WO2018143469A1

WO2018143469A1 - Gene amplification system, flow path chip, rotary driving mechanism, and gene amplification method

Info

Publication number: WO2018143469A1
Application number: PCT/JP2018/003953
Authority: WO
Inventors: 真人齋藤; 高橋　和也; 民谷　栄一
Original assignee: 国立大学法人大阪大学
Priority date: 2017-02-06
Filing date: 2018-02-06
Publication date: 2018-08-09
Also published as: JPWO2018143469A1

Abstract

A gene amplification system (100) comprises a flow path chip (1) and a rotary driving mechanism (3). The flow path chip (1) has a meandering flow path (15) that meanders toward one side and the other side. The rotary driving mechanism (3) causes the flow path chip (1) to rotate around a rotation axis. The rotary driving mechanism (3) causes the flow path chip (1) to rotate and causes a buoyancy force to act on a liquid droplet (91a) inside the meandering flow path (15) such that the liquid droplet (91a) moves along the meandering flow path (15). The rotary driving mechanism (3) includes a heater stage (30). The heater stage (30) has a plurality of heater surfaces (31a, 32a). The plurality of heater surfaces (31, 32a) are set to different temperatures. The rotary driving mechanism (3) holds the flow path chip (1) such that the meandering flow path (15) is disposed so as to straddle the plurality of heater surfaces (31a, 32a).

Description

Gene amplification system, flow channel chip, rotation drive mechanism, and gene amplification method

The present invention relates to a gene amplification system, a channel chip, a rotation drive mechanism, and a gene amplification method.

As a system for amplifying a gene (or nucleic acid), a digital droplet PCR (hereinafter referred to as “ddPCR”) system is known. The ddPCR system is a system that amplifies DNA molecules by a polymerase chain reaction (PCR) in a droplet (micro compartment) surrounded by oil. According to ddPCR, after completion of the PCR step, the presence or absence of amplified DNA molecules in each droplet is determined, and the number of droplets containing DNA is counted, so that the DNA to be detected is statistically likely (or It is possible to measure absolute quantification of nucleic acids such as RNA. The presence / absence of amplified DNA molecules is determined by detecting fluorescence.

The droplet surrounded by oil is produced using a microchannel. For example, Non-Patent Document 1 discloses a method of rotating a microchannel and producing a droplet in a centrifugal field. The microchannel is a channel having a width and depth (height) on the order of micrometers. Specifically, the microchannel has a width and depth of 1 μm or more and less than 1 mm.

However, a general ddPCR system controls the temperature of the entire reaction system. For this reason, it takes 75 seconds or more to complete one cycle of PCR. Furthermore, a typical ddPCR system performs a PCR step after completing the creation of a droplet. As a result, it takes 2 to 3 hours to complete the entire PCR cycle. One cycle of PCR includes two steps. Specifically, one cycle of PCR includes a heating step and a cooling step. In the heating step, the PCR solution is heated. In the cooling step, the heated PCR solution is cooled.

The present invention has been made in view of the above problems, and an object thereof is to provide a gene amplification system and a gene amplification method capable of performing ddPCR more rapidly. Another object of the present invention is to provide a channel chip and a rotation drive mechanism that can be used in a gene amplification system.

The gene amplification system of the present invention includes a flow path chip and a rotation drive mechanism. The channel chip has a meandering channel. The meandering channel meanders to one side and the other side. The rotation drive mechanism has a rotation shaft and rotates the flow channel chip around the rotation shaft. The rotation drive mechanism rotates the flow channel chip to impart buoyancy to the droplets in the meandering channel, thereby moving the droplets along the meandering channel. The rotation driving mechanism includes a heater stage and a driving device. The heater stage has a plurality of heater surfaces set at different temperatures. The drive device rotates the heater stage around the rotation axis. The rotation drive mechanism holds the flow path chip so that the meandering flow path is disposed across the plurality of heater surfaces.

In one embodiment, the plurality of heater surfaces include a first heater surface and a second heater surface. The first heater surface is set to a first temperature. The second heater surface is set to a second temperature different from the first temperature.

In one embodiment, the rotational drive mechanism has an end on the one side of the meandering channel facing the first heater surface, and an end on the other side of the meandering channel is the second heater surface. The flow channel chip is held so as to face the surface.

In one embodiment, the meandering channel includes a first inclined channel and a second inclined channel. The first inclined channel extends to the one side. The second inclined channel extends to the other side.

In one embodiment, the first inclined channel and the second inclined channel are inclined with respect to a radial direction orthogonal to the rotation axis.

In one embodiment, the meandering channel includes a plurality of the first inclined channels and a plurality of the second inclined channels.

In one embodiment, the first inclined flow path and the second inclined flow path are alternately arranged along the radial direction.

In one embodiment, the flow channel chip further includes a droplet supply chamber. The droplet supply chamber supplies the droplet to the starting end of the meandering flow path.

In one embodiment, the droplet supply chamber has a shape whose width becomes narrower toward a start end of the meandering flow path.

In one embodiment, the droplet supply chamber has a triangular shape.

In one embodiment, a triangular top of the droplet supply chamber is connected to a starting end of the meandering channel.

In one embodiment, the flow channel chip further includes a liquid introduction chamber and an introduction flow channel. A liquid is introduced into the liquid introduction chamber. The introduction channel communicates with the liquid introduction chamber and the meandering channel.

In one embodiment, the introduction flow path includes a meandering flow path at an intermediate portion thereof.

The channel chip of the present invention includes a meandering channel. The meandering channel meanders to one side and the other side. When the channel chip is rotated around the rotation axis, buoyancy is applied to the droplets in the meandering channel, and the droplets move along the serpentine channel.

The rotation drive mechanism of the present invention rotates the flow channel chip around the rotation axis. The channel chip has a meandering channel. The meandering channel meanders to one side and the other side. The rotational drive mechanism includes a heater stage and a drive device. The heater stage has a plurality of heater surfaces set at different temperatures. The drive device rotates the heater stage around the rotation axis. The rotation drive mechanism holds the flow path chip so that the meandering flow path is disposed across the plurality of heater surfaces.

In one embodiment, the first heater surface extends along a radial direction orthogonal to the rotation axis.

In one embodiment, the second heater surface is arranged side by side with the first heater surface and extends along the radial direction.

The gene amplification method of the present invention is a method of amplifying a gene using a channel chip. The channel chip has a meandering channel and a liquid introduction chamber. The meandering channel meanders on one side and the other side. The liquid introduction chamber communicates with the meandering flow path. The gene amplification method includes a step of holding the flow channel chip with respect to a heater stage so that the meandering flow channel is disposed across a plurality of heater surfaces, and the plurality of heater surfaces are set to different temperatures. A step of introducing a polymerase chain reaction solution into the liquid introduction chamber, and rotating the heater stage around a rotation axis to impart buoyancy to the droplets in the meandering flow path. Moving the droplet along the meandering flow path.

In one embodiment, in the step of holding the flow channel chip with respect to the heater stage, the end portion on the one side of the meandering channel faces the first heater surface, and the other side of the meandering channel is on the other side. The flow path chip is held with respect to the heater stage so that the end faces the second heater surface. In the step of setting the plurality of heater surfaces to different temperatures, the first heater surface is set to a first temperature, and the second heater surface is set to a second temperature different from the first temperature. .

According to the present invention, ddPCR can be performed more rapidly.

It is a top view of a channel chip concerning an embodiment of the present invention. It is a front view of a channel chip concerning an embodiment of the present invention. It is a figure which expands and shows a part of flow-path chip | tip which concerns on embodiment of this invention. It is a figure which shows the structure of the rotational drive mechanism which concerns on embodiment of this invention. It is a schematic diagram which shows a part of gene amplification system which concerns on embodiment of this invention. It is a flowchart which shows the gene amplification method which concerns on embodiment of this invention. It is a schematic diagram which shows the gene amplification method using the gene amplification system which concerns on embodiment of this invention. It is a schematic diagram which shows the gene amplification method using the gene amplification system which concerns on embodiment of this invention. It is a schematic diagram which shows the gene amplification method using the gene amplification system which concerns on embodiment of this invention. It is a schematic diagram which shows the gene amplification method using the gene amplification system which concerns on embodiment of this invention. It is a schematic diagram which shows the gene amplification method using the gene amplification system which concerns on embodiment of this invention. It is a schematic diagram which expands and shows a part of meandering flow path which concerns on embodiment of this invention. It is a figure which expands and shows a part of meandering flow path which concerns on embodiment of this invention. (A) is a figure which expands and shows a part of board | substrate which concerns on embodiment of this invention. FIG. 14B is a sectional view taken along line XIVB-XIVB shown in FIG. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention. It is a figure which shows a part of introduction channel, droplet supply chamber, and a meandering channel which concern on embodiment of this invention. It is a top view of the channel chip concerning other embodiments of the present invention. It is a figure which shows the result of Example 1 of this invention. (A) And (b) is a figure which shows the movement of the droplet which concerns on Example 2 of this invention. (A) And (b) is a figure which shows the movement of the droplet which concerns on Example 3 of this invention. (A) And (b) is a figure which shows the movement of the droplet which concerns on Example 4 of this invention. 6 is a graph showing the relationship between the rotation speed and the movement speed of droplets according to Examples 2 to 4 of the present invention. 6 is a graph showing the relationship between the rotation speed and the time required to complete one cycle of PCR according to Examples 2 to 4 of the present invention. (A) is a figure which shows the detection result of the fluorescence which concerns on Example 5. FIG. (B) is a figure which shows the detection result of the fluorescence which concerns on a comparative example.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the following embodiments. In the drawings, the same or corresponding parts are denoted by the same reference numerals, and description thereof will not be repeated.

First, the flow channel chip 1 according to the present embodiment will be described with reference to FIG. FIG. 1 is a plan view of the flow path chip 1. The flow channel chip 1 includes a substrate 11. The substrate 11 includes a liquid introduction chamber 12, an introduction channel 13, a droplet supply chamber 14, a meandering channel 15, and a storage chamber 16.

The introduction flow path 13 and the meandering flow path 15 are typically micro flow paths. However, the introduction flow path 13 and the meandering flow path 15 are not limited to micro flow paths. The introduction channel 13 and the meandering channel 15 may have a channel width of 1 mm and a depth (height) of 1 mm, for example.

The liquid introduction chamber 12 and the like (the liquid introduction chamber 12, the introduction flow path 13, the droplet supply chamber 14, the meandering flow path 15, and the storage chamber 16) are formed on the substrate 11 by, for example, a fine processing technique. For example, the liquid introduction chamber 12 and the like can be formed by soft lithography. Soft lithography is a technique for molding a relatively soft material such as silicone rubber. Alternatively, the liquid introduction chamber 12 or the like can be formed by photolithography and a dry process (dry etching) or a wet process (wet etching). Alternatively, the liquid introduction chamber 12 and the like can be formed by injection molding or cutting.

The shape and size of the substrate 11 are not particularly limited as long as the liquid introduction chamber 12 and the like can be formed. In the present embodiment, the substrate 11 has a rectangular shape. Further, the length of the substrate 11 in the longitudinal direction LD is, for example, 70 mm, and the length of the width direction WD perpendicular to the longitudinal direction LD of the substrate 11 is, for example, 50 mm.

The material of the substrate 11 is not particularly limited as long as the liquid introduction chamber 12 and the like can be formed, but is preferably a material that does not inhibit PCR. The material of the substrate 11 is preferably a material that does not hinder the observation (detection) of fluorescence. For example, the substrate 11 may be made of silicone rubber, glass, cycloolefin polymer (COP), or polycarbonate (PC).

The liquid introduction chamber 12 is formed at one end of the substrate 11 in the longitudinal direction LD. In FIG. 1, one side of the substrate 11 in the longitudinal direction LD is the upper side, and the other side of the substrate 11 in the longitudinal direction LD is the lower side.

Liquid is introduced into the liquid introduction chamber 12. Specifically, the liquid introduction chamber 12 has an inlet (opening), and the liquid is introduced into the liquid introduction chamber 12 through the inlet. The inlet is formed in the ceiling of the liquid introduction chamber 12. In this embodiment, the liquid is an oil such as fluorine oil and a PCR solution. Specifically, the PCR solution is introduced after the oil is introduced. More specifically, after the introduction channel 13, the droplet supply chamber 14, and the meandering channel 15 are filled with oil and the oil is supplied to the storage chamber 16, the PCR solution is introduced.

The PCR solution typically includes a sample solution, a primer, a DNA polymerase, a DNA molecule to be amplified (target sequence), deoxynucleotide triphosphate (dNTP), and a buffer solution. PCR solutions can also contain fluorescent dyes or probe DNA. The sample liquid contains DNA, and the DNA has DNA molecules to be amplified. The sample liquid is a turbid liquid containing, for example, influenza virus, norovirus, or other infectious disease virus. Alternatively, the sample liquid is a turbid liquid containing drug-resistant bacteria, enterohemorrhagic E. coli, and other infectious bacteria. Alternatively, the sample liquid is a turbid liquid containing microorganisms or animal cells. Alternatively, the sample solution is an extract of DNA or RNA of influenza virus, norovirus, or other infectious disease virus. Alternatively, the sample liquid is an extract of DNA or RNA of drug-resistant bacteria, enterohemorrhagic Escherichia coli, or other infectious bacteria. Alternatively, the sample liquid is an extract of DNA or RNA of microorganisms or animal cells. When the detection target is RNA, the PCR solution may be prepared after the reverse transcription process is performed on the sample liquid. Alternatively, a reverse transcription process may be included in the PCR step. The reverse transcription process is a process of synthesizing complementary DNA for RNA by reverse transcriptase.

The depth (height) of the liquid introduction chamber 12 is not particularly limited. For example, the liquid introduction chamber 12 may have a depth selected from a range of 10 μm to 1 mm. The volume of the liquid introduction chamber 12 is preferably larger than the total value of the volume of the introduction channel 13, the volume of the droplet supply chamber 14, and the volume of the meandering channel 15. Under this condition, the introduction flow path 13, the droplet supply chamber 14, and the meandering flow path 15 can be filled with oil, and the oil can be supplied to the storage chamber 16.

The introduction channel 13 allows the liquid introduction chamber 12 and the droplet supply chamber 14 to communicate with each other. In other words, the start end of the introduction flow path 13 is connected (communication) with the liquid introduction chamber 12, and the end of the introduction flow path 13 is connected (communication) with the droplet supply chamber 14. The introduction channel 13 includes a first introduction channel 13a and a second introduction channel 13b.

The first introduction flow path 13 a includes the start end of the introduction flow path 13. The first introduction flow path 13 a extends from the liquid introduction chamber 12 to the other end portion in the longitudinal direction LD of the substrate 11 and then extends in the width direction WD of the substrate 11. The channel width and depth of the first introduction channel 13a are not particularly limited. For example, the first introduction channel 13a may have a channel width and depth selected from a range of 10 μm to 1 mm.

The second introduction channel 13 b includes the end of the introduction channel 13. The second introduction channel 13 b extends from the end of the first introduction channel 13 a in the longitudinal direction LD of the substrate 11 and is connected to the droplet supply chamber 14. The channel width and depth of the second introduction channel 13b may be the same as or different from the channel width and depth of the first introduction channel 13a. For example, the second introduction channel 13b may have a channel width selected from a range of 10 μm to 1 mm. Typically, the second introduction channel 13b has a narrower channel width than the first introduction channel 13a. Further, the second introduction flow path 13b may have a depth selected from a range of 10 μm to 1 mm. Typically, the second introduction channel 13 b has a depth shallower than that of the droplet supply chamber 14.

The droplet supply chamber 14 is formed at the other end of the substrate 11 in the longitudinal direction LD. The droplet supply chamber 14 communicates (connects) with the starting end of the meandering channel 15. The droplet supply chamber 14 supplies a PCR solution droplet generated at the terminal end of the introduction channel 13 (near the interface between the introduction channel 13 and the droplet supply chamber 14) to the meandering channel 15. The droplet of the PCR solution is generated when the droplet supply chamber 14 is filled with oil.

The shape of the droplet supply chamber 14 is not particularly limited, but preferably includes a shape for guiding the droplet of the PCR solution to the meandering channel 15. In the present embodiment, the shape of the droplet supply chamber 14 on the side of the meandering channel 15 is a triangle, and the top of the triangle is connected to the starting end of the meandering channel 15. With this shape, a droplet of the PCR solution can be guided to the meandering channel 15. The depth of the droplet supply chamber 14 is not particularly limited. For example, the droplet supply chamber 14 may have a depth selected from a range of 10 μm to 1 mm. Typically, the droplet supply chamber 14 has a deeper depth than the second introduction channel 13b.

The meandering flow path 15 extends to one side in the longitudinal direction LD of the substrate 11 while meandering in the width direction WD of the substrate 11. The end of the meandering channel 15 communicates (connects) with the accommodation chamber 16. In the present embodiment, the meandering flow path 15 is formed at the other end of the substrate 11 in the longitudinal direction LD. The width and depth of the meandering channel 15 are not particularly limited as long as the PCR solution droplets can pass through the meandering channel 15. For example, the meandering channel 15 may have a channel width and depth selected from a range of 10 μm to 1 mm.

The storage chamber 16 is formed between the liquid introduction chamber 12 and the meandering channel 15. Therefore, the liquid introduction chamber 12, the storage chamber 16, and the meandering flow path 15 are arranged in this order along the longitudinal direction LD of the substrate 11.

The storage chamber 16 stores oil and droplets of the PCR solution. The storage chamber 16 has an outlet (opening) for discharging gas. The outlet is formed in the ceiling portion of the storage chamber 16. The depth of the storage chamber 16 is not particularly limited. For example, the storage chamber 16 may have a depth selected from a range of 10 μm to 1 mm. The volume of the storage chamber 16 is typically larger than the liquid introduction chamber 12. Since the storage chamber 16 has a larger volume than the liquid introduction chamber 12, it is possible to suppress leakage of liquid such as oil from the outlet of the storage chamber 16.

As shown in FIG. 1, the flow channel chip 1 can further include at least one spacer 17 disposed in the liquid introduction chamber 12. The flow channel chip 1 may further include at least one spacer 17 disposed in the storage chamber 16. When the material of the substrate 11 is a soft material, the ceiling of the liquid introduction chamber 12 may bend depending on the area of the liquid introduction chamber 12. By arranging the spacer 17 in the liquid introduction chamber 12, it is possible to prevent the ceiling portion of the liquid introduction chamber 12 from being bent. Similarly, by arranging the spacer 17 in the storage chamber 16, it is possible to prevent the ceiling portion of the storage chamber 16 from being bent.

Subsequently, the flow channel chip 1 according to the present embodiment will be further described with reference to FIG. FIG. 2 is a front view of the flow path chip 1. The flow channel chip 1 further includes a floor member 18.

The floor member 18 is disposed below the substrate 11 and constitutes a floor portion such as the liquid introduction chamber 12. The material of the floor member 18 is not particularly limited, but is preferably a material that does not inhibit PCR. The material of the floor member 18 is preferably a material that does not hinder the observation (detection) of fluorescence.

When the material of the substrate 11 is a relatively soft material such as silicone rubber, the rigidity of the floor member 18 is preferably higher than the rigidity of the substrate 11. For example, the floor member 18 can be a glass substrate. When the rigidity of the floor member 18 is higher than the rigidity of the substrate 11, the floor member 18 supports the substrate 11. Therefore, it is possible to suppress the substrate 11 (flow channel chip 1) from being bent by the floor member 18. In other words, the rigidity of the channel chip 1 can be increased. Therefore, when moving the flow path chip 1 to the fluorescence detection field after the PCR step is completed, it is possible to reduce the risk of liquid (oil etc.) leaking from the flow path chip 1.

When the material of the substrate 11 is a material having relatively high rigidity such as glass, cycloolefin polymer, or polycarbonate, the rigidity of the floor member 18 may be higher or lower than the rigidity of the substrate 11. Alternatively, the rigidity of the floor member 18 may be the same as the rigidity of the substrate 11. For example, the floor member 18 can be glass, cycloolefin polymer, polycarbonate, or a crimp seal.

In addition, when the rigidity of the substrate 11 is relatively high, the flow path chip 1 may include a lid member instead of the floor member 18. When the flow path chip 1 includes a lid member, the top and bottom of the substrate 11 are reversed and the lid member is disposed above the substrate 11. In other words, the lid member constitutes a ceiling portion of the liquid introduction chamber 12 or the like. Therefore, when the flow channel chip 1 includes the lid member, the inlet of the liquid introduction chamber 12 and the outlet of the storage chamber 16 are formed in the lid member. The material of the lid member is not particularly limited, but is preferably a material that does not inhibit PCR, like the floor member 18. The material of the lid member is preferably a material that does not hinder the observation of fluorescence.

Subsequently, the flow path chip 1 according to the present embodiment will be further described with reference to FIG. FIG. 3 is an enlarged view showing a part of the flow path chip 1. Specifically, FIG. 3 shows an enlarged view of the meandering flow path 15 of the flow path chip 1.

As shown in FIG. 3, the meandering flow path 15 meanders to one side and the other side of the width direction WD of the substrate 11. In FIG. 3, one side of the width direction WD of the substrate 11 is the right side, and the other side of the width direction WD of the substrate 11 is the left side. One end of the meandering channel 15 is disposed on the first heater surface 31a, and the other end of the meandering channel 15 is disposed on the second heater surface 32a.

The first heater surface 31a is set to the first temperature. The second heater surface 32a is set to a second temperature different from the first temperature. In the present embodiment, the first heater surface 31a (first temperature) is set to a temperature for executing the PCR heating step, and the second heater surface 32a (second temperature) executes the PCR cooling step. Set temperature for. Therefore, the second heater surface 32a is set to a temperature lower than that of the first heater surface 31a. Typically, the 1st heater surface 31a (1st temperature) is set to the temperature selected from the range of 90 to 98 degreeC. The temperature of the second heater surface 32a is set according to the amplification target DNA molecule. Typically, the 2nd heater surface 32a (2nd temperature) is set to the temperature selected from the range of 50 to 70 degreeC.

According to the present embodiment, the droplets of the PCR solution reciprocate between the heating field and the cooling field by moving through the meandering flow path 15. One cycle of PCR is completed when the droplet of PCR solution passes through the heating field and the cooling field once. The meandering flow path 15 shown in FIG. Therefore, the number of PCR cycles is 30. In addition, the frequency | count of folding back the meandering flow path 15 is not limited to 60 times. The number of times the meandering channel 15 is folded back can be changed as necessary.

The length L1 of the meandering channel 15 along the longitudinal direction LD of the substrate 11 is changed according to the number of PCR cycles. Specifically, the length L1 of the meandering channel 15 becomes longer as the number of PCR cycles is increased. For example, when the number of PCR cycles is 30, the length L1 of the meandering flow path 15 may be 20 mm.

In general, the PCR cooling step requires a longer time than the PCR heating step. Therefore, in the meandering flow path 15, the meandering flow path 15 is formed in the first heater surface 31 a and the second heater surface so that the area overlapping the second heater surface 32 a is larger than the area overlapping the first heater surface 31 a. It is made to oppose 32a (temperature control field). For example, as shown in FIG. 3, the meandering flow path 15 is disposed such that a portion extending from the center of the width direction WD of the substrate 11 to the other end faces the second heater surface 32 a.

Next, the rotation drive mechanism 3 according to the present embodiment will be described with reference to FIG. FIG. 4 is a diagram showing a configuration of the rotation drive mechanism 3. The rotation drive mechanism 3 includes a heater stage 30, a disk-shaped base 40, and a drive device 50. The heater stage 30 is fixed to the base 40, and the driving device 50 rotates the heater stage 30 and the base 40 about the rotation axis AX (center axis). The heater stage 30 and the base 40 are made of, for example, aluminum or copper, and the driving device 50 is typically a motor. The driving device 50 is preferably capable of controlling the rotation speed (number of rotations). In addition, although the material of the heater stage 30 and the base 40 is not limited to aluminum or copper, it is preferable that it is a material with high heat conductivity. Hereinafter, the heater stage 30 will be further described.

The heater stage 30 has the first heater surface 31a and the second heater surface 32a described with reference to FIG. As shown in FIG. 4, the first heater surface 31 a and the second heater surface 32 a extend along a radial direction orthogonal to the rotation axis AX. Moreover, the 1st heater surface 31a and the 2nd heater surface 32a are located in a line at intervals.

As described with reference to FIG. 3, one end of the meandering channel 15 is disposed on the first heater surface 31a, and the other end of the meandering channel 15 is on the second heater surface 32a. Be placed. Accordingly, the flow channel chip 1 described with reference to FIGS. 1 to 3 is set with respect to the rotational drive mechanism 3 such that the longitudinal direction LD thereof is along the radial direction of the rotational drive mechanism 3. As a result, the liquid introduction chamber 12, the storage chamber 16, and the meandering channel 15 are arranged in this order along the radial direction. Specifically, among the liquid introduction chamber 12, the storage chamber 16, and the meandering channel 15, the liquid introduction chamber 12 is closest to the rotation axis AX (rotation center).

In the present embodiment, the heater stage 30 includes a first heater stage 31 and a second heater stage 32. The first heater stage 31 has a first heater surface 31a, and the second heater stage 32 has a second heater surface 32a. The first heater stage 31 heats the first heater surface 31a to the first temperature. The second heater stage 32 heats the second heater surface 32a to the second temperature.

The first heater stage 31 includes an annular connecting part 311 and a plurality of first heating parts 312. The connecting portion 311 is disposed around the rotation axis AX. The plurality of first heating units 312 extend radially from the coupling unit 311 around the rotation axis AX. Accordingly, each of the first heating units 312 is disposed along the radial direction. The first heater stage 31 shown in FIG. 4 has four first heating units 312. The four first heating units 312 are typically provided with an angular interval of 90 degrees.

Each first heating unit 312 includes a protrusion 313 that protrudes upward. The protrusion 313 extends in the radial direction. In the present embodiment, the ridge portion 313 has a rectangular parallelepiped shape, and the upper surface of the ridge portion 313 constitutes the first heater surface 31a.

The length of the protrusion 313 in the radial direction is determined according to the length L1 of the meandering channel 15 described with reference to FIG. For example, when the length L1 of the meandering channel 15 is 20 mm, the length in the radial direction of the protrusion 313 is 20 mm or more.

The second heater stage 32 includes a disk-shaped main body 321 and a plurality of second heating units 322. The main body 321 is disposed below the first heater stage 31 and separated from the first heater stage 31.

Each of the second heating parts 322 protrudes upward from the main body part 321 and extends in the radial direction. The second heater stage 32 shown in FIG. 4 has four second heating units 322. The four second heating units 322 are typically provided with an angular interval of 90 degrees.

Each of the second heating sections 322 has a second heater surface 32a. In this embodiment, the 2nd heating part 322 is a rectangular parallelepiped shape, and the upper surface of the 2nd heating part 322 comprises the 2nd heater surface 32a.

The length of the second heating unit 322 in the radial direction is determined according to the length L1 of the meandering flow path 15 described with reference to FIG. For example, when the length L1 of the meandering flow path 15 is 20 mm, the radial length of the second heating unit 322 is 20 mm or more.

As already described, the PCR cooling step generally requires a longer time than the PCR heating step. Therefore, the width of the second heater surface 32a is preferably larger than the width of the first heater surface 31a. Since the second heater surface 32a has a larger width than the first heater surface 31a, the region of the meandering flow path 15 that overlaps the second heater surface 32a is made larger than the region that overlaps the first heater surface 31a. Becomes easier.

The heater stage 30 shown in FIG. The separation member 33 is fixed to the center portion of the main body portion 321 of the second heater stage 32, and supports the first heater stage 31 so that the first heater stage 31 is separated from the main body portion 321 of the second heater stage 32. The material of the separation member 33 is not particularly limited, but is preferably a material having high heat resistance. For example, the material of the spacing member 33 may be a resin such as polycarbonate or polyphenylene sulfide (PPS).

In the present embodiment, the separation member 33 includes a large diameter portion 331 and a small diameter portion 332. The small diameter portion 332 is fixed on the large diameter portion 331, and the large diameter portion 331 is fixed to the central portion of the main body portion 321 of the second heater stage 32. A step shape (step) is formed by the large diameter portion 331 and the small diameter portion 332, and the connecting portion 311 of the first heater stage 31 is fixed around the small diameter portion 332 (step). As a result, the first heater stage 31 is disposed at a position separated from the main body portion 321 of the second heater stage 32 by the height of the large diameter portion 331. The large diameter portion 331 and the small diameter portion 332 may be formed integrally.

Further, the separation member 33 supports the first heater stage 31 so that the first heater surface 31a and the second heater surface 32a are arranged with a space therebetween. Preferably, the separation member 33 supports the first heater stage 31 such that thermal interference between the adjacent first heater surface 31a and the second heater surface 32a is reduced. For example, the distance between adjacent first heater surface 31a and second heater surface 32a may be 1 mm.

Subsequently, the gene amplification system 100 according to the present embodiment will be described with reference to FIG. FIG. 5 is a schematic diagram showing a part of the gene amplification system 100. The gene amplification system 100 includes the flow path chip 1 described with reference to FIGS. 1 to 3 and the rotation drive mechanism 3 described with reference to FIG. The rotation drive mechanism 3 rotates the channel chip 1 around the rotation center X (around the rotation axis AX). In the present embodiment, the rotation direction RD is a clockwise direction. As shown in FIG. 5, the rotation drive mechanism 3 holds the flow channel chip 1 so that the liquid introduction chamber 12 in the liquid introduction chamber 12 and the like is closest to the rotation center X. The gene amplification system 100 rotates the channel chip 1 around the rotation center X in a state where the meandering channel 15 is opposed to the first heater surface 31a and the second heater surface 32a (temperature control field), and ddPCR is performed. Execute.

Subsequently, a gene amplification method using the gene amplification system 100 will be described with reference to FIGS. FIG. 6 is a flowchart showing the gene amplification method according to this embodiment. As shown in FIG. 6, the gene amplification method according to this embodiment includes steps S601 to S607. Hereinafter, steps S601 to S607 will be described.

In the gene amplification method according to the present embodiment, first, the flow path chip 1 is held by the rotation drive mechanism 3 in step S601. After the flow path chip 1 is held by the rotational drive mechanism 3, oil is introduced into the liquid introduction chamber 12 in step S602.

After the oil is introduced, in step S603, the rotation drive mechanism 3 rotates the flow path chip 1 around the rotation axis AX (rotation center X). As a result, the introduction channel 13, the droplet supply chamber 14, and the meandering channel 15 are filled with oil, and the oil is supplied to the storage chamber 16.

After the rotation of the flow path chip 1, the temperatures of the first heater surface 31a and the second heater surface 32a are set in step S604. Specifically, the first heater surface 31a is set to a temperature for executing a PCR heating step, and the second heater surface 32a is set to a temperature for executing a PCR cooling step.

After setting the temperatures of the first heater surface 31a and the second heater surface 32a, the PCR solution is introduced into the liquid introduction chamber 12 in step S605. After introducing the PCR solution, in step S606, the rotation driving mechanism 3 rotates the flow path chip 1 around the rotation axis AX (rotation center X). As a result, a droplet of the PCR solution is generated at the end portion of the introduction channel 13, and ddPCR is performed in the meandering channel 15. The droplets of the PCR solution that have passed through the meandering flow path 15 are stored in the storage chamber 16.

After completion of the rotation of the channel chip 1, in step S607, the channel chip 1 is moved to the fluorescence detection field, and the presence or absence of DNA in each droplet is determined by detecting fluorescence.

Subsequently, the gene amplification method described with reference to FIG. 6 will be described in detail with reference to FIGS. 7 to 11 are schematic diagrams showing a gene amplification method using the gene amplification system 100. FIG.

First, as shown in FIG. 7, the flow path chip 1 is set in the rotation drive mechanism 3 (step S601). Specifically, the flow path is such that one end of the meandering flow path 15 is disposed on the first heater surface 31a and the other end of the meandering flow path 15 is disposed on the second heater surface 32a. The chip 1 is held on the heater stage 30. Thereafter, the oil 71 is introduced into the liquid introduction chamber 12 (step S602). For introducing the oil 71, a pipetter 72 is typically used.

After introducing the oil 71 into the liquid introduction chamber 12, the rotation drive mechanism 3 rotates the flow channel chip 1 in the rotation direction RD about the rotation axis AX (rotation center X) (step S603). As a result, the oil 71 flows from the liquid introduction chamber 12 to the storage chamber 16. As shown in FIG. 8, the rotation of the flow path chip 1 is continued until all the oil 71 is discharged from the liquid introduction chamber 12. By the rotation of the flow channel chip 1, the introduction flow channel 13, the droplet supply chamber 14, and the meandering flow channel 15 are filled with the oil 71 and the oil 71 is supplied to the storage chamber 16.

After the rotation of the flow path chip 1, the first heater surface 31a and the second heater surface 32a are heated, the first heater surface 31a is set to the first temperature, and the second heater surface 32a is set to the second temperature. (Step S604). Typically, the first heater surface 31a (first temperature) is set to a temperature selected from the range of 90 ° C. to 98 ° C., and the second heater surface 32a (second temperature) is 50 ° C. to 70 ° C. It is set to a temperature selected from the range of ℃ or less.

After setting the temperatures of the first heater surface 31a and the second heater surface 32a, the PCR solution 91 is introduced into the liquid introduction chamber 12 as shown in FIG. 9 (step S605). For introducing the PCR solution 91, a pipetter 92 is typically used.

After introducing the PCR solution 91 into the liquid introduction chamber 12, the rotation drive mechanism 3 rotates the flow path chip 1 in the rotation direction RD about the rotation axis AX (rotation center X) (step S606). As a result, as shown in FIG. 10, the PCR solution 91 flows through the introduction channel 13 and becomes a droplet 91 a at the end portion of the introduction channel 13. The droplet 91 a is supplied to the meandering channel 15 via the droplet supply chamber 14 filled with the oil 71, moves in the meandering channel 15 filled with the oil 71, and is accommodated in the accommodation chamber 16.

Specifically, when the PCR solution 91 reaches the droplet supply chamber 14 filled with the oil 71, the interface between the oil 71 and the PCR solution 91 is between the oil 71 (oil phase) and the PCR solution 91 (water phase). A pressure difference due to the density difference occurs. Using this pressure difference as a driving force, a droplet 91a is generated. Note that a pressure based on centrifugal force is applied to the oil 71 and the PCR solution 91. Therefore, the gene amplification system 100 and the gene amplification method according to the present embodiment generate the droplet 91a by the centrifugal field.

The droplet 91a moving through the meandering channel 15 is heated by the first heater surface 31a and then cooled by the second heater surface 32a. By this heating and cooling, one cycle of PCR (ddPCR) is completed in the droplet 91a. Specifically, when the first heater surface 31a heats the droplet 91a, the double-stranded DNA contained in the droplet 91a is separated and single-stranded DNA is generated. Thereafter, the second heater surface 32a cools the droplet 91a. As a result, first, the primer binds to the amplification target DNA molecule in the single-stranded DNA. Thereafter, dNTP binds to the primer by DNA polymerase, and a DNA strand extends from the primer to generate double-stranded DNA.

The rotation of the channel chip 1 is continued until a desired amount of the PCR solution 91 is discharged from the liquid introduction chamber 12. After completion of the rotation of the flow path chip 1, the flow path chip 1 is removed from the rotation drive mechanism 3 and moved to the fluorescence detection field as shown in FIG. 11 (step S607). In the fluorescence detection field, the presence or absence of amplified DNA molecules in each droplet 91a is determined by detecting fluorescence. By counting the number of droplets 91a containing DNA, it is possible to measure the absolute quantification of the DNA (or nucleic acid such as RNA) that is statistically likely to be detected.

Specifically, in the fluorescence detection field, the excitation light emitted from the excitation light source is applied to the accommodation chamber 16. The excitation light excites the fluorescent dye. The excited fluorescent dye emits fluorescence. The emitted fluorescence is detected by a fluorescence detector. Since the droplet 91a that emits fluorescence contains DNA and the droplet 91a that does not emit fluorescence does not contain DNA, the presence or absence of DNA in each droplet 91a can be determined. The excitation light source can be, for example, a laser light source or a light emitting diode (LED). The fluorescence detector includes, for example, a photomultiplier detector, a condenser lens, and a fluorescence filter.

Fluorescence detection methods include intercalator method and hybridization method. In the intercalator method, a fluorescent dye (SYBR green I) that specifically inserts into double-stranded DNA and emits fluorescence is used. In other words, the PCR solution 91 to which a fluorescent dye is added is used. On the other hand, the TagMan probe method is the most common hybridization method, and a probe DNA in which a fluorescent dye is bound to an oligonucleotide specific to the DNA sequence is used. In other words, the PCR solution 91 to which the probe DNA is added is used. The fluorescent dye used in the TagMan probe method can be, for example, FAM (Carboxyfluorescein).

If the detection target is RNA, the reverse transcription process is performed before the PCR step. For example, the PCR solution may be prepared after the reverse transcription process is performed on the sample liquid. Alternatively, a reverse transcription process may be included in the PCR step.

Subsequently, the principle of the droplet 91a moving through the meandering channel 15 will be described with reference to FIG. FIG. 12 is an enlarged schematic view showing a part of the meandering flow path 15. As shown in FIG. 12, the meandering flow path 15 includes a first inclined flow path 15a and a second inclined flow path 15b.

The first inclined channel 15a extends to one side of the substrate 11 in the width direction WD and is connected (communicated) to one end of the second inclined channel 15b. The second inclined channel 15b extends to the other side in the width direction WD of the substrate 11 and is connected (communicated) to the other end of the first inclined channel 15a. The first inclined channel 15 a and the second inclined channel 15 b are inclined with respect to the longitudinal direction LD of the substrate 11. In other words, the first inclined channel 15a and the second inclined channel 15b are inclined with respect to the radial direction orthogonal to the rotation axis AX. The first inclined channel 15a and the second inclined channel 15b are alternately arranged along the longitudinal direction LD (radial direction).

When the droplet 91a surrounded by the oil 71 having different densities is rotated around the rotation axis AX (rotation center X), the buoyancy F toward the rotation axis AX (rotation center X) with respect to the droplet 91a is caused by centrifugal force. Is granted. Since the first inclined flow path 15a and the second inclined flow path 15b are inclined with respect to the radial direction, the droplet 91a has a meandering flow path 15 (first inclined flow path 15a and It moves toward the rotation axis AX (rotation center X) along the second inclined flow path 15b).

The magnitude of the buoyancy F depends on the rotational speed (rotational speed) of the drive device 50 described with reference to FIG. Specifically, the greater the number of revolutions, the greater the buoyancy F and the faster the moving speed of the droplet 91a. Therefore, the moving speed of the droplet 91a can be controlled by controlling the rotation speed of the driving device 50. Therefore, by increasing the number of rotations, it is possible to speed up heat exchange with respect to the droplet 91a, that is, speed up PCR (ddPCR). However, depending on the channel lengths of the first inclined channel 15a and the second inclined channel 15b, if the moving speed of the droplet 91a becomes too fast, appropriate heat exchange may not be performed and PCR may not be performed. is there. Therefore, it is preferable to determine the rotation speed of the drive device 50 according to the flow path lengths of the first inclined flow path 15a and the second inclined flow path 15b.

Subsequently, the meandering flow path 15 will be further described with reference to FIG. FIG. 13 is an enlarged view showing a part of the meandering flow path 15. As shown in FIG. 13, the first inclined channel 15 a and the second inclined channel 15 b have an inclination of an angle θ with respect to the longitudinal direction LD of the substrate 11. When the angle θ is larger than 0 °, the droplet 91a moves in the meandering flow path 15 using the buoyancy F described with reference to FIG.

The angle θ and the length L2 of the meandering channel 15 in the width direction WD of the substrate 11 are not particularly limited as long as the PCR heating step and the cooling step can be performed. For example, the angle θ can be selected from a range of 10 ° or less. Further, the length L2 of the meandering channel 15 can be selected from a range of 1 mm or more and 20 mm or less.

Subsequently, the substrate 11 will be further described with reference to FIGS. 14 (a) and 14 (b). FIG. 14A is an enlarged view showing a part of the substrate 11. Specifically, FIG. 14A shows an enlarged view of the vicinity of the second introduction flow path 13b. FIG. 14B is a cross-sectional view taken along line XIVB-XIVB shown in FIG.

As shown in FIGS. 14A and 14B, the substrate 11 further includes a terrace structure 19. Specifically, the introduction flow path 13 includes a terrace portion 13c. The terrace portion 13 c is formed between the end of the second introduction flow path 13 b and the droplet supply chamber 14. The terrace structure 19 includes a second introduction flow path 13b (straight line portion) and a terrace portion 13c.

The width W2 of the terrace portion 13c along the width direction WD of the substrate 11 is larger than the flow path width W1 of the second introduction flow path 13b. In other words, the channel width of the terminal portion of the second introduction channel 13b is expanded by the terrace portion 13c. Further, the depth of the terrace portion 13c is equal to the depth h1 of the second introduction channel 13b, and is shallower than the depth h2 of the droplet supply chamber 14. By forming the terrace structure 19 that satisfies these conditions at the end portion of the introduction channel 13, the droplet 91a can be generated more reliably.

The dimensions of each part of the terrace structure 19 are not particularly limited as long as the droplet 91a can be generated. For example, when the channel width W1 of the second introduction channel 13b is 30 μm, the width W2 of the terrace portion 13c can be 100 μm. Further, the terrace length L3 of the terrace portion 13c along the longitudinal direction LD of the substrate 11 may be 30 μm. When the depth h2 of the droplet supply chamber 14 is 100 μm, the depth h1 of the second introduction flow path 13b and the terrace portion 13c may be 30 μm.

The diameter of the droplet 91a can be controlled by the dimensions of each part of the terrace structure 19. Typically, the diameter of the droplet 91a is not less than 10 μm and not more than 300 μm. The channel width W1 of the second introduction channel 13b is 30 μm, the width W2 of the terrace portion 13c is 100 μm, the terrace length L3 is 30 μm, and the depth h1 of the second introduction channel 13b and the terrace portion 13c. Is 30 μm, a droplet 91a having a diameter of about 95 μm is generated.

Next, with reference to FIGS. 15 to 20, the droplet 91a moving in the meandering flow path 15 will be described. 15 to 20 are views showing a part of the introduction flow path 13, the droplet supply chamber 14, and a part of the meandering flow path 15. Specifically, FIGS. 15 to 20 show the state in which the droplet 91a is supplied from the droplet supply chamber 14 to the meandering flow path 15 in time series.

As shown in FIGS. 15 to 20, the speed at which the droplet 91a moves through the droplet supply chamber 14 is faster than the speed at which the droplet 91a moves through the meandering channel 15. This is because the droplet 91 a moves in the direction of the buoyancy F in the droplet supply chamber 14.

Further, the droplet supply chamber 14 according to the present embodiment has a shape whose width becomes narrower toward the start end of the meandering flow path 15. With this shape, the droplet 91a can be supplied to the meandering channel 15 more smoothly. More preferably, the width of the droplet supply chamber 14 is narrowed along the direction of the buoyancy F. By narrowing the width of the droplet supply chamber 14 along the direction of the buoyancy F, the droplet 91a can be guided more smoothly toward the starting end of the meandering channel 15. The shape for guiding the droplet 91a to the meandering channel 15 is not limited to a triangle. For example, the droplet supply chamber 14 may include an arc shape (a part of the circumferential surface) whose width becomes narrower toward the starting end of the meandering flow path 15.

The embodiment of the present invention has been described above with reference to the drawings. According to this embodiment, the droplet 91a reciprocates between the heating field (first heater surface 31a) and the cooling field (second heater surface 32a) using the buoyancy F as a driving force. Therefore, ddPCR can be performed more rapidly than the configuration in which the temperature of the temperature control field is transitioned between two temperatures.

Further, according to the present embodiment, the moving speed of the droplet 91a can be controlled by controlling the rotation speed. Therefore, ddPCR can be performed more quickly by controlling the number of rotations and increasing the moving speed of the droplet 91a. Furthermore, by controlling the rotation speed, it is possible to control the time during which the droplet 91a moves through the heating field and the time during which the droplet 91a moves through the cooling field. Therefore, ddPCR can be performed reliably.

Further, according to the present embodiment, the droplet 91a moves in the meandering flow path 15 using the buoyancy F as a driving force. Therefore, it is not necessary to feed the oil phase (oil 71). As a result, the amount of oil phase (oil 71) used can be reduced. Therefore, the running cost can be reduced. Furthermore, since a solution driving unit such as a micropump is not required, the gene amplification system 100 (ddPCR system) can be downsized.

Further, according to the present embodiment, since the droplet 91a is surrounded by the oil 71, the adsorption of the droplet 91a to the channel wall surface can be suppressed. Therefore, sample loss can be reduced and efficiency can be increased.

Further, according to the present embodiment, the generation of the droplet 91a in the centrifugal field and the PCR in the temperature control field can be achieved by one apparatus (rotation drive mechanism 3). Therefore, ddPCR can be performed simply by dripping the oil 71 and the PCR solution 91 onto the flow path chip 1 and the rotation drive mechanism 3 rotating the flow path chip 1. Can do.

Further, according to the present embodiment, the generation of the droplet 91a and the PCR can be performed within one chip. Therefore, ddPCR can be performed more simply and rapidly.

Note that the present invention is not limited to the above-described embodiment, and can be implemented in various modes without departing from the gist thereof.

For example, in the embodiment of the present invention, the PCR solution 91 is introduced into the liquid introduction chamber 12 after the temperatures of the first heater surface 31 a and the second heater surface 32 a are set, but the PCR solution 91 is introduced into the liquid introduction chamber 12. After that, the temperature of the first heater surface 31a and the second heater surface 32a may be set.

In the embodiment of the present invention, the channel chip 1 is rotated in the clockwise direction, but the channel chip 1 may be rotated in the counterclockwise direction.

In the embodiment of the present invention, the rotation drive mechanism 3 includes four sets (temperature control fields) of the first heater surface 31a and the second heater surface 32a, but the present invention is not limited to this form. The rotational drive mechanism 3 may include one, two, or three pairs of the first heater surface 31a and the second heater surface 32a, or five pairs of the first heater surface 31a and the second heater surface 32a. You may have more than one.

Further, the introduction channel 13 of the channel chip 1 may include a meandering channel 13d as shown in FIG. FIG. 21 is a plan view of a channel chip 1 according to another embodiment of the present invention. In the flow channel chip 1 shown in FIG. 21, the introduction flow channel 13 includes a meandering flow channel 13d. The meandering flow path 13 d is formed between the liquid introduction chamber 12 and the droplet supply chamber 14. More specifically, the meandering flow path 13d is formed in the first introduction flow path 13a. By providing the meandering channel 13d, the moving speed of the PCR solution 91 from the liquid introduction chamber 12 to the droplet supply chamber 14 can be adjusted, and the frequency (throughput) of the droplets 91a can be adjusted.

Further, in the embodiment of the present invention, the heater stage 30 has two heater surfaces (the first heater surface 31a and the second heater surface 32a), and the meandering channel 15 is disposed across the two heater surfaces. However, the heater stage 30 may have three or more heater surfaces. Three or more heater surfaces are set to different temperatures. In this case, the meandering channel 15 is disposed across three or more heater surfaces. Since the heater stage 30 has three or more heater surfaces, the temperature gradient of the temperature control field can be controlled more freely.

Hereinafter, examples of the present invention will be described. However, the present invention is not limited to the examples described below.

[Example 1]
First, the flow channel chip 1 used in Example 1 will be described. In Example 1, the flow channel chip 1 shown in FIG. 1 was used. The substrate 11 was made of silicone rubber. The depths of the liquid introduction chamber 12, the droplet supply chamber 14, and the storage chamber 16 were 100 μm. The channel width and depth of the first introduction channel 13a were both 100 μm. In the terrace structure 19 described with reference to FIGS. 14A and 14B, the flow path width W1 of the second introduction flow path 13b is 30 μm, the width W2 of the terrace portion 13c is 100 μm, and the terrace length L3 is 30 μm, the depth h1 of the second introduction flow path 13b and the terrace portion 13c was 30 μm. The length L1 of the meandering flow path 15 along the longitudinal direction LD of the substrate 11 was 20 mm. The length L2 of the meandering channel 15 along the width direction WD of the substrate 11 was 3.5 mm. The channel width and depth of the meandering channel 15 (the first inclined channel 15a and the second inclined channel 15b) were both 100 μm. The angle θ at which the first inclined channel 15a and the second inclined channel 15b are inclined with respect to the radial direction (longitudinal direction LD) was 4 °.

In Example 1, fluorine oil was used as the oil 71. Further, instead of the PCR solution 91 (aqueous phase), water colored in red was used. In Example 1, the channel chip 1 was rotated around the rotation axis AX at a rotation speed of 440 rpm. FIG. 22 is a diagram showing the results of Example 1. FIG. 22 was obtained by imaging the meandering flow path 15 with a high speed camera. As shown in FIG. 22, it was confirmed that the droplet S was generated by rotating the flow channel chip 1 around the rotation axis AX. Further, it was confirmed that the droplet S moved along the meandering channel 15 toward the rotation axis AX, and the meandering channel 15 was filled with the droplet S. In Example 1, a droplet S having a diameter of about 95 μm was generated.

[Examples 2 to 4]
In Examples 2 to 4, the moving speed of the droplet S was measured using the channel chip 1 having the same configuration as that of Example 1. In Examples 2 to 4, as in Example 1, fluorine oil was used as the oil 71. Further, instead of the PCR solution 91 (aqueous phase), water colored in red was used.

In Example 2, the channel chip 1 was rotated around the rotation axis AX at a rotation speed of 440 rpm as in Example 1. In Example 3, the flow channel chip 1 was rotated around the rotation axis AX at a rotation speed of 1000 rpm. In Example 4, the flow channel chip 1 was rotated around the rotation axis AX at a rotation speed of 1320 rpm. In Examples 3 and 4, a droplet S having a diameter of about 95 μm was also generated.

FIG. 23A and FIG. 23B are diagrams illustrating the movement of the droplet S according to the second embodiment. FIG. 23A and FIG. 23B were obtained by imaging the meandering flow path 15 with a high-speed camera. FIG. 23B shows the meandering flow path 15 when 30 seconds have elapsed from the time when FIG. 23A was imaged. In FIG. 23B, the arrow indicates the distance that the droplet S has moved in 30 seconds.

FIGS. 24A and 24B are diagrams illustrating movement of the droplet S according to the third embodiment. 24A and 24B are obtained by imaging the meandering flow path 15 with a high-speed camera. FIG. 24B shows the meandering flow path 15 when 30 seconds have elapsed from the time when FIG. 24A was imaged. In FIG. 24B, the arrow indicates the distance that the droplet S has moved in 30 seconds.

FIG. 25A and FIG. 25B are diagrams illustrating the movement of the droplet S according to the fourth embodiment. FIG. 25A and FIG. 25B were obtained by imaging the meandering flow path 15 with a high speed camera. FIG. 25B shows the meandering flow path 15 when 30 seconds have elapsed from the time when the image of FIG. In FIG. 25B, the arrow indicates the distance that the droplet S has moved in 30 seconds.

As shown in FIG. 23 (a) and FIG. 23 (b) to FIG. 25 (a) and FIG. 25 (b), the moving distance of the droplet S increases as the rotational speed rpm increases.

FIG. 26 is a graph showing the relationship between the rotational speed and the moving speed of the droplet S. In FIG. 26, the horizontal axis indicates the rotational speed, and the vertical axis indicates the moving speed of the droplet S. The graph shown in FIG. 26 is created by plotting the moving speed of the droplet S acquired from the imaging results shown in FIGS. 23 (a) and 23 (b) to FIG. 25 (a) and FIG. 25 (b). As shown in FIG. 26, it was confirmed that the moving speed of the droplet S increased as the rotational speed rpm increased.

FIG. 27 is a graph showing the relationship between the rotation speed and the time required to complete one cycle of PCR. In FIG. 27, the horizontal axis indicates the rotation speed. The vertical axis indicates the time required to complete one cycle of PCR.

The time required to complete one cycle of PCR indicates the time required for the droplet S to reciprocate once through the meandering channel 15. The time required for the droplet S to make one round trip through the meandering channel 15 is the liquid obtained from the imaging results shown in FIGS. 23 (a) and 23 (b) to 25 (a) and 25 (b). It calculated | required based on the moving speed of the droplet S, and the length L2 (3.5 mm) of the meandering flow path 15. As shown in FIG. 27, it was confirmed that the time required for the droplet S to reciprocate once through the meandering flow path 15 was shortened by increasing the rotation speed rpm.

Example 5 and Comparative Example
The channel chip 1 used in Example 5 and the comparative example has the same configuration as that of Example 1 except that it includes six types of thermo seals A to F. Specifically, the thermo seals A to F are embedded in the substrate 11 so as to face the meandering flow path 15. Specifically, the thermo seals A to C are opposed to one end of the meandering flow path 15 in the width direction WD of the substrate 11. In other words, the thermo seals A to C are embedded in the substrate 11 so as to face the first heater surface 31a. The color of the thermo seal A becomes green at a temperature of 100 ° C. The color of the thermo seal B becomes green at a temperature of 95 ° C. The color of the thermo seal C becomes green at a temperature of 90 ° C. On the other hand, the thermo seals D to F face the other end of the meandering flow path 15 in the width direction WD of the substrate 11. In other words, the thermo seals D to F are embedded in the substrate 11 so as to face the second heater surface 32a. The color of the thermo seal D becomes green at a temperature of 65 ° C. The color of the thermo seal E becomes green at a temperature of 60 ° C. The color of the thermo seal F becomes green at a temperature of 55 ° C.

In Example 5, ddPCR was performed using PCR-6 as a target, using a PCR solution containing genomic DNA of drug-resistant bacteria (IMP-6 positive). In addition, fluorescence was detected after the ddPCR step. In the comparative example, the ddPCR step was performed using a solution to which no DNA was added. The solution used in the comparative example contains the same components as in Example 5 except that no DNA was added. Also in the comparative example, fluorescence was detected after the end of the ddPCR step.

In Example 5 and the comparative example, the flow path chip 1 was rotated around the rotation axis AX at a rotation speed of 800 rpm using fluorine oil. Among the thermo seals A to C, the color of the thermo seal B is green, and among the thermo seals D to F, the color of the thermo seal E is green. Therefore, it was confirmed that the temperature (first temperature) of the first heater surface 31a can be controlled to about 95 ° C. Moreover, it has confirmed that the temperature (2nd temperature) of the 2nd heater surface 32a was controllable to about 60 degreeC.

FIG. 28 (a) is a diagram showing a fluorescence detection result according to Example 5. FIG. 28B is a diagram showing a fluorescence detection result according to the comparative example. As shown in FIG. 28 (a) and FIG. 28 (b), the droplet containing the drug-resistant bacterial genomic DNA (Example 5) is associated with DNA amplification compared to the droplet not containing DNA (Comparative Example). Strong fluorescence was observed (detected). Therefore, it was confirmed that ddPCR can be performed by the gene amplification system described in the embodiment.

According to the present invention, ddPCR can be performed more rapidly, which is useful for a system for measuring absolute quantification of genes.

DESCRIPTION OF SYMBOLS 1 Channel chip 3 Rotation drive mechanism 11 Substrate 12 Liquid introduction chamber 13 Introduction channel 13a First introduction channel 13b Second introduction channel

13c Terrace part

13d Meander channel 14 Droplet supply chamber 15 Meander channel 15a First inclination Channel 15b Second inclined channel 16 Storage chamber 19 Terrace structure 30 Heater stage 31 First heater stage 31a First heater surface 32 Second heater stage 32a Second heater surface 50 Driving device 71 Oil 91 PCR solution 91a Droplet 100 Gene Amplification system AX Rotating shaft F Buoyancy

Claims

A flow path chip having a meandering flow path meandering to one side and the other side;
Rotation drive that has a rotation shaft and moves the droplet along the meandering channel by rotating the channel chip around the rotation axis to impart buoyancy to the droplet in the meandering channel With a mechanism and
The rotational drive mechanism is
A heater stage having a plurality of heater surfaces set at different temperatures;
A drive device for rotating the heater stage around the rotation axis,
The rotation drive mechanism is a gene amplification system that holds the flow path chip so that the meandering flow path is disposed across the plurality of heater surfaces.
The plurality of heater surfaces include a first heater surface set to a first temperature and a second heater surface set to a second temperature different from the first temperature,
The rotational drive mechanism is configured such that the one end of the meandering channel faces the first heater surface and the other end of the meandering channel faces the second heater surface. The gene amplification system according to claim 1, wherein the flow path chip is held.
The meandering channel is
A first inclined channel extending to the one side;
A second inclined channel extending to the other side,
The gene amplification system according to claim 1 or 2, wherein the first inclined channel and the second inclined channel are inclined with respect to a radial direction orthogonal to the rotation axis.
The meandering channel includes a plurality of the first inclined channels and a plurality of the second inclined channels,
The gene amplification system according to claim 3, wherein the first inclined flow path and the second inclined flow path are alternately arranged along the radial direction.
The channel chip further includes a droplet supply chamber for supplying the droplet to the start end of the meandering channel,
The gene amplification system according to any one of claims 1 to 4, wherein the droplet supply chamber has a shape whose width becomes narrower toward a start end of the meandering flow path.
The droplet supply chamber has a triangular shape;
The gene amplification system according to claim 5, wherein a triangular top of the droplet supply chamber is connected to a start end of the meandering flow path.
The channel chip is
A liquid introduction chamber into which liquid is introduced;
An introduction flow path communicating with the liquid introduction chamber and the meandering flow path;
The gene amplification system according to any one of claims 1 to 6, wherein the introduction flow path includes a meandering flow path at an intermediate portion thereof.
A channel chip,
Comprising a meandering flow path meandering to one side and the other side;
A flow channel chip in which, when the flow channel chip is rotated around a rotation axis, buoyancy is imparted to the droplets in the meandering channel, and the droplets move along the serpentine channel.
A rotation drive mechanism for rotating the flow path chip around the rotation axis,
The flow path chip has a meandering flow path that meanders to one side and the other side,
The rotational drive mechanism is
A heater stage having a plurality of heater surfaces set at different temperatures;
A drive device for rotating the heater stage around the rotation axis,
The rotation drive mechanism is a rotation drive mechanism that holds the flow path chip so that the meandering flow path is disposed across the plurality of heater surfaces.
The plurality of heater surfaces include a first heater surface set to a first temperature and a second heater surface set to a second temperature different from the first temperature,
The rotational drive mechanism is configured such that the one end of the meandering channel faces the first heater surface and the other end of the meandering channel faces the second heater surface. The rotation drive mechanism according to claim 9, wherein the flow path chip is held.
The first heater surface extends along a radial direction orthogonal to the rotation axis,
The rotational drive mechanism according to claim 10, wherein the second heater surface is arranged side by side with the first heater surface and extends along the radial direction.
A gene amplification method for amplifying a gene using a channel chip,
The flow channel chip has a meandering channel meandering on one side and the other side, and a liquid introduction chamber communicating with the meandering channel,
The gene amplification method includes:
Holding the flow path chip with respect to the heater stage such that the meandering flow path is disposed across a plurality of heater surfaces;
Setting the plurality of heater surfaces to different temperatures;
Introducing a polymerase chain reaction solution into the liquid introduction chamber;
A gene amplification method comprising: rotating the heater stage around a rotation axis to impart buoyancy to the droplet in the meandering channel, thereby moving the droplet along the meandering channel.
In the step of holding the flow channel chip with respect to the heater stage, the one end of the meandering channel faces the first heater surface, and the other end of the meandering channel is the second. Holding the flow path chip against the heater stage so as to face the heater surface,
In the step of setting the plurality of heater surfaces to different temperatures, the first heater surface is set to a first temperature, and the second heater surface is set to a second temperature different from the first temperature. Item 13. The gene amplification method according to Item 12.