Nucleic acid amplification chip, nucleic acid amplification device, and nucleic acid amplification method
The nucleic acid amplification chip with a gravity-aligned flow path design and gas transfer device addresses the issues of slow transfer and bubble formation in PCR methods, facilitating faster and more accurate nucleic acid amplification without fluorescence detection.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE & TECHNOLOGY
- Filing Date
- 2022-12-16
- Publication Date
- 2026-06-19
Smart Images
Figure 0007876200000001 
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Abstract
Description
Technical Field
[0001] The present invention relates to a nucleic acid amplification chip, a nucleic acid amplification device, and a nucleic acid amplification method.
Background Art
[0002] The detection of nucleic acids is central in various fields such as pharmaceutical research and development, forensic medicine, clinical examinations, and the identification of types of agricultural crops and pathogenic microorganisms. The ability to detect various diseases including cancer, microbial infections, gene markers based on molecular phylogeny, etc. has become a universal technology for disease and risk-of-onset diagnosis, marker discovery, safety assessment in food and the environment, criminal proof, and many other technologies.
[0003] The PCR method is a powerful technique for selectively amplifying a specific region of DNA. Using PCR, millions of copies of a DNA fragment can be generated from a single template DNA for the target DNA sequence in the template DNA. PCR repeats three-phase or two-phase temperature conditions called thermal cycles, and individual reactions such as denaturation of DNA into single strands, annealing of the denatured single-stranded DNA and primers, and extension of primers by a thermostable DNA polymerase enzyme are sequentially repeated.
[0004] Thus, the PCR method is a powerful technique for exponentially amplifying genes by thermal cycling, but general-purpose thermal cycler devices used for PCR have a problem that temperature control is slow due to the large heat capacity of the aluminum block part which is a heater, and it takes time for PCR operations of 30 to 40 cycles. Therefore, a speedup of PCR operations has been desired, and various methods have been developed to achieve the speedup.
[0005] For example, performing PCR operations on a chip with microchannels has been investigated because it reduces the amount of sample, thereby decreasing the heat capacity and accelerating the thermal cycle. However, when using microchannels, if the desired amount of solution is not accurately transferred to a predetermined temperature range where a heater or other device is installed, it can lead to insufficient heating or, conversely, overheating.
[0006] The applicant has proposed a nucleic acid amplification device that incorporates a microfluidic chip with a liquid delivery mechanism using a microblower, enabling real-time PCR while detecting the movement of the solution by fluorescence detection (Patent Document 1). [Prior art documents] [Patent Documents]
[0007] [Patent Document 1] Patent No. 6226284 [Overview of the project] [Problems that the invention aims to solve]
[0008] However, some PCR methods, such as the Sanger method, only require DNA amplification and do not require a fluorescent probe for quantification, making it impossible to detect solution movement using fluorescence detection. If one were to attempt to measure solution movement using fluorescence detection, it would either require the addition of unnecessary fluorescent dyes or a separate detection method to confirm the rate of solution movement. Furthermore, in microchannels, the entrainment of air when the fluid flows through the microchannel, and the generation of bubbles due to the temperature during heating, can create stagnant areas within the microchannel, hindering the movement of the solution.
[0009] In view of these factors, the present invention aims to provide a nucleic acid amplification chip that enables faster solution transfer more easily without measuring solution transfer by fluorescence detection, and also suppresses the generation of bubbles. [Means for solving the problem]
[0010] To solve the above problems, the nucleic acid amplification chip of the present invention has a flow path between a first connecting tube having a first opening and a second connecting tube having a second opening, the first convex tube having a first convex shape in a first direction that communicates with the first connecting tube, the second convex tube having a first convex shape in a first direction that communicates with the second connecting tube, and a tubular bridge portion that fluidly communicates the first convex tube and the second convex tube.
[0011] Furthermore, another embodiment of the present invention relates to a nucleic acid amplification apparatus including a nucleic acid amplification chip according to one embodiment of the present invention, a first heater, a second heater, and a gas transfer device.
[0012] Furthermore, another embodiment of the present invention relates to a nucleic acid amplification method using a nucleic acid amplification device according to one embodiment of the present invention. [Effects of the Invention]
[0013] This invention provides a nucleic acid amplification chip that enables faster solution transfer more easily without measuring solution transfer by fluorescence detection, and also suppresses the generation of bubbles. [Brief explanation of the drawing]
[0014] [Figure 1] This is a schematic diagram of a nucleic acid amplification device according to one embodiment of the present invention. [Figure 2] This is a modified example of the nucleic acid amplification chip of the present invention. [Figure 3] This is a modified example of the nucleic acid amplification chip of the present invention. [Figure 4] This is a modified example of the nucleic acid amplification chip of the present invention. [Figure 5] This is a modified example of the nucleic acid amplification chip of the present invention. [Figure 6] This figure shows nucleic acid amplification being performed using a nucleic acid amplification device according to one embodiment of the present invention. [Modes for carrying out the invention]
[0015] The embodiments of the present invention will be described in detail below, but the present invention is not limited to these embodiments.
[0016] (Nucleic Acid Amplification Device) FIG. 1 is a schematic diagram of a nucleic acid amplification device 1000 according to an embodiment of the present invention. As shown in FIG. 1, the nucleic acid amplification device 1000 includes a nucleic acid amplification chip 100, a first heater 14, a second heater 24, and a gas transfer device (not shown). The nucleic acid amplification chip 100 includes a first connecting tube 12 having a first opening 11, a first convex tube (also simply referred to as the "first convex tube") 13 convex in a first direction, a second connecting tube 22 having a second opening 21, a second convex tube (also simply referred to as the "second convex tube") 23 convex in the first direction, and a bridge portion 30, and includes a flow path 200. In the nucleic acid amplification device 1000, the first direction of the nucleic acid amplification chip 100 coincides with the direction of gravity. Hereinafter, referring to the nucleic acid amplification device 1000 in FIG. 1, each component will be described in detail.
[0017] (Nucleic Acid Amplification Chip) The nucleic acid amplification chip 100 is a chip that holds a PCR solution containing a gene to be amplified in the flow path 200 and exponentially amplifies the gene by repeating thermal cycling.
[0018] The nucleic acid amplification chip 100 includes a first connecting tube 12 having a first opening 11, a first convex tube 13 convex in a first direction, a second connecting tube 22 having a second opening 21, a second convex tube 23 convex in the first direction, and a bridge portion 30, and includes a flow path 200.
[0019] The first connecting tube 12 has a first opening 11 and communicates with one end of the first convex tube 13.
[0020] The first opening 11 is provided in the first connecting tube 12 and opens the flow path 200 to the outside of the nucleic acid amplification chip 100. From the first opening 11, liquids such as gas and PCR solution can be injected into the flow path 200 using a gas transfer device, a dispenser, etc. described later. Conversely, gas and liquid in the flow path 200 can also be discharged from the first opening 11.
[0021] The connection angle R1 between the first connecting tube 12 and the first convex tube 13 is preferably 90° or more. Furthermore, the first connecting tube 12 preferably extends parallel to the first direction. With such a structure, when the nucleic acid amplification chip 100 is installed in the nucleic acid amplification device 1000 such that the first direction is the direction of gravity, the liquid injected into the first connecting tube 12 flows into the first convex tube 13 along the direction of gravity. Furthermore, if there is gas in the flow path 200, the bubbles pass through the first connecting tube 12 to the first opening 11 and are discharged.
[0022] The first convex tube 13, which is convex in the first direction, holds the PCR solution and is heated to a first temperature range by the first heater 14 (described later), and is the part where the first PCR reaction is performed.
[0023] The first convex tube 13 is formed to have a portion (convex part) that is convex in a first direction, and the PCR solution injected from the first connecting tube 12 can be retained in the convex part. The convex part may be, for example, U-shaped or V-shaped.
[0024] Since the PCR solution accumulates in the first convex tube 13 and is heated to carry out the reaction, it is preferable that the size of the first convex tube 13 be large enough to hold the solution required to carry out the desired PCR reaction.
[0025] The second opening 21, second connecting tube 22, and second convex tube 23 are the same as the first opening 11, first connecting tube 12, and first convex tube 13, respectively, except that the temperature of the PCR reaction performed in the second convex tube 23 is different from the first temperature range. The second opening 21, second connecting tube 22, and second convex tube 23 may have a symmetrical shape to the first opening 11, first connecting tube 12, and first convex tube 13, or they may have different shapes. As will be described later, the PCR solution is repeatedly transferred alternately between the first convex tube 13 and the second convex tube 23, so if the first direction is the direction of gravity, it is preferable that the heights of the first convex tube 13 and the second convex tube 23 in the direction of gravity are the same.
[0026] The bridge section 30 is tubular and provides fluid communication between the first convex tube 13 and the second convex tube 23. The bridge section 30 is positioned away from the first and second convex tubes in the direction opposite to the first direction. When the nucleic acid amplification chip 100 is installed such that the first direction is the direction of gravity, the bridge section 30, sandwiched between the adjacent first and second convex tubes 13 and 23, is positioned at a higher height in the direction of gravity than these two convex tubes. Therefore, when the PCR solution is transferred from one convex tube and passes over the bridge section 30, it can flow into the other convex tube along with gravity.
[0027] The bridge section 30 may be a bridge shape extending perpendicularly to the first direction (Figures 2 and 3), or it may be a convex shape in the opposite direction to the first direction (Figures 1 and 4). For example, it may be a mountain shape convex in the opposite direction to the first direction (Figure 1), or it may have a semicircular shape convex in the opposite direction to the first direction (Figure 4). When the bridge section 30 is convex in the opposite direction to the first direction, it is preferable because the solution is automatically transferred to the other convex tube once it is transferred to the midpoint of the bridge section 30 (the inflection point of the convex portion). Furthermore, if the shape includes a bridge section between adjacent convex tubes, three or more convex tubes may be provided between the first connecting tube 12 and the second connecting tube 22. In Figure 5, a first convex tube 13, a bridge section 30, a second convex tube 23, a second bridge section 40, and a third convex tube 63 are provided in order between the first connecting tube 12 and the second connecting tube 22. Adjacent convex tubes can be heated in different temperature ranges, and PCR reactions can be performed on each of them. Non-adjacent convex tubes, for example, the first convex tube 13 and the third convex tube 63, may be heated in different temperature ranges or in the same temperature range.
[0028] The connection angles between the bridge section 30 and the first convex tube 13 and the second convex tube 23, and between the second bridge section 40 and the second convex tube 23 and the third convex tube 63, are preferably 90° or greater. By setting such angles, air bubbles can be removed from the solution without accumulating at the connection points.
[0029] The channel 200 of the nucleic acid amplification chip 100 is preferably formed in a plane parallel to the first direction. This allows the solution in the channel 200 to move along gravity when the first direction is the direction of gravity.
[0030] The channel 200 of the nucleic acid amplification chip 100 may have a cylindrical shape with a circular or elliptical internal cross-sectional shape perpendicular to the central axis (hereinafter simply referred to as "cross-sectional shape"), or it may be a tube with a polygonal cross-sectional shape. Furthermore, the channel 200 may have a cross-sectional shape that combines curved and polygonal sections. Preferably, the cross-sectional shape of the channel 200 is uniform throughout the channel.
[0031] The channel 200 is preferably a microchannel. It is preferable that the maximum length between two points on the outer circumference of the cross-sectional shape of the channel 200 is 0.5 mm or more. For example, if the cross-sectional shape is circular, its diameter is preferably 0.5 mm or more. If the cross-sectional shape is elliptical, its major axis radius is preferably 0.25 mm or more. If the cross-sectional shape is polygonal, it is preferable that the maximum diagonal length is 0.5 mm or more. Having such dimensions allows the gravitational effect to be greater than the frictional force between the solution and the inner wall of the channel, enabling the solution to move to the desired position according to gravity. The velocity of movement in the direction of gravity within the channel 200 is preferably 5 mm / s or more.
[0032] Furthermore, it is preferable that the microchannel size be such that the solution can be maintained in a plug-like state within the channel 200. It is preferable that the maximum length between two points on the outer circumference of the cross-sectional shape of the channel 200 is 1 mm or less. For example, if the cross-sectional shape is circular, it is preferable that its diameter is 1 mm or less. Also, if the cross-sectional shape is elliptical, it is preferable that its major axis radius is 0.5 mm or less. If the cross-sectional shape is polygonal, it is preferable that the maximum diagonal length is 1 mm or less.
[0033] The material used in the nucleic acid amplification chip 100 is preferably one that has relatively high thermal conductivity, is stable within the temperature range required for PCR, is resistant to erosion by electrolyte solutions and organic solvents, and does not adsorb nucleic acids or proteins. For example, glass, quartz, silicon, resin, and metal can be used as materials. Preferably, the material is a resin such as polymethyl methacrylate (PMMA) or cycloolefin polymer (COP).
[0034] The nucleic acid amplification chip 100 can be formed by injection molding or MEMS processing.
[0035] (heater) The heater heats the solution in the convex tube to a specific temperature range. The nucleic acid amplification apparatus 1000 in Figure 1 includes a first heater 14 and a second heater 24.
[0036] The first heater 14 is a heater that heats the solution in the first convex tube 13 to a first temperature range. In order to heat the first convex tube 13, the first heater 14 is installed so as to cover the back surface of the first convex tube 13 of the nucleic acid amplification chip 100.
[0037] The second heater 24 is a heater that heats the solution in the second convex tube 23 to a second temperature range different from the first temperature. In order to heat the second convex tube 23, the second heater 24 is installed so as to cover the back surface of the second convex tube 23 of the nucleic acid amplification chip 100.
[0038] The first and second temperatures are determined by the thermal cycle of the PCR reaction (denaturation temperature zone and extension / annealing temperature zone), respectively. If there are three or more heaters as shown in Figure 5, adjacent heaters are set to different temperature zones. Non-adjacent heaters, for example, the first heater 14 and the third heater 64, may be heated at different temperature zones or at the same temperature zone.
[0039] (Gas transfer device) The nucleic acid amplification apparatus 1000 includes at least one gas transfer device. The gas transfer device is connected to at least one of the first opening 11 and the second opening 21 and injects gas into the flow path 200 or discharges gas from the flow path 200. By injecting or discharging gas with the gas transfer device, the PCR solution in the flow path 200 can be moved.
[0040] The gas transfer device can be any device capable of injecting or discharging a desired amount of gas into or from the flow path 200, but the pressure in the flow path 200 is released when the device is stationary. For example, a microdispenser or a microblower can be used as the gas transfer device. For example, a microdispenser such as the Burkert Microdosing Unit Type-7615 and a microblower such as the Murata Manufacturing Microblower MZB100T02 can be used.
[0041] The gas transfer device may be connected to either the first opening 11 or the second opening 21, or it may be connected to both. If connected to both openings, the gas transfer device will operate alternately in accordance with the direction of liquid transfer. Even when connected to both openings, the pressure in the flow path 200 will be released when the gas transfer device is stationary.
[0042] If the gas transfer device is connected to only one of the openings, the gas transfer device can move the solution by introducing gas into the flow path 200, or by drawing gas out of the flow path 200 and moving the solution. In either case, when stationary, the pressure is released and the solution moves through the flow path 200 along with gravity.
[0043] (Nucleic acid amplification method) Next, a nucleic acid amplification method using the nucleic acid amplification device 1000 will be described. The following description will use an embodiment in which a gas transfer device is provided at the first opening 11, but the method is not limited to this embodiment. (1) The nucleic acid amplification device 1000 is installed such that the first direction of the nucleic acid amplification chip 100 is the direction of gravity. (2) Using a micropipette or the like, 5 to 25 μL of PCR solution is measured and injected into the flow path 200 through the first opening 11. The PCR solution flows into the first convex tube 13 via the first connecting tube 12 and is held there (Figure 5(a)). The PCR solution can be any solution that can be amplified by the Sanger method and does not need to contain a fluorescent dye. Alternatively, it may contain a fluorescent dye. As an example of PCR reagents, Takara Bio's SpeedSTAR® HS DNA polymerase can be used at a final concentration of 0.025 U / μL, mixed with the included FAST Buffer I and dNTP Mixture at the concentrations specified in the manual, and then mixed with 0.5 μM primer to create a PCR premixture. The composition of the PCR reagent is not limited to this, and any reagent composition suitable for PCR is acceptable. (3) The PCR solution in the first convex tube 13 is heated to the denaturation temperature (approximately 95°C) by the first heater 14, causing the double-stranded DNA to dissociate into single strands. (4) After the reaction is complete, gas is injected using a gas transfer device to transfer the PCR solution so that it exceeds the bridge section 30, and the gas transfer device is stopped. Once the pressure in the flow path 200 is released, the solution that has exceeded the bridge section 30 moves into the second convex tube 23 by gravity and is held there (Figure 5(b)). (5) The PCR solution in the second convex tube 23 is heated to the extension and annealing temperature (50-70°C) by the second heater 24, the primers are bound, and the region between the two primer sequences is replicated from the single-stranded template DNA into double-stranded DNA. (6) The gas transfer device draws gas from the flow path 200 and transfers the PCR solution so that it passes from the second convex tube 23 over the bridge section 30, and then stops the gas transfer device. As in the forward journey, once the pressure in the flow path 200 is released, the solution that has passed the bridge section 30 moves into the first convex tube 13 by gravity and is held there.
[0044] By repeating steps (3) to (6) and changing the temperature, the DNA of the target sequence can be amplified exponentially.
[0045] In this embodiment, as described above, once the PCR solution exceeds the bridge portion 30, the solution flows into the convex tube at the destination due to gravity and can be retained, so precise adjustment of solution delivery is not necessary. According to this embodiment, by repeatedly injecting or aspirating gas into the flow path 200 to the extent that the solution exceeds the bridge portion 30, the liquid can be transferred to the desired position and a thermal cycle can be performed.
[0046] Furthermore, in this embodiment, since the nucleic acid amplification chip 100 is installed so that its first orientation is in the direction of gravity, the generated bubbles move in the opposite direction to gravity, and as a result move toward the first opening 11, the second opening 21, or the bridge portion 30, and can be removed from within the flow path 200. By removing the bubbles, accidental errors in the thermal cycle can be avoided.
[0047] Although various embodiments and modifications have been described above, the present invention is not limited to these. Other embodiments conceivable within the scope of the technical idea of the present invention are also included within the scope of the present invention.
[0048] Furthermore, one or more of the above-described embodiments and variations may be combined as appropriate. [Explanation of symbols]
[0049] 11 First opening 12 1st connecting pipe 13 1st convex tube 14. First heater 21 Second opening 22 2nd connecting pipe 23 Second convex tube 24. Second heater 30 Bridge section 100 nucleic acid amplification chips 200 flow paths
Claims
1. Between the first connecting pipe having a first opening and the second connecting pipe having a second opening A first convex pipe, which is convex in the first direction and communicates with the first connecting pipe, A second convex pipe that is convex in the first direction and communicates with the second connecting pipe, A tubular bridge portion that fluidly connects the first convex pipe and the second convex pipe, It has a flow channel having the following When the plane including the first connecting pipe, the second connecting pipe, the first convex pipe, the second convex pipe, and the bridge portion is arranged along the direction of gravity, the first direction coincides with the direction of gravity. The bridge portion is a nucleic acid amplification chip positioned away from the first convex tube and the second convex tube in the direction opposite to the first direction.
2. The nucleic acid amplification chip according to claim 1, wherein the first connecting tube, the first convex tube, the second connecting tube, the second convex tube, and the bridge portion have the same shape in their internal cross-section perpendicular to the central axis of the flow path.
3. The nucleic acid amplification chip according to claim 1, wherein the connection angle of the pair of communicating tubes is 90° or more.
4. The nucleic acid amplification chip according to claim 2, wherein the maximum length of the channel connecting two points on the outer circumference of the inner cross-section is 0.5 mm or more.
5. The nucleic acid amplification chip according to claim 4, wherein the flow channel has a maximum value of 1 mm or less.
6. The nucleic acid amplification chip according to claim 1, wherein the bridge portion is convex in the direction opposite to the first direction.
7. A nucleic acid amplification chip according to claim 1, installed in a nucleic acid amplification device, wherein the first direction is the direction of gravity, and the speed of movement in the first direction within the flow path is 5 mm / s or more.
8. The nucleic acid amplification chip according to claim 1, further comprising a third convex tube and a second bridge portion.
9. A nucleic acid amplification device, A nucleic acid amplification chip according to any one of claims 1 to 8 The first heater, The second heater, Equipped with a gas transfer device, The first heater heats the first convex tube to a first temperature, The second heater heats the second convex tube at a second temperature different from the first temperature. The gas transfer device transfers the gas in the flow path. The nucleic acid amplification device is installed such that the nucleic acid amplification chip is positioned in the first direction, which is the direction of gravity.
10. The nucleic acid amplification apparatus according to claim 9, wherein the gas transfer device is a microdispenser or a microblower.
11. A nucleic acid amplification device, The nucleic acid amplification chip according to claim 8 and The first heater, The second heater, The third heater, Equipped with a gas transfer device, The first heater heats the first convex tube to a first temperature, The second heater heats the second convex tube at a second temperature different from the first temperature. The third heater heats the third convex tube at a third temperature. The gas transfer device transfers the gas in the flow path. The nucleic acid amplification device is installed such that the nucleic acid amplification chip is positioned in the first direction, which is the direction of gravity.
12. A step of installing the nucleic acid amplification chip in the nucleic acid amplification apparatus according to claim 9 such that the first direction is the direction of gravity, The process involves injecting the nucleic acid amplification solution into the first convex tube through the first opening, The process involves heating the nucleic acid amplification solution in the first convex tube to a first temperature using the first heater, The process involves transferring the gas using the gas transfer device, transferring the nucleic acid amplification solution to a position where it crosses the bridge section, and allowing the nucleic acid amplification solution to flow into the second convex tube, The process involves heating the nucleic acid amplification solution in the second convex tube to a second temperature using the second heater, A nucleic acid amplification method that includes [the following].
13. The nucleic acid amplification method according to claim 12, further comprising the steps of transferring gas using the gas transfer device, transferring the nucleic acid amplification solution to a position where it crosses the bridge portion, and allowing the nucleic acid amplification solution to flow into the first convex tube.
14. A step of installing the nucleic acid amplification chip in the nucleic acid amplification apparatus according to claim 11 such that the first direction is the direction of gravity, The process involves injecting the nucleic acid amplification solution into the first convex tube through the first opening, The process involves heating the nucleic acid amplification solution in the first convex tube to a first temperature using the first heater, The process involves transferring the gas using the gas transfer device, transferring the nucleic acid amplification solution to a position where it crosses the bridge section, and allowing the nucleic acid amplification solution to flow into the second convex tube, The process involves heating the nucleic acid amplification solution in the second convex tube to a second temperature using the second heater, The process involves transferring the gas using the gas transfer device, transferring the nucleic acid amplification solution to a position where it crosses the second bridge section, and allowing the nucleic acid amplification solution to flow into the third convex tube. The process involves heating the nucleic acid amplification solution in the third convex tube to the third temperature using the third heater, A nucleic acid amplification method that includes [the following].
15. The nucleic acid amplification method according to claim 14, further comprising the steps of transferring gas with the gas transfer device to transfer the nucleic acid amplification solution to a position where the nucleic acid amplification solution exceeds the second bridge portion, and allowing the nucleic acid amplification solution to flow into the second convex tube.
16. The nucleic acid amplification method according to claim 14 or 15, further comprising the steps of transferring gas with the gas transfer device to transfer the nucleic acid amplification solution to a position where the nucleic acid amplification solution crosses the bridge portion, and allowing the nucleic acid amplification solution to flow into the first convex tube.