharge section 8 Serpentine Channel 11 Branch position 31 Chamber inlet 32 downstream end of chamber 51,52 Secondary flow path connection A, B, B1, B2, B3, C Microfluidic Device

Claims

1. A microchannel dispensing device includes a main channel that allows a fluid to flow downward in one direction, a branch channel that branches off from the main channel, and a chamber connected to the branch channel, and dispenses a fluid into the chamber, the main flow path includes a first flow path resistance portion located downstream of a branch position in the vicinity of the branch position where the main flow path branches into the branch flow path, the first flow path resistance portion reducing a cross-sectional area of ​​the flow path and temporarily obstructing a downward flow of the fluid; a secondary flow path disposed between the main flow path and the chamber, the secondary flow path being separate from the branch flow path; the secondary flow path has a flow path cross-sectional area smaller than the flow path cross-sectional area of ​​the first flow path resistance portion, and is connected to a range from the vicinity of a downstream end of the chamber to a downstream side of the vicinity of the first flow path resistance portion provided in the main flow path, a connection portion between the secondary flow path and the chamber constitutes a second flow path resistance portion that inhibits the inflow of fluid into the chamber, The connection between the secondary flow path and the main flow path constitutes a third flow path resistance portion that inhibits the inflow of fluid from the main flow path to the secondary flow path. A dispensing device for a microchannel.

2. 2. The microchannel dispensing device according to claim 1, wherein the secondary channel is formed by a uniform channel cross-sectional area and is connected to the chamber at any position of the secondary channel, and the second breakthrough pressure for releasing the temporary obstruction to inflow caused by the second channel resistance portion and the third breakthrough pressure for releasing the temporary obstruction to inflow caused by the third channel resistance portion are sufficiently larger than the first breakthrough pressure for releasing the temporary obstruction to downstream flow caused by the first channel resistance portion.

3. 2. The microchannel dispensing device according to claim 1, wherein the secondary channel has a smaller channel cross-sectional area than the main channel by at least reducing the channel height.

4. 4. The microchannel dispensing device according to claim 3, wherein the main channel, the branch channel, and the chamber all have a uniform channel height, and the secondary channel is configured at a predetermined channel height from the same bottom or ceiling as the main channel.

5. A microchannel device using the microchannel dispensing device according to any one of claims 1 to 4, the system comprises an inlet for supplying a fluid, the main flow path connected to the inlet, a discharge part connected to an end of the main flow path, a plurality of branch flow paths provided at appropriate intervals from the main flow path, chambers connected to each of the plurality of branch flow paths, and secondary flow paths connecting each of the chambers to the main flow path, the main flow path is provided with a first flow path resistance portion downstream of each of the branching positions in the vicinity of each of the branching positions into the plurality of branch flow paths, the first flow path resistance portion temporarily obstructing a downward flow of the fluid; a second flow path resistance portion that inhibits the inflow of fluid into the chamber is formed at a connection portion between the secondary flow path and the chamber, A third flow path resistance portion is formed at a connection portion between the secondary flow path and the main flow path, the third flow path resistance portion inhibiting the inflow of fluid from the main flow path to the secondary flow path. A microchannel device characterized by:

6. the plurality of branch flow paths are provided only on one side wall of the main flow path, 6. The microchannel device according to claim 5, wherein a connection portion of the secondary channel connected between the chamber and the upstream side of the main channel is located upstream of the next branch channel provided adjacent to the downstream side of the main channel.

7. the plurality of branch flow paths are alternately provided on both side walls of the main flow path, 6. The microchannel device according to claim 5, wherein a connection portion of the secondary channel connected between the chamber and the upstream side of the main channel is located upstream of the next branch channel provided on the opposite side wall on the downstream side of the main channel.

8. 6. The microchannel device according to claim 5, wherein the main channel is configured by a plurality of supply channels to which fluid is supplied from a single inlet portion and which branch off, and the discharge portions are individually provided at the ends of the supply channels.

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