WO2016158831A1

WO2016158831A1 - Heat-convection-generating device and heat-convection-generating system

Info

Publication number: WO2016158831A1
Application number: PCT/JP2016/059847
Authority: WO
Inventors: 浩史麻生; 忠宣関矢; 真人齋藤; 民谷　栄一
Original assignee: コニカミノルタ株式会社; 国立大学法人大阪大学
Priority date: 2015-03-30
Filing date: 2016-03-28
Publication date: 2016-10-06
Also published as: JP6596800B2; JPWO2016158831A1

Abstract

[Problem] To provide a heat-convection-generating device and heat-convection-generating system having high reproducibility of heat convection and robustness superior to that of the prior art, whereby heat convection such as thermal cycling in PCR, for example, can be rapidly performed with good efficiency. [Solution] A heat convection generation chip 10, a shaft 41 capable of rotatably fixing the heat convection generation chip 10, first and second heaters 31, 32 having first and second heat source parts 31b, 32b for heating or cooling a liquid in an annular flow channel 14, and a motor 40 for rotating the shaft 41 and thereby causing the entire annular flow channel 14 to rotate about an axis AX of the shaft 41 are provided, and in plan view of the annular flow channel 14 in the direction orthogonal to the direction in which the liquid flows through the annular flow channel 14, the center of gravity (center of the annular flow channel) Q of the annular flow channel 14 in the plane and the center of rotation of the shaft 41 which is the point of intersection of the axis AX and the plane do not coincide, at least one heat source part 32b of the second heater 32 is positioned in only one range among the ranges of 30° to 150° or 210° to 330° about the center of gravity (center of the annular flow channel) Q with respect to a straight line Z connecting the center of rotation and the center of gravity (center of the annular flow channel) Q, and heats or cools a flow channel area of the annular flow channel 14 in the one range, and the first heater 31 heats or cools a flow channel area other than the flow channel area of the annular flow channel 14 heated or cooled by the second heater 32.

Description

Thermal convection generating device and thermal convection generating system

The present invention relates to a thermal convection generating device and a thermal convection generating system for performing, for example, a PCR method.

As a gene amplification method, the polymerase chain reaction (Polymerase Chain Reaction, hereinafter referred to as “PCR”) is known. PCR is a method that can amplify a large amount of a specific DNA fragment from a very small amount of DNA sample in a short time. Because of its simplicity of scanning, not only basic research but also clinical genetic diagnosis, food hygiene inspection, criminal investigation Has been applied to a wide range of fields.

Patent Document 1 discloses a PCR thermal convection generator that performs PCR by generating thermal convection by heat supplied from the bottom of the container in an upright cylindrical container. This thermal convection generation device performs solution driving by convection, and has an advantage that a PCR solution can be fed without using an external pump. However, there is a problem that stable thermal convection cannot be obtained and reproducibility is poor because the convection state changes greatly due to a slight deviation in the position of the heat supply to the PCR solution and a slight difference in the inclination of the container containing the PCR solution. .

Patent Document 2 discloses a thermal convection generating device for PCR, and includes a plurality of rectangular parallelepiped first to third heat sources (1 ^st to 3 ^rd Heat Source) stacked with gaps (Gap). A hole in which a reaction tube is accommodated is formed, and a PCR reaction tube (Reaction Vessel) is inserted into the hole and fixed. In a state where the upper surface of the laminated body is inclined so as to form a predetermined angle with respect to a line in the radial direction, a centrifugal force is imparted to the liquid in the reaction tube by rotating the laminated body around the vertical axis to heat the laminated body. It is configured to promote convection. The heat source temperature is set such that the first heat source> the second heat source> the third heat source.

However, in the PCR thermal convection generating apparatus, as described above, since the stack of heat sources that rotate around the vertical axis is inclined at a predetermined angle, it is moved to the bottom of the reaction tube (Reaction Vessel) under centrifugal force. When the reaction liquid splits from the bottom in two directions, the resultant force in the direction of the rotation axis acting on the liquid in the reaction tube differs between when flowing upward and when flowing downward, making it difficult for the liquid to flow smoothly and with reproducibility. There was a problem that good thermal convection could not be obtained.

In Patent Document 3, a liquid is introduced into an annular channel of a thermal convection generating chip provided along a plane perpendicular to the rotation axis direction, and the liquid is heated around a rotation axis while being heated by a heat source. A thermal convection generating device for PCR that applies centrifugal force to the liquid by rotating a convection generating chip is disclosed. This thermal convection generating device can realize thermal convection which is excellent in reproducibility compared with Patent Documents 1 and 2. However, it is more robust than the device of Patent Document 3 (the ability to prevent changes in external factors by an internal mechanism), has high reproducibility of thermal convection, and can perform thermal cycle of PCR more efficiently and quickly. Devices and thermal convection generation systems are needed.

International Publication No. 2002/072267 International Publication No. 2011/086497 JP 2014-39498 A

The present invention has been made in view of the above problems, and has excellent robustness, high reproducibility of heat convection, and heat convection capable of more efficiently and quickly performing heat convection such as PCR thermal cycle. It is an object to provide a generation device and a thermal convection generation system.

In order to achieve at least one of the above-described objects, a thermal convection generation device reflecting one aspect of the present invention is provided.
A rotating shaft capable of rotatably fixing a chip for generating heat convection having an annular flow path for circulating a liquid;
A first temperature adjustment unit having a first heat source unit for heating or cooling the liquid in the annular channel;
A second temperature adjustment unit having a second heat source unit for heating or cooling the liquid in the annular channel;
A rotation drive means for rotating the entire annular flow path around the rotation axis by rotationally driving the rotation shaft, and a direction in which the liquid flows through the annular flow path (a circle presented by the annular flow path) When the annular flow path is viewed in plan in a direction orthogonal to the matching plane), the center of gravity of the annular flow path (center of the circular flow path) on the plane is the intersection of the axis and the plane. A thermal convection generating device whose rotational axis does not coincide with the rotational center,
With respect to a straight line connecting the rotation center and the center of gravity (center of the annular channel), the center of gravity (center of the annular channel) is 30 ° to 150 ° or 210 ° to 330 °. In any one of the ranges, the at least one second heat source part is positioned and the flow passage area of the annular flow passage is heated or cooled in the range,
The first heat source unit is a heat convection generating device that heats or cools a channel area other than the channel area of the annular channel heated or cooled by the second heat source unit.

In order to achieve at least one of the above-described objects, a thermal convection generation system reflecting one aspect of the present invention is provided.
The thermal convection generator;
A chip for generating heat convection having an annular flow path for circulating a liquid,
The thermal convection generating chip has a liquid supply path communicating with the annular flow path, and the liquid in the liquid supply path is supplied to the annular flow path by centrifugal force applied to the thermal convection generating chip by the rotational drive. A thermal convection generation system.

In order to achieve at least one of the above-described objects, a convection PCR method reflecting one aspect of the present invention is a convection PCR method using the thermal convection generation apparatus or the thermal convection generation system described above,
A liquid introduction step for introducing a sample solution, a reaction reagent solution, and other liquids necessary for a PCR reaction into a circular channel individually or integrally into a PCR reaction solution;
A PCR reaction step of performing convection PCR by refluxing the PCR reaction solution at a predetermined speed in a circular channel,
This is a convection PCR method in which the temperature difference between the heat source part of the first temperature control part and the heat source part of the second temperature control part is maintained at 10 ° C. or higher during the PCR reaction.

According to the present invention, there is provided a thermal convection generation device and a thermal convection generation system that are more robust than conventional ones, have high reproducibility of thermal convection, and can perform thermal convection more efficiently and quickly, for example, a PCR thermal cycle. Can be offered.

FIG. 1 is a diagram illustrating an appearance of the thermal convection generation system according to the first embodiment. 1A is an exploded perspective view of a thermal convection generating device of the thermal convection generating system of FIG. FIG. 2A shows a part (back surface) of the thermal convection generating chip shown in FIG. 1, and includes the annular flow path of the thermal convection generating chip and the first and second heaters of the thermal convection generating device. It is a figure explaining the positional relationship. FIG. 2B is a view of the cross section along the line AA in FIG. FIG. 3 is a diagram showing an example of another thermal convection generation chip that can be substituted for the thermal convection generation chip of the first embodiment. FIG. 4 is a block diagram of a control system of the thermal convection generation system according to the first embodiment. FIG. 5 is a diagram illustrating a state in which fluorescence emitted from the annular flow path is detected by the thermal convection generation system according to the first embodiment. FIG. 6 is a diagram illustrating a thermal convection generation chip of the thermal convection generation system according to the second embodiment. FIG. 7 is an exploded perspective view of the thermal convection generating chip of FIG. FIG. 8A is an enlarged view of a part of the thermal convection generating chip of FIG. FIG. 8B is an enlarged view of the back surface of the thermal convection generating chip substrate (the uppermost one of the stacked plates) of FIG. FIG. 9 is a partial cross-sectional view of the thermal convection generating chip taken along the line BB in FIG. FIG. 10 is an enlarged perspective view of the cover portion of the thermal convection generating chip shown in FIG. FIG. 11 is a view schematically showing the guide passage of the thermal convection generating chip shown in FIG. FIG. 12 is a diagram illustrating a thermal convection generation chip of the thermal convection generation system according to the third embodiment. 13 is an exploded perspective view of the thermal convection generating chip shown in FIG. FIG. 14 is a diagram showing the structure of the liquid supply path of the thermal convection generating chip shown in FIG. 12 and the positional relationship between the first heater, the second heater, and the annular flow path. FIG. 15 is a partially enlarged perspective view of the first substrate of the thermal convection generating chip shown in FIG. FIG. 16 is a partially enlarged perspective view of the second substrate of the thermal convection generating chip shown in FIG. FIG. 17 is a partially enlarged perspective view of the third substrate of the thermal convection generating chip shown in FIG. 18 is a cross-sectional view taken along the line CC of the thermal convection generating chip shown in FIG. 12 as viewed in the direction of the arrow. FIG. 19 is an enlarged perspective view of the cover portion of the thermal convection generating chip shown in FIG. FIG. 20 is a graph showing the relationship between the voltage of the power source of the motor used in the first embodiment and the rotational speed of the motor, and the relationship between the speed and the centrifugal force (F = mv ² / r, g = (2πN)). obtained relation between the rotational speed of the motor using from ² r) with a relative acceleration of gravity in the first embodiment, which was developed by further determining the relationship between the voltage and the relative acceleration of gravity. FIG. 21 is a graph showing the relationship between the relative gravitational acceleration and the voltage of the motor power supply. FIG. 22A is a graph showing the results of Example 1. FIG. FIG. 22B is a graph showing the results of Comparative Example 1. FIG. 22C is a graph showing the results of Example 2. FIG. 22D is a graph showing the results of Comparative Example 2. FIG. 22E is a graph showing the results of Example 3. FIG. 22F is a graph showing the results of Comparative Example 3.

Hereinafter, a thermal convection generation device and a thermal convection generation system according to the present invention will be described with reference to the drawings.

[First Embodiment]
FIG. 1 shows an external appearance (front surface) of a thermal convection generation system 100 according to the first embodiment of the present invention, and FIG. 1A shows an exploded perspective view of the thermal convection generation apparatus 1 and the like of FIG.

The thermal convection generation system 100 according to the first embodiment includes a thermal convection generation chip 10 having an annular flow path 14 for circulating a liquid, and a thermal convection capable of rotatably attaching and detaching the thermal convection generation chip 10. And the generation device 1 (see FIG. 1A).

The thermal convection generating device 1 includes a shaft 41 for rotatably fixing the thermal convection generating chip 10 and a first heater (first temperature) for heating or cooling the liquid in the annular flow path 14 of the thermal convection generating chip 10. An adjustment unit) 31 and a second heater (second temperature adjustment unit) 32, and a motor (rotation drive means) 40 that rotates the shaft 41 around the shaft 41 by rotating the shaft 41. Have.

As will be described later, during the convection PCR according to the present invention, the temperature (denaturation temperature) of the second temperature adjustment unit 32 is higher than the temperature (annealing temperature) of the first temperature adjustment unit 31. When viewed relatively, the liquid in the annular flow path 14 that has been heated by the second temperature adjusting unit 32 is “cooled” by the first temperature adjusting unit 31, but the temperature of the second temperature adjusting unit 32 is also the first temperature. Since the temperature of the temperature adjusting unit 31 is also higher than room temperature (room temperature), a function of “heating” is required to obtain the denaturation temperature and the annealing temperature, respectively. In this sense, both the first temperature adjusting unit 31 and the second temperature adjusting unit 32 can be referred to as “heating” members, that is, “heaters”. Therefore, in the present specification, “first temperature adjusting unit”. Is replaced by “first heater”, and “second temperature control unit” is replaced by “second heater”, and the detailed description of the invention and specific embodiments are described.

As shown in FIG. 1 and FIG. 1A, the heat convection generating chip 10, the stage 20, the first heater 31, the second heater 32, and the like can be rotated integrally with the shaft 41 protruding from the motor 40. Is provided. The stage 20 is in a state where a specific portion of the annular flow path 14 is in contact with the first heater (first temperature adjustment unit) 31 and the second heater (second temperature adjustment unit) 32, and the thermal convection generation chip 10. Is a member on which can be placed. The central axis AX of the shaft 41 becomes the rotation axis of the rotation.

≪Tip for thermal convection generation≫
FIG. 2A shows the back surface of the substrate 11 of the thermal convection generating chip 10. FIG. 2B shows a cross-sectional view of the thermal convection generating chip 10 along the line AA in FIG.

2 (A) and 2 (B), the thermal convection generating chip 10 has at least an annular channel 14 for circulating the liquid. The thermal convection generating chip 10 includes a disk-shaped substrate 11 in which a groove forming a part of the annular flow path 14 is formed, and a lid body 13 bonded to the surface of the substrate 11 in which the groove is formed. An annular flow path 14 is formed by joining the substrate 11 and the lid 13.

The substrate 11 is formed with a center hole 17 for fixing the substrate 11 to the shaft 41 and a fixing portion (screw hole) (not shown) formed for fixing the substrate 11 to the stage 20. The lid 13 has a substantially same diameter as the substrate 11 and is formed in a disk shape thinner than the substrate 11. The lid 13 is detachably fixed to the substrate 11 by appropriate fixing means while being laminated (bonded) to the lower surface of the substrate 11.

The material of the chip 10 for generating heat convection needs to be a material that can withstand the temperature of the first heater 31 and the second heater 32. In addition, a transparent material is preferable from the viewpoint of visually recognizing liquid existing in the annular flow path 14 of the thermal convection generating chip 10. As such a material, for example, cyclic olefin, polypropylene, polycarbonate, a composite of polydimethylsiloxane and glass, or acrylic is preferable. Among the above materials, cyclic olefins are most preferable, and then polypropylene or polycarbonate is preferable in terms of excellent degassing property and heat resistance, and low gas permeability, water absorption, and autofluorescence. In consideration of the heat conduction from the first heater 31 and the second heater 32, it is preferable to select a material having a thermal conductivity of 0.1 to 1.0 W / (m · K). The substrate 11 and the bottom plate 12 are preferably made of synthetic resin.

The substrate 11 of the heat convection generating chip 10 is not limited to a disk shape, but may be other shapes such as a rectangular plate shape. From the viewpoint of easy rotation, the disk-shaped (disk-shaped) substrate 11 and the lid 13 are preferable. A thin adhesive resin sheet (eg, a sheet made of the above material) having airtightness and liquid-tightness is sandwiched between the substrate 11 and the lid 13, and the annular flow path 14, a liquid supply described later. The passage 15 and the gas discharge passage 16 may be sealed.

<Annular channel>
The annular flow channel 14 is for introducing a liquid such as a mixed solution (details will be described later) of the sample liquid and the reaction reagent solution to cause thermal convection in the annular flow channel 14. The annular channel 14 has a predetermined positional relationship with the rotation axis AX of the shaft 41 so that the liquid in the annular channel 14 circulates in the annular channel 14 when the thermal convection generating chip 10 is driven to rotate. Is provided.

In the present invention, the distance from the rotation axis AX of the annular flow path 14 is not particularly limited, but the compactness of the thermal convection generating device 1 when considering actual work and the efficiency of thermal convection performed using the present device. It is preferable to set the distance to 1 cm or more and 10 cm or less in relation to the property.

Further, as illustrated in the first embodiment of FIG. 1, the annular flow path 14 is a circular plane drawn when the thermal convection generating chip 10 is driven to rotate about the rotation axis AX (example of FIG. 2). In this case, the lower surface of the substrate or the like is preferably parallel to the lower surface of the substrate. In other words, as shown in FIG. 2B, it is preferable that the angle θ ′ formed between the annular flow path 14 and the rotation axis AX is 90 °. The angle θ ′ is not limited to 90 °, and may be set to an arbitrary angle in the range of 80 ° to 100 ° as long as the liquid circulates in the annular flow path 14.

(Surface roughness of the annular channel)
Occurrence of wet residue of the annular channel 14 (part where the liquid does not adhere due to the water-repellent wall repellent property such as the annular channel) by suppressing the surface roughness of the wall of the annular channel 14 to a predetermined level or less. From the viewpoint of suppressing the surface roughness Ra, the surface roughness Ra of the wall surface of the annular flow path 14 is preferably 100 nm or less, more preferably 50 nm or less, and further preferably 30 nm or less.

As one of the causes of the generation of bubbles in the liquid in the annular flow path 14, for example, the wet residue generated when the liquid is filled in the annular flow path 14 becomes gas as it is. If bubbles are generated in the liquid in the annular flow path 14, the thermal convection of the liquid in the annular flow path 14 is hindered, so it is desirable to reduce the remaining wet as much as possible.

By reducing the amount of wet residue as much as possible, the flow of the liquid in the annular flow path 14 becomes smooth, and switching between the first temperature adjustment and the second temperature adjustment is suitably performed, and the liquid convects in the annular flow path 14. There is an advantage that it becomes easy.

In order to make the surface roughness Ra of the annular channel 14 in the above range, the material of the base material 11 and the lid 13 (in some cases, the resin sheet in some cases) of the chip 10 for generating heat convection satisfying the requirements of the surface roughness. In addition to the method of selecting, the base material 11 and the lid body 13 (or resin sheet portion) constituting the wall surface of the annular flow path may be polished by a predetermined method. As a polishing method, for example, a tape-shaped polishing tool (polishing tape) in which abrasive grains having a particle size of submicron to several tens of μm are uniformly applied with an adhesive on a polyester film having a thickness of about 25 to 75 μm. ).

A test for actually checking the presence or absence of wet residue when the surface roughness of the wall surface of the annular channel 14 is within the above range and outside the above range (polyethylene glycol is applied to the surface of the annular channel 14, When water containing red food is filled in the annular channel 14 and heated at 95 ° C., a test is performed to check whether bubbles are generated or not. In the former case, bubbles are generated, and in the latter case, bubbles are generated. Result.

(Ring channel shape)
The shape of the annular channel is typically a direction in which the liquid flows through the annular channel 14 (a circle presented by the annular channel 14 as illustrated in the first embodiment of FIGS. 1 and 1A). When the annular flow path 14 is viewed in a plane (when viewed as a plane) in a direction orthogonal to the plane that coincides with the plane, it is preferably a perfect circular belt shape. By making the circular channel 14 into a perfect circle, the channel length can be minimized, and convection PCR can be performed efficiently in a short time. However, if thermal convection occurs, the shape of the annular flow path is not limited to a perfect circle, but other shapes that allow the liquid in the flow path to circulate in the above-described plan view, for example, an oval shape or an elliptical shape It may be formed in a polygonal shape (eg, triangular shape, quadrangular shape, or more polygonal shape).

In addition, the circle | round | yen exhibited by the annular flow path is, for example, presented by the edge portion of the annular flow path 14 (the apex portion of the polygon appearing in the cross-sectional shape of the annular flow path 14 (see FIG. 2B)). When the cross section of the circular flow path 14 is not polygonal, it indicates a portion corresponding to the edge portion (for example, when the cross section of the circular flow path 14 is circular, it indicates the top of the circle).

In the first embodiment, as shown in FIGS. 1 and 2, a plurality of annular flow paths 14 are provided on the peripheral edge of the lower surface of the substrate 11. These annular flow paths 14 are provided around the axis of the disk-shaped substrate 11 at predetermined equal angular intervals, and are arranged symmetrically with respect to the axis AX. The axis of the substrate 11 coincides with the rotational axis AX of the shaft 41 in a state where the thermal convection generating device 1 is assembled and the thermal convection generating chip 10 is attached. Moreover, although it is preferable to provide the annular flow path 14 at equiangular intervals, it does not need to be provided at equiangular intervals.

The number, processing method, dimensions of each part, etc. of the annular channel 14 are not particularly limited. For example, the outer diameter (D) is 30 to 70 mm, the depth (S) of the annular channel is 300 to 500 μm, and the width of the annular channel. An example is shown in which (W) is set in the range of 400 to 600 μm (see FIG. 2B). As a typical example, as in the first embodiment (FIG. 1), four channels having the same shape and size are formed on a substrate having a diameter (D) of 40 mm by microfabrication technology. (D) An example in which a 5 mm perfect circular groove (depth: 300 μm, width: 500 μm) is formed.

《Liquid supply path》
The thermal convection generating chip may optionally be provided with a liquid supply path communicating with the annular flow path for supplying liquid to the annular flow path. In the thermal convection generation system 100 according to the first embodiment (see FIG. 2A), the direction perpendicular to the direction in which the liquid flows through the annular channel (a plane that coincides with the circle presented by the annular channel 14). When the annular flow path 14 is viewed in plan (when viewed as a plane), at a portion of the annular flow path 14 that intersects the radial line Z connecting the rotation center AX and the center of gravity (center of the annular flow path) Q. A liquid supply path 15 is provided so as to communicate with each other. The “flow channel area” means an area of the annular flow channel 14 when the annular flow channel 14 is viewed in plan view.

The liquid supply path 15 includes, in order from the upper side of FIG. 2A, an elongated extending portion 15a that communicates with a liquid supply hole 15d formed in the substrate 11, and a teardrop-shaped liquid reservoir that communicates with one end thereof. 15b, and a communication portion 15c having a narrow width for connecting the tip portion thereof and the annular flow path 14 in communication. The capacity of the liquid reservoir 15b is set larger than the capacity of the annular channel 14.

The liquid supply path 15 is configured so that the liquid in the liquid supply path 15 is supplied to the annular flow path 14 by centrifugal force applied to the thermal convection generation chip when the thermal convection generation chip 10 is rotationally driven. Yes. In the thermal convection generating system according to the first embodiment, as described above, the liquid is introduced and stored in the liquid reservoir 15b via the extending portion 15a of the liquid supply path 15 by a micropipette or the like. When the chip 10 is driven to rotate about the rotation axis AX, the liquid in the liquid supply path 14 (extension part 15a, liquid reservoir part 15b) is caused to flow through the communication part 15c by the centrifugal force applied to the thermal convection generation chip 10 through the communication part 15c. 14 (see FIG. 2A).

《Gas discharge passage》
The heat convection generating chip may optionally be provided with a gas discharge path communicating with the annular flow path for extracting gas contained in the liquid in the annular flow path. In the thermal convection generating system of the first embodiment, the gas discharge path 16 communicates with the hole 16d and the hole 16d for discharging the gas from the annular flow path 14 to the outside in order from the upper side of FIG. The elongated elongated portion 16a extending along the radial line Z of the disk-shaped substrate 11, the teardrop-shaped gas reservoir portion 16b connected to one end thereof, and the tip portion thereof and the annular channel 14 are connected to each other. Narrow communication portion 16c (see FIG. 2A).

By providing the gas discharge path 16, the gas generated when the liquid in the annular flow path 14 is in thermal convection and the gas (such as bubbles) generated when the liquid is injected into the liquid flow path 15 are caused by centrifugal force to the gas discharge path 16. Since the gas can be removed from the liquid by being discharged through the extending portion 16a and the hole 16d, thermal convection of the liquid can be performed smoothly.

"stage"
The stage 20 supports the heat source 30 (the first heater 31 and the second heater 32) and transmits the rotational force of the motor 40 to the thermal convection generating chip 10 disposed on the stage 20. . As the material of the stage 20, a synthetic resin such as cycloolefin polymer or polycarbonate, or a metal can be used. The stage 20 is preferably formed in a disk shape as illustrated in the first embodiment of FIG. 2 in consideration of ease of rotation, but is not particularly limited as long as it is rotatable.

(Connection between stage and chip for generating thermal convection)
As shown in FIG. 1A, the stage 20 used in the first embodiment positions the thermal convection generating chip 10 concentrically with respect to the stage 20 and relatively rotates the stage 20 and the thermal convection generating chip 10 relative to each other. It is impossible to connect by connecting means. The structure of the connecting means is not particularly limited. For example, a concave portion or a convex portion is formed at the position of the lower surface of the thermal convection generating chip that is eccentric with respect to the central axis of the thermal convection generating chip 10, and is formed on the central axis of the stage. Alternatively, a structure may be employed in which a convex portion or a concave portion is formed at a position on the upper surface of the stage that is eccentric, and the concave portion or convex portion of the stage and the convex portion or concave portion of the thermal convection generating chip are fitted. The central axis of the thermal convection generating chip 10 coincides with the axis AX in a state where the thermal convection generating device is assembled with an axis passing through the center of the central hole 17.

(Heater mounting hole)
As shown in FIG. 1A, the stage 20 is provided with a heater mounting hole 21 for inserting and mounting the first heater 31 and the second heater 32. A plurality of heater mounting holes 21 may be provided according to the number of sets of the first heater 31 and the second heater 32, and it is preferable that the heater mounting holes 21 are arranged at equiangular intervals around the axis AX. As shown in FIG. 1A, four arc-shaped heater mounting holes 21 are provided around the axis AX of the shaft 41 at an angular interval of 90 °, and are arranged on the target with respect to the axis AX.

《Heat source》
In the example of the heat convection generating system 100 of the first embodiment, the heat source 30 includes a ring-shaped second heater 32 and another ring-shaped second concentrically arranged inside the second heater 32, as shown in FIG. 1A. 1 heater 31. The first heater 31 and the second heater 32 are individually temperature controlled and maintained.

(First heater)
The first heater heats or cools the liquid in a predetermined flow path area of the annular flow path. Here, “heating” means that the temperature of the liquid in the annular flow path 14 located above is raised by the heat source portion 31b of the first heater 31. “Cooling” here means It means that the temperature of the liquid in the annular flow path 14 located above the heat source part 31b of the first heater 31 is lowered.

In the example of the thermal convection generation system 100 of the first embodiment (see FIGS. 1 and 1A), the first heater 31 has a ring-shaped connecting portion 31a and a transverse cross section provided at equal intervals in the circumferential direction. It has a U-shaped columnar heat source 31b. The connecting portion 31a of the first heater 31 is provided with a pair of

screw holes

31c, 31c, which are through holes, at an angular interval of 180 ° around the axis AX. A pair of

screw holes

31c, 31c is joined with shaft portions of screws passing through the second screw insertion holes 22, 22 of the stage 20, so that the first heater 31 is fixed and supported on the stage 20 by the engagement. It is configured.

When the first heater 31 is fixed and supported on the stage 20, the upper end portions of the heat source portions 31 b of the first heater 31 protrude from the upper surface of the stage 20, and the annular flow path 14 is viewed in plan (described later). It contacts the position of the lower surface of the chip 10 for generating heat convection that overlaps a part of the annular channel 14 (see FIG. 1). In the state of contact, the first heater 31 is supplied with power and generates heat or cold, so that the upper end portion of the heat source of the first heater 31 is in contact with the flow path area portion of the annular flow path 14. The liquid is adjusted to a predetermined temperature. Fixing / supporting is not limited to the above-described mode, and other fixing / supporting modes may be used.

(Second heater)
The second heater heats or cools the liquid in a predetermined flow path area of the annular flow path. Here, “heating” means that the temperature of the liquid in the annular flow path 14 located above is raised by the heat source portion 32b of the second heater 32, and “cooling” here means It means that the temperature of the liquid in the annular flow path 14 located above the heat source portion 32b of the first heater 32 is lowered. In the example of the heat convection generating device according to the first embodiment (see FIGS. 1 and 1A), the second heater 32 is provided with a ring-shaped connecting portion 32a at equal intervals in the circumferential direction (four ) L-shaped columnar heat source 32b.

A pair of

screw holes

32c and 32c are provided in the connecting portion 32a of the second heater 32 at an angular interval of 190 ° around the axis AX. The pair of

screw holes

32c, 32c are configured so that shaft portions of screws that penetrate the first screw insertion holes 23 of the stage 20 are combined, and the second heater 32 is fixed and supported by the stage 20 by the combination. Has been.

When the second heater 32 is fixed and supported on the stage 20, the upper ends of the heat source portions 32b of the second heater 32 protrude from the upper surface of the stage, and the annular flow path 14 is viewed in plan (described later). It contacts the position of the lower surface of the thermal convection generating chip that overlaps a part of the annular channel 14 (see FIG. 1). The flow area of the annular flow path 14 in which the upper end portion of the heat source portion 32b of the second heater 32 is in contact with the second heater 32 when the second heater 32 is supplied with power and generates heat or cold in this state of contact. The liquid in the part is adjusted to a predetermined temperature.

(Positional relationship between the first and second heaters and the annular flow path)
With reference to FIGS. 2A and 3, the positional relationship between the annular flow path 14, the first heater 31, and the second heater 32 will be described.

As shown in FIG. 2A, the first heater 31 and the second heater 32 are orthogonal to the direction in which the liquid flows through the annular flow path 14 (a plane coinciding with the circle presented by the annular flow path 14). When the annular channel 14 is viewed in plan in a direction, the center of rotation (center of the annular channel) Q of the annular channel 14 on the plane and the center of rotation of the rotating shaft that is the intersection of the axis AX and the plane And a straight line connecting the rotation center AX and the center of gravity (center of the annular flow path) Q (FIG. 2 (A), one-dot chain line Z (partially not shown) in FIG. 3). , Centered on the center of gravity Q of the annular flow path 14 (center of the annular flow path) Q, any one of the ranges in which the angle (θ) is 30 ° to 150 ° or 210 ° to 330 ° In addition, at least one second heat source part 32b is located and the flow of the annular flow path 14 is within the range. The area is heated or cooled, and the first heat source unit 31b heats or cools the flow channel area other than the flow channel area of the annular flow channel 14 heated or cooled by the second heat source unit 32b (FIG. 2A) or Refer to FIG. 3, hereinafter referred to as “heater installation conditions”). Here, you may provide a clearance gap (henceforth "clearance") between the 1st heat source part 31b and the 2nd heat source part 32b.

The heat source portions of the heaters are formed and arranged so that the clearance CL, which is the gap between the first heater 31 and the second heater 32, surrounds the heat source portion 32b of the second heater 32 (the first heater and the second heater). Are preferably formed and arranged) (see FIGS. 2A and 3). For example, there is an example in which the first heater has a U-shape or a C-shape in a plan view and surrounds the second heater with a clearance CL.

(Area ratio of the first heater)
The area of the channel area that overlaps the upper surface of the heat source portion 31b of the first heater 31 when the annular channel 14 is viewed in plan as described above (when viewed in plan as shown in FIG. 2A or FIG. 3). The ratio of the total flow area to the entire flow path area is not particularly limited as long as the above-mentioned conditions (heater installation conditions) are satisfied. PCR reaction conditions (especially annealing time, annealing temperature, and liquid flow rate in the circular flow path) ) Can be changed as appropriate. Details will be described in “Area ratio between first heater and second heater” described later. As a specific example, as in the first embodiment shown in FIGS. 2A and 3, the area of the first heater 31 that matches the flow area of the annular flow path 14 is set to 70 with respect to the entire flow path area. An example of setting to about 75% is given.

(Flow area area of the second heater)
When the annular flow path 14 is viewed in plan (when viewed in plan as shown in FIG. 2A or FIG. 3), the area of the flow path area that overlaps the upper surface of the heat source portion 32b of the second heater 32 is the flow path area. The proportion of the total area is not particularly limited as long as the above conditions (heater installation conditions) are satisfied. PCR reaction conditions (particularly, denaturation time, denaturation temperature in PCR, and flow rate of liquid in the circular channel) ) Can be changed as appropriate. Details will be described in “Area ratio between first heater and second heater” described later. As a specific example, as in the first embodiment shown in FIGS. 2A and 3, the area of the second heater 32 that matches the flow area of the annular flow path 14 is set to the area of the entire flow path area. An example of about 15 to 20% is given.

(Area ratio between the first heater and the second heater)
The ratio of the area where the heat source part of the second heater 32 overlaps with the channel area of the annular channel 14 to the area where the first heater 31 overlaps with the channel area of the annular channel is 1:11 to 1: 2. It is preferable. It is preferable that the first heater 31 and the second heater 32 are provided so that the clearance CL has an area ratio of the above ratio in the flow path area excluding the flow path area overlapping the annular flow path 14 in plan view.

(Area ratio of clearance)
The clearance CL is such that when the annular flow path 14 of the thermal convection generating system 100 is viewed in plan view, the area of the flow path area of the annular flow path 14 that overlaps the clearance CL is 0.1 to It is preferable to set so as to occupy a range of 15%.

The liquid that passes through the flow passage area of the annular flow passage 14 that overlaps the gap between the first heater 31 and the second heater 32 is air-cooled because air is present instead of the heat source 30 and the like below. It becomes. The cooling of the liquid by this air cooling is considered to have higher cooling efficiency at first glance than cooling by a heat medium (such as the first heater) that is higher than the air and lower than the liquid. Since the rate is low, cooling with the above-described heat medium that is actually formed of a material having a higher thermal conductivity than air can provide higher cooling efficiency.

Therefore, when the annular flow path 14 of the thermal convection generation system 100 is viewed in plan view, the ratio of the area of the annular flow path area 14 that overlaps the clearance CL to the total area of the flow path area is the first heater. 31 is sufficient to prevent or suppress mutual heat conduction through the air between the first heater 32 and the second heater 32 (air in the clearance portion), and cooling efficiency is reduced due to air cooling in the clearance CL portion. However, it may be set within an allowable range, so it is preferable to set the ratio as small as possible within the range of 0.1 to 15%. In the thermal convection generation system 100 of the first embodiment, the clearance CL shown in FIGS. 2A and 3 is set to about 10 to 15% with respect to the entire flow path area.

(Temperature of the first heater)
When convection PCR is performed by the thermal convection generation system 100, the temperature of the first heater 31 is set to the annealing temperature in the PCR method. The annealing temperature is a temperature determined based on the denaturation temperature (Tm value) of the primer, and generally (Tm value −5 ° C.) is appropriate. Here, the Tm value can be calculated from, for example, the following formula (1) based on the designed base sequence of the primer for PCR.
Tm value (° C) = 2 (nA + nT) +4 (nC + nG) + 35-2 (nA + nT + nC + nG) (1)
(In the calculation formula, nA, nT, nC and nG represent the number of bases of adenine, thymine, cytosine and guanine contained in the primer, respectively.)
The Tm values of the forward and reverse primers are designed to be as close as possible, but if they are separated, the annealing temperature may be set based on the lower Tm value. In the case of RT-PCR, the Tm value is set to, for example, 50 ° C. or higher so as not to be lower than the reverse transcription reaction temperature. Typically, the temperature of the first heater 31 is set to a temperature of 55 ° C. to 65 ° C. that is often used as the annealing temperature.

(Temperature of the second heater)
When convection PCR is performed by the thermal convection generation system 100, the temperature of the second heater 32 of the thermal convection generation system 100 is a temperature necessary for applying heat to the double-stranded nucleic acid portion to form a single-stranded nucleic acid. Generally, it is set to about 95 ° C. The second heater 32 is maintained at the above temperature at least during the convection PCR.

It is desirable to control the temperature of the first heater and the second heater of the heat source 30 so as to satisfy the following conditions (1) to (3). The condition (1) is for efficiently promoting thermal convection.
(1) Temperature of the second heater−temperature of the first heater ≧ 10 ° C.
(2) The temperature of the first heater minus the temperature of the clearance portion) ≧ 10 ° C.
(3) The temperature of the second heater—the temperature of the clearance portion) ≧ 10 ° C.

<< Rotation drive means >>
The rotation driving means is means for rotating the rotating shaft and rotating the heat convection generating chip fixed to the rotating shaft in a predetermined manner. In the first embodiment, as described above, the motor 40 or the like that has the shaft 41 as the rotation shaft and whose operation is controlled by the control means and rotates the rotation shaft in a desired manner corresponds to the rotation drive means. Any means can be used as long as it is not limited to a motor and can be driven in a desired manner.

"heatsink"
As shown in FIG. 1A, a heat sink 60 that radiates heat generated in the first heater 31 and cools the first heater 31 may be provided. The heat sink 60 can remove excess heat at a low manufacturing cost, and can improve the accuracy of thermal convection PCR.

[assembly]
Assembly will be described with reference to FIGS. 1 and 1A. As described above, with the first heater 31 and the second heater 32 fixed and supported with respect to the stage 20, the thermal convection generating chip 10 and the stage 20 are fixed to each other by the connecting means described above. Then, in the state in which the chip 10 for heat convection generation, the stage 20, the first heater 31 and the second heater 32 are laminated, the shaft 41 of the motor 40 is connected to the center hole 24 of the stage 20 as shown in FIGS. And the center hole 17 of the chip 10 for generating heat convection. In this state, the shaft 41 of the motor 40 and the stage 20 are fixed by appropriate means (such as fitting), and the assembly is completed. Such assembly may be performed before or after the liquid introduction step (details will be described later).

≪Control means≫
As shown in FIG. 4, the control means 50 of the thermal convection generation system 100 includes an arithmetic control unit 51, a display unit 52, and an input unit 53, and includes a first heater 31, a second heater 32, a motor 40, depending on circumstances. , The excitation light source 91, the fluorescence detector 92, the detection light source 93, the detection light detector 94) and the like are electrically connected to each other. The arithmetic control unit 51 is configured by a microcomputer including a CPU, a ROM, a RAM, and the like. The CPU supplies power to the first heater 31, the second heater 32, and the motor 40 according to information input from the input unit and a program stored in the ROM, and controls its operation. The program includes an operation program for PCR. The display unit 52 includes a liquid crystal display device, and the input unit 53 includes input devices such as a keyboard and a mouse. The temperature of the first heater 31 and the second heater 32 and the rotational driving speed of the heat convection generating chip 10 by the motor 40 can be adjusted via the input unit 53 of the control means 50.

≪Optical detection system≫
As illustrated in FIG. 5, the thermal convection generation system 100 optionally includes an optical detection system 90 that receives and quantifies fluorescence emitted from the annular channel 14. The optical detection system 90 includes an excitation light source 91, a fluorescence detector 92, a detection light source 93, and a detection light detector 94. The optical detection system 90 is controlled by the control unit 50 to perform various operations, and transmits various data to the arithmetic control unit of the control unit 50. In the vicinity of the annular channel 14, a detected part P 1 that reflects or scatters the detection light emitted from the detection light source 93 is provided.

The excitation light source 91 is a light source that emits light that excites the fluorescent dye contained in the liquid in the annular flow path 14 of the thermal convection generating chip 10, and is provided with an LED and a fluorescent filter that extracts only light of a desired wavelength. A white light source or the like is used as an excitation light source.

The fluorescence detector 92 detects fluorescence L1 ′ emitted from the annular flow path, and includes, for example, a photomultiplier detector, a condensing lens, a fluorescence filter, and the like.

The detection light source 93 irradiates the detection light L2 toward the detection unit P1 on the thermal convection generation chip 10, and for example, a light source (LED or the like) that emits laser light is used as the detection light source. The detection part P1 is a fixed point set on a rotation locus formed by the detected part as the thermal convection generation chip 10 rotates. When the detection light L2 from the detection light source 93 is irradiated, the detected portion is configured to reflect or scatter the detection light L2. The detection light detector 93 includes, for example, a photomultiplier detector, a condensing lens, a band pass filter, and the like, and detects the detection light L2 ′ reflected or scattered.

≪PCR≫
Hereinafter, a convection (thermal convection) PCR method using the thermal convection generation system of the first embodiment will be described. The convection PCR method according to the present invention includes a liquid introduction step, a PCR reaction step, and a detection step in this order. The “PCR” includes various PCRs such as reverse transcription PCR (RT-PCR) and real-time PCR which is quantitative PCR.

(PCR reaction solution)
The PCR reaction solution used in the present invention is composed of a sample solution and a reaction reagent solution. Note that it is not necessary to mix the sample solution and the reaction reagent solution at the time before performing PCR.

(Sample liquid)
The sample liquid means a liquid containing a nucleic acid to be amplified by PCR. The sample liquid includes a solution containing the target DNA, RNA, pseudo nucleic acid, and the like, and a liquid containing the sample. For example, when RT-PCR is performed, influenza virus, norovirus, other infectious disease viruses in general, extracts of expressed RNA from cells, and the like are used as sample liquids. When performing PCR by eluting the nucleic acid to be amplified from the sample in one step, influenza virus, norovirus, and other infectious disease viruses in general, cells suspended in an appropriate solution such as buffer or water It can be used as a sample liquid.

(Reaction reagent solution)
The reaction reagent solution means a solution containing a reagent and an enzyme necessary for amplifying the nucleic acid to be amplified contained in the sample solution by the PCR method. When the sample solution is a solution containing nucleic acid, the reaction reagent solution means a solution containing dNTP, MgCl ₂ , various polymerases and the like.

When the sample solution is a suspension of RNA virus and reverse transcription PCR is performed in one step including elution of RNA to be amplified from the sample, as a reaction reagent solution, for example, the product name “SuperScriptIII OneStep RT-PCR System "(Product No. 12574-018, Life Technology Co., Ltd.) can be used. “SuperScript” is a registered trademark of Life Technology Corporation. Furthermore, for example, the product name “GeneAmp Ez rTth RNA PCR Kit” (product number N8080179, manufactured by Applied Biosystems), the product name “PrimeScriptII High Fidelity One Step RT-PCR kit” (product code R026A or R026B, Takara Bio Inc. Product name "Platinum (registered trademark) Quantitative RT-PCR ThermoScript (application trademark) One-Step System" 製品 (product code 11731-015, manufactured by lifetechnologies), product name "SpeedSTAR (application trademark) HS DNA Polymerase" ( A product code RR070A, manufactured by Takara Bio Inc., a product name “Ampdirect (registered trademark) Plus (product code 241-208800-98, manufactured by Shimadzu Corporation)”, and the like can be used.

<< Liquid introduction process >>
The liquid introduction process is a process for introducing the sample liquid, the reaction reagent solution, and other liquids necessary for the PCR reaction, which are included in the PCR reaction solution, individually or integrally into the annular flow path.

When PCR is performed by the thermal convection generation system 100 of the first embodiment, first, the reaction reagent solution is injected into the liquid supply hole 15d formed on the surface of the substrate 11 of the thermal convection generation chip 10 on the side opposite to the lid 13. . The injected reagent solution passes through the extended portion 15a of the liquid supply path 15 and flows into the liquid reservoir 15b by capillary action. Next, the sample liquid is injected into the liquid supply hole 15d. The injected specimen liquid passes through the extension part 15a of the liquid supply path 15 and flows into the liquid reservoir part 15b by capillary action. The reaction reagent solution and the sample liquid that have flowed into the liquid reservoir 15b stay in the liquid reservoir 15b while being mixed in a stopped state in which the thermal convection generating chip 10 is not rotated. Finally, an oil is optionally introduced as described above to prevent evaporation of the reaction reagent solution. Note that the reaction reagent solution, the sample liquid, the oil, and the like may be pushed into the liquid reservoir 15b using a pipetter or the like regardless of the capillary phenomenon.

When the user operates the input unit 53 of the control means 50 to drive the motor 40 to rotate the stage 20 and the thermal convection generating chip 10 placed thereon around the axis AX of the shaft 41, centrifugal force Thus, the sample solution and the reaction reagent solution are introduced into the annular channel 14. On the other hand, oil having a low specific gravity stays in the liquid reservoir 15b and functions as a lid for preventing the liquid in the annular channel 14 from flowing back from the communication portion 15c to the liquid reservoir 15b. Prevents the liquid in the annular channel 14 from evaporating.

<< PCR reaction process >>
The PCR reaction step is a step for performing convection PCR by refluxing the PCR reaction solution at a predetermined rate in the circular flow path. Prior to the liquid introduction step, it is desirable to set the second heater 32 to the above-described predetermined PCR denaturation temperature and set the first heater 31 to the predetermined PCR annealing temperature.

Subsequent to the liquid introduction process, when the motor 40 is driven to rotate the stage 20 and the thermal convection generating chip 10 around the axis AX of the shaft 41, the centrifugal force due to the rotation and the temperature difference between the second heater and the first heater The PCR reaction solution circulates in the circular channel 14 due to the thermal convection caused by. When the double-stranded nucleic acid in the PCR reaction solution passes through the flow path area above the second heater 32, it exchanges heat with the heat source portion 32b of the second heater 32 to a high temperature (eg, 90 to 98 ° C.). It is denatured by heating to form a single-stranded nucleic acid. Thereafter, when the PCR reaction solution enters the flow path area above the first heater 31, this time, heat exchange is performed with the heat source portion 31 b of the first heater 31, and the liquid temperature decreases at a faster rate than air cooling. (Eg, 55 ° C. to 60 ° C.), and annealing and extension (nucleic acid molecule extension by polymerase) occur.

If the PCR reaction solution in the annular channel 14 contains a gas, the gas moves by the centrifugal force described above and flows into the gas discharge path 16, so that the gas in the PCR reaction solution is removed. Can be removed. As a result, the PCR reaction solution can be smoothly convected.

In addition, when performing reverse transcription PCR using the thermal convection generation apparatus 1 and the thermal convection generation chip 10 according to the present invention, for example, the following two methods (a) and (b) can be performed. Hereinafter, the two methods (a) and (b) will be described with reference to FIGS.

(A) The reaction reagent solution and the sample solution are mixed in advance to produce a mixed solution. Next, the mixed solution is injected into the liquid supply path 15 of the thermal convection generating chip 10, and the thermal convection generating chip 10 is rotated to allow the mixed solution to enter the annular flow path 14. Thereafter, the rotation of the thermal convection generating chip 10 is stopped, the first heater 31 and the second heater 32 are set to the same temperature (for example, 40 to 60 ° C.), and the mixed solution in the annular flow path 14 is kept for a certain time ( For example, the reverse transcription reaction is performed by heating for 60 seconds.

(B) First, the reaction reagent solution is filled in the annular flow path 14 of the thermal convection generation chip 10, and then the sample liquid is injected into the liquid supply path 15, and the thermal convection generation chip 10 is rotated. The specimen liquid in the liquid supply path 15 is caused to enter the annular flow path 14. Then, the temperature of the first heater 31 and the temperature of the second heater 32 are made different from each other, and the liquid in the annular flow path 14 is convectively mixed to mix the reaction reagent solution and the sample liquid, thereby generating a mixed solution. Thereafter, the rotation of the thermal convection generating chip 10 is stopped, the first heater 31 and the second heater 32 are set to the same temperature (for example, 40 to 60 ° C.), and the mixed solution in the annular flow path 14 is kept for a certain time ( For example, the reverse transcription reaction is performed by heating for 60 seconds.

A template DNA (cDNA) is synthesized from RNA by reverse transcription reaction by any of the methods (a) and (b) described above. Then, the first heater 31 and the second heater 32 are set to temperatures suitable for PCR (for example, the temperature of the first heater 31 is set to 60 ° C. and the temperature of the second heater 32 is set to 95 ° C.), and the thermal convection PCR reaction is performed. Cause it to occur.

(Rotational speed of chip for generating heat convection)
The rotational speed of the thermal convection generating chip 10 is such that the time required for the double-stranded DNA (nucleic acid molecule) contained in the PCR reaction solution in the circular channel 14 to denature and become single-stranded DNA is increased. 2 It is set so as to pass through the flow channel area above the first heater 31 and to pass through the flow channel area above the first heater 31 over a time required for primer annealing and nucleic acid molecule extension.

Specifically, the time for the PCR reaction solution in the annular channel 14 to pass through the channel area above the second heater 32: the denaturation time (A) is generally 5 to 60 seconds, preferably 10 to 20 Second, time to pass through the flow path area above the first heater 31: annealing time + extension time (B) is generally (5-30) seconds + (number of nucleic acid bases ÷ 60 bases) seconds Time passing through the clearance portion: It is preferable that the air cooling time (C) satisfies the relational expression of 0.001 <(C) / (A) + (B) + (C) <0.15. Therefore, the rotational speed of the thermal convection generating chip 10 is preferably set so that the PCR reaction solution passes through each flow channel area during the time (A) to (C). In this case, it should be considered that the

heat source portions

31b and 32b of the first heater and the second heater 32 described above adjust the area of the flow path area where the annular flow path 14 overlaps. In addition, since the nucleic acid extension speed varies depending on the type of polymerase (Taq), the extension speed (60 b / sec) used in the above calculation formula of annealing time + extension time (B) is 60 b / sec to 500 b / sec. You may change in the range of second. Note that “b” in the unit “b / sec” is an abbreviation for a base such as DNA.

(Measurement of liquid velocity)
FIG. 20 is a graph showing the relationship between the voltage of the power source of the motor used in the first embodiment and the rotational speed of the motor, and the relationship between the speed and the centrifugal force (F = mv ² / r, g = (2πN)). obtained relation between the rotational speed of the motor using from ² r) with a relative acceleration of gravity in the first embodiment, which was developed by further determining the relationship between the voltage and the relative acceleration of gravity. FIG. 21 is a graph showing the relationship between the relative gravitational acceleration and the voltage of the power source of the motor 40.

Water is supplied in advance from the liquid supply hole 15d with a micropipette, and then the motor 40 is rotated so that the annular channel 14 is filled with water. When the motor 40 is rotated with a voltage equivalent to 1 G (2.12 V from FIG. 20) in a state where the second heater 32 and the first heater 31 are energized, the red colored liquid in the annular channel 14 is 30. About half a second in seconds. Thus, the speed at which the liquid moves in the annular channel 14 can be measured by a method using food red and water. In addition, by performing the trial operation of the thermal convection generating device in this way, the relationship between the rotational speed of the thermal convection generating chip 10 and the rotational speed of the motor 40 can also be determined. Furthermore, as described above, since the relationship between the power supply voltage of the motor 40 and the rotation speed of the motor 40 is known, what level of power supply voltage is applied to the motor 40 and how much liquid is in the annular flow path 14. Can be determined and the speed can be determined. By controlling the rotational speed of the thermal convection generating chip 10 to control the thermal convection speed, it is possible to adjust the time for passing above the liquid heat source section 32b and the heat source section 31b.

(Rotation drive time)
The drive time of the motor 40 for rotating the heat convection generating chip 10 is set so that the heating by the second heater 32 and the cooling by the first heater 31 are the same as the number of PCR thermal cycles.

<< Detection process >>
The detection step is a step of detecting and quantifying a substance contained in the liquid in the annular channel. For example, in the PCR method, since a fluorescent substance is intercalated in a double-stranded nucleic acid molecule that proliferates, the nucleic acid in the PCR reaction solution can be detected and quantified by detecting this fluorescence. Further, for example, in the case of real-time PCR, after a chimera probe containing a predetermined fluorescent substance is bound to a nucleic acid as a template, the chimera probe is cleaved by RNase H at the time of nucleic acid extension by a polymerase. Since the substance that emits fluorescence increases, the fluorescence can be detected and quantified.

As shown in FIG. 5, the excitation light source 91 irradiates light (excitation light) L 1 that excites the fluorescence toward the annular flow path 14 of the rotating thermal convection generating chip 10. The excitation light L1 is applied to the fluorescent substance in the annular channel 14, and when the fluorescent substance is excited, fluorescence L1 ′ having a predetermined wavelength is emitted. The fluorescence L1 ′ is detected and quantified by the fluorescence detector 92. Further, the detection light L2 is emitted from the detection light source 93 to the detected portion 95, and the detection light detector 93 detects the light L2 ′ reflected and scattered by the detected portion P1, thereby generating heat convection. The position of the annular flow path 14 during rotation of the chip 10 is detected.

Hereinafter, functions and effects of the thermal convection generating device 1 and the thermal convection generating system 100 according to the first embodiment of the present invention will be described.

(1) The thermal convection generating apparatus 100 of the first embodiment is
It has the shaft 41 which can fix rotatably the chip | tip 10 for thermal convection generation | occurrence | production which has the annular flow path 14 for circulating the liquid, and the 1st heat-source part 31b which heats or cools the liquid in the annular flow path 14. The first heater 31, the second heater 32 having the second heat source part 32 b that heats or cools the liquid in the annular flow path 14, and the shaft 41 is rotated to drive the entire annular flow path 14 along the axis of the shaft 41. A motor 40 (rotation drive means) that rotates around the axis AX, and the annular channel 14 is seen in a plan view in a direction orthogonal to the direction in which the liquid flows through the annular channel 14 (circle exhibited by the annular channel). The center of gravity (center of the annular flow path 14) Q of the annular flow path 14 on the plane and the rotation center of the rotation axis AX that is the intersection of the axis AX and the plane (the shaft in FIG. 2A) 41 center) In which the rotation center and the center of gravity (center of the annular channel 14) Q are connected to the straight line Z, the center of gravity (center of the annular channel 14) Q is 30 °. As shown in FIG. 2 (A) or FIG. 3, at least one second heat source part 32b is located in only one of the ranges of −150 ° or less or 210 ° or more and −330 ° or less. Then, the flow passage area of the annular flow passage is heated or cooled within the range, and the first heat source portion 31b is a flow other than the flow passage area of the annular flow passage 14 heated or cooled by the second heat source portion 32b. Since this is a thermal convection generating device that heats or cools the road area (the channel area excluding the area where the clearance CL and the annular channel 14 overlap in plan view), the thermal convection generating chip 10 is rotated to rotate the annular channel. 14 such as PCR reaction solution When the liquid is circulated, the liquid has two temperature regions in the annular channel 14 (the channel area of the annular channel 14 above the first heater 31 and the channel of the annular channel 14 above the second heater 32). Stable thermal convection generation that is repeated many times can be obtained with almost no time other than the area (see FIG. 2A or FIG. 3). Further, the same heat convection can be stably obtained even if the temperature of the environment in which the heat convection generating device is operated changes.

Here, the clearance CL means a minimum clearance that does not cause heat conduction between the first heat source unit 31b and the second heat source unit 32b or does not adversely affect the PCR reaction even if heat conduction is performed. .

Here, even when the clearance CL is not provided (for example, when the first heat source unit 31b and the second heat source unit 32b are in contact with each other), the first heat source unit 31b can be separated from the first heat source unit 31b by another temperature control unit or the like. Since the second heat source unit 32b only needs to be maintained at a predetermined temperature (even forcibly), in this case, the flow path area heated or cooled by the first heat source unit 31b is different from the above. In addition, the entire flow path area other than the flow path area of the annular flow path 14 heated or cooled by the second heat source unit 32b is provided.

Further, as described above, it is more efficient to adjust the temperature of the liquid by cooling or heating the liquid in the annular flow path 14 with a heat medium than adjusting the temperature of the liquid in the annular flow path 14 by air cooling. Is expensive. For example, for a liquid that has passed through the flow path area above the second heater 32 and has reached a high temperature (eg, 90 ° C. to 95 ° C.), the heat is lower than that of the liquid (eg, 50 ° C. to 60 ° C.). In many cases, the efficiency of temperature adjustment is higher when the liquid is cooled by the first heater 31 that is a medium than when the liquid is cooled by air. Therefore, when the clearance CL is provided between the first heat source part 31b and the second heat source part 32b, the liquid is air-cooled by not providing a gap exceeding the minimum clearance CL. The flow channel area can be reduced as much as possible. As a result, the efficiency of temperature adjustment of the liquid circulating in the annular flow channel can be increased, and rapid temperature control is realized.

Furthermore, since the temperature adjustment of the liquid in the annular channel is not based on air cooling, the temperature adjustment of the liquid is not easily influenced by the outside air temperature as in the case of air cooling, and is excellent in robustness and reproducibility of heat convection.

Further, as shown in FIG. 2A or 3, the first heater 31 and the second heater 32 are installed on the left and right of the line Z, and the second heater 32 is higher in temperature than the first heater 31. For example, a temperature difference occurs in the liquid in the annular channel on the left and right of the line Z, and the density is also different. That is, the density of the liquid that passes through the flow path area above the first heater 31 is higher than that of the liquid that passes through the flow path area above the second heater 32, and the way the liquid is subjected to centrifugal force differs from left to right. As a resultant force, a force that promotes the circulation of the liquid in the annular flow path is applied. Even when the temperature settings of the first heater 31 and the second heater 32 are reversed, it is possible to promote the circulation of the liquid through the annular flow path 14 for the same reason.

(2) If the area of the clearance CL portion in the plan view is in the range of 0.1 to 15% with respect to the entire area of the annular flow path 14, the liquid is above the clearance CL. The time for passing through the flow channel area in the annular flow channel is within a range that does not adversely affect the temperature control of the liquid, and the temperature of the liquid can be suitably adjusted.

(3) The heat source portion 31b of the first heater 31 and the heat source portion 32b of the second heater 32 have a flat plate portion that is flush with the second heat source portion 32. Since the

heat source portions

31b and 32b are formed and arranged so as to surround, the heat convection generating chip 10 that does not overlap the annular flow path 14 when viewed in plan (see FIG. 2A or FIG. 3). The first heater 31 and the second heater 32 are also in contact with the portion. As a result, the periphery and the inner side (the inner side of the ring) of the annular channel 14 are not air-cooled, and the liquid temperature can be adjusted more suitably. In addition, since each heat source part should just be formed and arrange | positioned so that the said clearance may surround the heat source part 32b of a 2nd heater in the state of the said planar view, a U-shape and a c-shape are the same. An effect is obtained.

(4) When viewed in plan, the channel area of the annular channel 14 in contact with the second heat source unit 32b of the second heater 32 is in contact with the first heat source unit 31b of the first heater 31. Since the area is smaller than the flow path area, the liquid heated by the second heater 32 can be efficiently cooled, adjusted to a predetermined temperature, and stably maintained at the temperature.

For example, when the PCR reaction is performed using the thermal convection generating apparatus 100, the PCR reaction solution that has reached the denaturation temperature by the second heater 32 is efficiently cooled from the denaturation temperature to the predetermined annealing extension temperature by the first heater 31. It will be stably maintained at the temperature, and the PCR reaction (annealing extension) will be stable.

(5) including the thermal convection generating device 1 and the thermal convection generating chip 10 having the annular flow path 14 for circulating the liquid, and the thermal convection generating chip 10 is a liquid that communicates with the annular flow path 14. If the thermal convection generating system has a liquid supply path 15 and the liquid in the liquid supply path 15 is supplied to the annular flow path 14 by the centrifugal force applied to the thermal convection generation chip 10 by the rotation driving, It is possible to immediately perform another PCR reaction by exchanging only the convection generating chip 10. Further, when performing the PCR reaction, the PCR reaction solution as a liquid is supplied into the annular flow path 14 simultaneously with the addition of the centrifugal force to start the reaction. Therefore, the PCR reaction start time and the heat convection start time are determined. Can be matched.

(6) A gas discharge passage 16 is formed on the rotation axis (axis AX) side as viewed from the flow passage area of the annular flow passage 14 above the second heater 32 and in communication with the annular flow passage 14 in the vicinity of the flow passage area. As a result, gas such as bubbles generated when the liquid is driven to rotate and the liquid is heated to a high temperature by the second heater easily moves to the gas discharge path.

(7) By arranging a plurality of the annular flow paths 14 so as to be symmetric with respect to the axis AX of the rotation axis, a balance is obtained when the thermal convection generating chip 10 is rotationally driven. The circulation and thermal convection of the liquid in the passage 14 are stabilized.

(8) Since the surface roughness Ra of the wall surface of the annular channel 14 is 100 nm or less, the remaining wetness of the liquid in the annular channel is reduced.

(9) When the annular channel is circular when viewed in plan, the liquid circulates smoothly in the annular channel and heat convection is suitably performed.

(10) Since the material of the wall surface of the annular channel is any one of cyclic olefin, polypropylene, and polycarbonate, since it is a resin, it is easy to adjust the surface roughness of the wall surface of the annular channel. It is easy to ensure the transparency of the thermal convection generating chip 10 necessary for optically detecting the fluorescent substance in the annular channel.

(11) The thermal convection generation system 100 applies an excitation light source that irradiates the liquid in the annular flow path 14A with excitation light that excites the fluorescent dye contained in the liquid in the annular flow path 14A, and the fluorescent dye. A fluorescence detector that detects fluorescence emitted by the fluorescent dye by irradiating the excitation light, and an arithmetic control unit that calculates a replication amount of the nucleic acid based on the fluorescence detected by the fluorescence detector. It is a waste.

For example, when the PCR reaction is performed by the thermal convection generation system 100, the temperature can be efficiently controlled as described in (1) above. Therefore, the PCR reaction solution has an unintended temperature in the PCR reaction (specifically, The range of 70 ° C. to 90 ° C.) is shortened, and unnecessary deactivation of an enzyme for PCR reaction such as Taq polymerase in a time zone that does not contribute to the PCR reaction can be reduced by shortening the time. As a result, the nucleic acid to be originally amplified is amplified with good reproducibility, the reproducibility of PCR itself is improved, and the fluorescence from the fluorescent substance labeled on the product (nucleic acid) of such a PCR reaction is detected. Accuracy in PCR and the like is increased.

(17) A convection PCR method using the thermal convection generating device or the thermal convection generating system according to any one of (1) to (16), wherein the second heat source of the second heater 32 is used during the PCR reaction. By maintaining the temperature difference between the part 32 b and the first heat source part 31 b of the first heater 31 at 10 ° C. or more, it is possible to promote thermal convection more efficiently.

[Second Embodiment]
Secondly, a thermal convection generation system 200 according to the embodiment will be described. The thermal convection generation system 200 of the second embodiment includes at least a thermal convection generation chip (see FIGS. 6 and 7) 10A and the thermal convection generation device 1 of the first embodiment. As described above, the thermal convection generating device 1 includes the shaft 41, the first heater 31, the second heater 32, the motor 40, and the like. Note that the configuration of the thermal convection generating device 1 itself is the same as that of the first embodiment, and thus the description thereof is omitted.

《Tip for generating heat convection》
As shown in FIG. 6, the thermal convection generating chip has a substrate 11A and a lid 13A laminated on the substrate 11A, and has a center hole 17 at the center of each member. FIG. 8A shows the front surface of the substrate 11A, and FIG. 8B shows the back surface of the substrate 11A. As shown in FIGS. 8A and 8B, the substrate 11A includes an annular flow path 14A and first to third

liquid supply paths

15A, 15B, and 15C. The plurality of liquid supply paths can be provided so as to store different liquids and supply them to the annular flow path 14A as described below. The material of each member such as the substrate 11A is as described in the first embodiment.

As shown in FIG. 8, the first liquid supply path 15A includes a first receiving portion 16A and a first suction path 17A. The second liquid supply path 15B includes a second receiving portion 16B and a second suction path 17B. The third liquid supply path 15C includes a third receiving portion 16C and a third suction path 17C. These first to third receiving portions 16A to 16C are for receiving a liquid.

The first to third suction passages 17A to 17C respectively connect the first to third receiving portions 16A to 16C and the annular flow path 14, and the liquids of the first to third receiving portions 16A to 16C are respectively capillary tubes. Suction by phenomenon.

Each suction passage has a first region T and a second region S. The first region T is located between the intermediate portion of each of the suction passages 17A to 17C and the annular flow path 14A. The second region S is located between the intermediate portions of the suction passages 17A to 17C and the receiving portions 16A to 16C. By rotating the thermal convection generating chip 10, the liquid in the first region T is separated from the liquid in the second region S and supplied to the annular flow path 14A.

The annular channel 14A is used for heat convection of a mixed solution (details will be described later) of the sample solution and the reaction reagent solution. The annular channel 14A is an annular belt-like channel in plan view (see FIGS. 8A and 8B). The annular channel 14A is configured by a groove (see FIG. 8B) formed on the lower surface of the substrate 11A and a part of the upper surface of the lid 13A (see FIG. 7). The dimensions of each part of the annular flow path 14A are not particularly limited. For example, the outer diameter of the annular flow path 14A is 60 mm, the depth is 400 μm, and the width is 500 μm, but the dimensions are set as described in the first embodiment. can do. In the second embodiment, a plurality of annular flow paths 14A are provided at predetermined angular intervals around the central axis of the substrate 11A (see FIGS. 6 and 8). The central axis coincides with the axis AX of the shaft 41 in a state where the thermal convection generating chip 10A is mounted on the thermal convection generating device 1.

As shown in FIG. 8A, the first receiving portion 16A is configured by a hole. The first suction passage 17A is bent at an acute angle at an intermediate portion thereof, the first region T extends in the radial direction of the substrate 11A, and the second region S has an angle θ between the first region T and the first region T. "Is extended in a direction that forms an acute angle (see FIG. 8B). In the first region T and the second region S, a groove that constitutes a part of the first suction passage 17A is formed. (See FIG. 8B) The second to third receiving

portions

16B and 16C are configured in the same manner as the first receiving portion 16A. The

third suction passages

17B and 17C are configured in the same manner as the first suction passage 17A.

By rotating the thermal convection generating chip 10A around its central axis, the liquid in each first region T is moved in the direction of the annular flow path 14A by centrifugal force, and the liquid in each second region S is centrifugal force. To move in the direction of each receiving part. The angle θ ″ is, for example, not less than 5 ° and not more than 85 °.

The suction passages 17A to 17C further have air holes 18A to 18C. This air hole introduces air into the intermediate portion of each of the suction passages 17A to 17C. The air holes 18A to 18C are constituted by holes, and each first region T and second region S are communicated with the space on the upper surface side of the substrate 11A. The air holes 18A to 18C promote separation of the liquid in each first region T and the liquid in the second region S.

That is, when the gap formed between the liquid in the first region T and the liquid in the second region S is in a vacuum state, each of the two liquids is sucked in the direction of the gap. It becomes difficult for the liquid in T and the liquid in 2nd area | region S to isolate | separate. By providing the air holes 18A to 18C, air is introduced into the gap, so that the liquid in the first region T and the liquid in the second region S are smoothly separated. If the liquid in the first region T and the liquid in the second region S can be smoothly separated without introducing air into the intermediate portions of the suction passages 17A to 17C, the air holes 18A to 18C are provided. It can be omitted.

The thermal convection generating chip 10A further includes an introduction chamber 19 and an introduction passage 19a. The introduction chamber 19 and the introduction passage 19a are provided between the first region T and the annular flow path 14A. The liquid discharged from the first region T of each of the supply paths 15A to 15C flows into the introduction chamber 19. Details of the introduction chamber 19 will be described later with reference to FIG. The liquid in the introduction chamber 19 flows into the annular channel 14A through the introduction passage 19a. The introduction passage 19 is constituted by a groove formed on the lower surface of the substrate 11A and the upper surface of the lid 13A (see FIGS. 8B and 6).

The first liquid supply path 15A supplies the sample liquid to the annular flow path 14. As the sample liquid, the same one as in the first embodiment can be used.

The dimensions (depth, width, length) of the first region T of the first liquid supply path 15A are, for example, as shown in Table 1 below. The dimensions (depth, width, length) of the second region S of the first liquid supply path 15A are, for example, as shown in Table 1 below.

The amount of the sample liquid filled in the first region T of the first liquid supply path 15A is equal to the amount of the sample liquid to be supplied to the annular flow path 14A. Further, the volume of the first receiving portion 16A of the first liquid supply path is larger than the volume of the first suction passage 17A.

The second liquid supply path 15B supplies a reaction reagent solution for performing PCR to the annular flow path 14A. As the reaction reagent solution, the same reaction reagent solution as that of the first embodiment can be used.

The dimensions (depth, width, length) of the first region T of the second liquid supply path 15B are, for example, as shown in Table 2 below. The dimensions (depth, width, length) of the second region S of the second liquid supply path 15B are, for example, as shown in Table 2 below.

The amount of the reaction reagent solution filled in the first region T of the second liquid supply path 15B is equal to the amount of the reaction reagent solution to be supplied to the annular flow path 14A. Further, the volume of the second receiving portion 16B of the second liquid supply path is larger than the volume of the suction passage 17B of the second liquid supply path. Note that the total volume of the first region T of the first liquid supply path 15A and the volume of the first region T of the second liquid supply path 15B is equal to the volume of the annular flow path 14A.

The third liquid supply path 15C is for supplying an evaporation suppression liquid (eg, mineral oil for PCR) to the heat convection flow path 11. The evaporation suppression liquid is a liquid that suppresses the evaporation of the liquid (for example, the sample liquid and the reaction reagent solution) in the annular channel 14A, and the maximum temperature of the heat source 30 (second heater) introduced into the annular channel 14. Those having a higher boiling point are used. Since the boiling point of the mineral oil is higher than the maximum temperature of the second heater 32 that heats the liquid in the annular channel 14A (eg, the sample liquid and the reaction reagent solution), the evaporation of the liquid in the annular channel 14A is suppressed. To do. Moreover, since the specific gravity of mineral oil is smaller than the specific gravity of the liquid in the annular flow path 14A, it functions as a lid that closes the introduction passage 19a. In addition, even if it is liquids other than mineral oil, specific gravity is smaller than the specific gravity of the liquid (for example; sample liquid and reaction reagent solution) in annular flow path 14A, and / or a boiling point is higher than the maximum temperature of the 2nd heater 32. If it is too high, it can be used as a liquid for suppressing evaporation.

When the thermal convection generating chip 10A rotates around its central axis, the annular channel 14A is filled with the sample liquid and the reaction reagent solution having a specific gravity greater than that of the mineral oil, and the mineral oil stays in the introduction passage 19a. The introduction passage 19a is blocked. As a result, evaporation of the sample solution and reaction reagent solution in the annular channel 14A and backflow into the introduction chamber 19 can be suppressed.

The dimensions (depth, width, length) of the first region T of the third liquid supply path 15C are, for example, as shown in Table 3 below. The dimensions (depth, width, length) of the second region S of the third liquid supply path 15B are, for example, as shown in Table 3 below. It should be noted that the positions and the dimensions of each of the first liquid supply path 15A, the second liquid supply path 15B, and the third liquid supply path 15C (see Tables 1 to 3) are the first to third liquid supply paths 15A to 15C. Are set so as not to interfere with each other.

The amount of mineral oil filled in the first region T of the third liquid supply passage 15C is an amount that can prevent the introduction passage 19a. The volume of the third receiving portion 16C of the third liquid supply path is larger than the volume of the third suction passage 17C of the third liquid supply path.

The thermal convection generating chip 10A further includes a cover portion 25 (see FIGS. 6, 7, 9, and 10). An opening of the introduction chamber 19 is formed on the upper surface of the thermal convection generating chip 10A. The cover portion 25 is provided on the substrate 11A of the thermal convection generating chip 10A and covers the opening (see FIGS. 6 and 7). Details of the introduction chamber 19 and the cover portion 25 will be described later with reference to FIGS. 9 and 10.

Each liquid supply passage 15A to 15C further includes a guide passage 26 (see FIG. 8A). That is, as shown in FIG. 11, a meniscus guide passage forming portion 26A is provided in each of the receiving portions 16A to 16C, and the guide passage 26 is formed. The guide passage 26 guides the liquid in each of the receiving portions 16A to 16C to each second region S.

9 and 10, the configuration of the introduction chamber 19 and the cover portion 25 will be described in detail. 9 is a cross-sectional view taken along line BB in FIG. 6, and FIG. 10 is a perspective view of the cover portion 25. As shown in FIG. As shown in FIG. 9, a groove 27 and a hole 28 are formed in the substrate 11A.

The introduction chamber 19 is formed by a groove 27 and a hole 28. The groove 27 has a semi-oval shape in plan view and is formed on the lower surface of the substrate 11A. The hole 28 has a trapezoidal shape in vertical section and is provided above the groove 27. The hole 28 and the groove 27 communicate with each other.

The introduction chamber 19 communicates with one end of the first region T of each of the supply passages 15A to 15C (the lower right end of the first region T in FIG. 8A) and heats through the opening 19B. It communicates with the space outside the convection generating chip 10A. The introduction chamber 19 communicates with the annular flow path 14A via an introduction passage 19a (see FIG. 8).

As shown in FIG. 10, the cover portion 25 is a substantially rectangular parallelepiped member, and is formed of a synthetic resin or the like. The cover portion 25 has a concave portion 25a, and the concave portion 25a communicates the space outside the thermal convection generating chip 10A with the introduction chamber 19.

The recess 25a includes a first inner wall surface 25b, a second inner wall surface 25c, and a boundary portion 25d. The first inner wall surface 25 b faces the opening of the introduction chamber 19. The second inner wall surface 25c intersects the first inner wall surface 25b and is inclined with respect to the first inner wall surface 25b. The angle formed by the first inner wall surface 25b and the second inner wall surface 25c is an obtuse angle (see FIG. 9). The boundary portion 25d is located between the first inner wall surface 25b and the second inner wall surface 25c.

The cover part 25 suppresses the liquid in the introduction chamber 19 from jumping out from the opening. Since the angle formed by the first inner wall surface 25b and the second inner wall surface 25c is an obtuse angle, the liquid adhering to the boundary portion 25d is difficult to stay. Therefore, the effect that it can suppress that the quantity of the liquid which flows in into 14 A of annular flow paths can be acquired can be acquired. Note that substantially the same effect can be obtained also when the boundary portion 25d is a curved surface having an arc cross section.

Referring to FIG. 11, the configuration of the guide passage 26 will be described in detail. FIG. 11 is a diagram schematically showing the guide passage 26. The guide passage 26 surrounds the entrance Sa of the second region S. That is, the guide passage 26 is formed by the arc-shaped inner peripheral wall surface of each of the receiving portions 16A to 16C, the substantially half-moon shaped bottom wall surface of the guide passage forming portion 25A, and the top wall surface of the bottom plate 13A (see FIG. 6). The guide passage 26 has a rectangular opening 29. The opening 29 faces the inlet Sa of the second region, and the area of the opening 29 is larger than the opening area of the inlet Sa.

The liquid in each of the receiving portions 16A to 16C flows into the inlet Sa while wetting the wall surface around the inlet Sa of each second region S. Since each inlet Sa is surrounded by three wall surfaces, the liquid easily flows into the inlet Sa.

Further, since the area of the opening 29 of the guide passage 26 is larger than the opening area of the inlet Sa, the inflow of liquid to the inlet 122ba due to capillary action is promoted compared to the case where the area of the opening 29 is equal to or smaller than the opening area of the inlet Sa. Therefore, the liquid easily flows into the inlet Sa.

Particularly, in the case where burrs are formed at the peripheral edge of each inlet Sa, it is effective from the viewpoint of introducing liquid into the inlet Sa if each of the receiving portions 16A to 16C is provided with a guide passage 26. That is, by providing the guide passage 26 in each of the receiving portions 16A to 16C, it is difficult for the liquid to flow into the inlet Sa even when there is a burr.

The thermal convection generating chip 10A includes a plurality of annular flow paths 14A and a plurality of first to third liquid supply paths 15A to 15C. The first to third liquid supply paths 15A to 15C include Liquid is supplied only to one annular channel 14A. Since each annular channel 14A is not in communication with the other annular channels 14A, by supplying the liquid individually to each of the plurality of annular channels 14A, a liquid containing a separate sample is individually introduced. (E.g. PCR reaction etc.)
Next, a method of using the thermal convection generating chip 10A will be described with reference to FIGS. First, the sample liquid is injected into the first receiving portion 16A of the first liquid supply path (see FIG. 8). The amount of the sample liquid injected into the first receiving portion 16A of the first liquid supply path is larger than the amount of the sample liquid filled in the first suction passage 17A of the first liquid supply path. It is not necessary to accurately weigh the amount.

The injected specimen fluid flows into the second region S of the first suction passage 17A and further into the first region T by capillary action. When the sample liquid reaches one end portion of the first region T located at the innermost position of the first suction passage 17A (the right lower end portion of the first region T in FIG. 8A), the liquid due to capillary action Flow stops. As a result, the sample liquid is filled over the entire length of the first suction passage 17A.

Similarly, the reaction reagent solution is injected into the second receiving portion 16B, and the second suction passage 17B is filled with the reaction reagent solution. Further, mineral oil is injected into the third receiving portion 16C, and the third suction passage 17C is filled with mineral oil.

Next, when the thermal convection generating chip 10A is mounted on the shaft 41 of the motor 40 of the thermal convection generating device 1 (see FIGS. 1 and 2) and the thermal convection generating chip 10A is rotated around the axis AX of the shaft 41. The centrifugal force is applied to the liquid in the suction passages 17A to 17C. As a result, in each of the supply paths 15A to 15C, the liquid in the first region T and the liquid in the second region S move away from each other, and the liquid in the first region T flows into the introduction chamber 19 Then, the liquid in the second region S returns to the receiving parts 16A to 16C. It is preferable to provide a structure (for example, a liquid absorbing member) so that the liquid returned to the receiving portions 16A to 16C does not scatter.

Of the liquid (sample liquid, reaction reagent solution, and mineral oil) that has flowed into the introduction chamber 19, the sample liquid and the reaction reagent solution flow into the annular flow path 14A via the introduction passage 19a, and the mineral oil passes through the introduction passage 19a. Stay in position. The sample liquid and the reaction reagent solution in the annular flow path 14A are heated by the second heater 32 and the first heater 31, thereby causing thermal convection and mixing the sample liquid and the reaction reagent solution. At that time, since the mineral oil blocks the introduction passage 19B, the evaporation of the liquid in the annular flow path 14A and the backflow to the introduction chamber 19 are suppressed.

<Positional relationship between thermal convection generating chip and heat source>
As described in the above method of using the thermal convection generating chip 10A, the first heater 31 and the second heater 32 are in thermal convection in the state where the assembly is completed by attaching the thermal convection generating chip 10A to the thermal convection generating device 1. Abutting on the lower surface of the generation chip 10 A, as shown in FIG. 8A, the arrangement relationship is the same as that of the annular flow path 14, the first heater 31, and the second heater 32 of the first embodiment. This arrangement relationship may be changed as shown in FIG. Also in the second embodiment, the operation and effect described in the first embodiment can be obtained by the above configuration.

Hereinafter, the operation and effect of the thermal convection generation system 200 of the second embodiment will be described.

(12) The liquid supply path 14A includes the receiving portions 16A to 16C that receive the liquid, communicates the receiving portions 16A to 16C with the annular flow path 14A, and supplies the liquid in the receiving portions 16A to 16C to the capillary tube. Each of the suction passages 17A to 17C for sucking according to a phenomenon, and each of the suction passages 17A to 17C includes a first region T positioned between the intermediate portion of the suction passages 17A to 17C and the annular flow path 14A, and A second region S located between the suction passages 17A to 17C and the receiving portions 16A to 16C;
By rotating the thermal convection generating chip 10A around its central axis, the liquid in the first region T is separated from the liquid in the second region S and supplied to the annular flow path 14. The user can supply a predetermined amount of liquid to the annular channel 14A without weighing the liquid introduced into the annular channel 14. Accordingly, it is possible to save the user from weighing the liquid. In this way, the liquid can be accurately weighed without labor and the heat convection generating device 200 of the second embodiment has the same heat source configuration as the heat convection generating device 100 of the first embodiment. Since the temperature control of the liquid flowing through the annular channel 14A can be performed efficiently as in the case, the heat convection with extremely high reproducibility and stability regardless of the operator's technique, and the heat convection PCR using this. Can be realized.

(13) Since each of the suction passages 17A to 17C is bent at an acute angle at the intermediate portion, it is easy to separate the liquid in the first region T, which is preferable from the viewpoint of weighing.

(14) Since the suction passages 17A to 17C further include air holes 18A to 18C for introducing air into the intermediate portions, the liquid near the intermediate portions is caused by the centrifugal force due to the rotation of the thermal convection generating chip 10A. When the two receiving portions 16A to 16C and the annular flow path 14A are moved in two directions, a suction pressure is generated in the vicinity of the intermediate portion, and air is introduced into the intermediate portion from the air holes 18A to 18C. Is separated from the liquid in the second region S.

(15) A plurality of liquid supply paths 15A to 15C are provided, and the plurality of liquid supply paths 15A to 15C supply different liquids (eg, reaction reagent solution, sample liquid) to the annular flow path for each liquid supply path. If it is a thing, thermal convection PCR of several types of liquid from which a component differs can be performed simultaneously.

[Third Embodiment]
Next, a thermal convection generation system 300 according to the third embodiment of the present invention will be described. The thermal convection generation system 300 according to the second embodiment includes at least the thermal convection generation chip (FIG. 8) 10 B and the thermal convection generation apparatus 1 according to the first embodiment. The thermal convection generating device 1 includes a shaft 41, a first heater 31, a second heater 32, a motor 40, and the like. Note that the configuration of the thermal convection generating device 1 itself is the same as that of the first embodiment, and thus the description thereof is omitted.

《Tip for generating heat convection》
FIG. 12 is a perspective view of a thermal convection generating chip 10B according to the second embodiment of the present invention, and FIG. 13 is an exploded perspective view of the thermal convection generating chip 10B. In the present embodiment, the same reference numerals are used for portions corresponding to those in the second embodiment, and the description overlapping with that in the first embodiment is omitted.

As shown in FIG. 12, the thermal convection generating chip 10B has a multilayer structure. That is, as shown in FIG. 13, the thermal convection generating chip 10B includes a first substrate 10Ba, a second substrate 10Bb, a third substrate 10Bc, and a lid 13B. The first substrate 10Ba, the second substrate 10Bb, the third substrate 10Bc, and the bottom plate 13B are stacked.

The structure of the supply paths 15Aa to 15Ca of the thermal convection generating chip 10B will be described with reference to FIG. FIG. 14 is a diagram schematically showing the structure of the supply paths 15Aa to 15Ca of the thermal convection generating chip 10B.

In FIG. 14, a light gray portion is formed on the first substrate 10Ba, a dark gray portion is formed on the second substrate 10Bb, and a colorless portion is formed on the third substrate 10Bc.

The thermal convection generating chip 10B has a plurality of annular channels 14B and a plurality of supply channels 15Aa to 15Ca. The first liquid supply path 15Aa includes a first receiving part 101A and a plurality of first suction paths 102A. The second liquid supply path 15Ba includes a second receiving part 101B and a plurality of second suction paths 102B. The third liquid supply path 15Ca includes a third receiving part 101C and a plurality of third suction paths 102C. Suction passages 102A to 102C are provided between each of the plurality of annular passages 14B and the receiving portions 101A to 101C of the supply passages 15Aa to 15Ca. The supply passages 15Aa to 15Ca intersect three-dimensionally and form a labyrinth-like fluid passage.

As shown in FIGS. 13 and 14, the first liquid supply path 15Aa is formed in the first substrate 10Ba. The first liquid supply path 15Ba is formed across the first substrate 10Ba and the second substrate 10Bb. The third liquid supply path 15Ca is formed across the first substrate 10Ba, the second substrate 10Bb, and the third substrate 10Bc. Each of the first to third suction passages 102A to 102C has a first region T ′ and a second region S ′ (see FIG. 14).

(First substrate)
FIG. 15 is a perspective view of the first substrate. As shown in FIG. 15, the first substrate 10Ba includes a first receiving portion 101A, a first suction passage 102A, air holes 103A, holes 104U and 105U, air introduction holes 103Bu and 103Cu, and a rectangular hole 106U. And have.

As shown in FIG. 18, a groove 109U forming a part of the first suction passage 102A is formed on the lower surface of the first substrate 10Ba, and the first suction passage 102A is formed by the groove 109U and the upper surface of the second substrate 10Bb. (See FIGS. 13 and 14).

(Second board)
FIG. 16 is a perspective view of the second substrate. The second substrate 10Bb has one hole 104M, a second suction passage 102B extending from the hole 104M, one hole 105M, an air hole 103Bm, an air hole 103Cm, and a rectangular hole 106M. . A second receiving portion 101B of the second liquid supply path 15Ba is formed by the hole 104M and the hole 104U of the first substrate (see FIG. 14). The air hole 103B of the second liquid supply path 15Ca is formed by the air introduction hole 103Bm and the air introduction hole 103Bu of the first substrate (see FIG. 14).

As shown in FIG. 18, a groove 109M forming a part of the second suction passage 102B is formed on the lower surface of the second substrate 10Bb, and the second suction passage 102C is formed by the groove 109M and the upper surface of the third substrate 15Bc. (See FIG. 14).

(Third substrate)
FIG. 17 is a perspective view of the third substrate. The third substrate 10Bc has one hole 105B, a third suction passage 102C, an air introduction hole 103Cb, a rectangular hole 106B, a groove 107, and an introduction passage 18.

The third receiving portion 101C of the third liquid supply path 15Ca is formed by the hole 105B, the hole 105M of the second substrate, and the hole 105U of the first substrate. An air hole 103C (see FIG. 14) of the third liquid supply path 15Ca is formed by the air introduction hole 103Cb, the air introduction hole 103Cm of the second substrate, and the air introduction hole 103Cu of the first substrate. A part of the introduction chamber 19 A is formed by the rectangular hole 106 B and the groove 107.

As shown in FIG. 18, a groove 109B forming a part of the third suction passage 102C is formed on the lower surface of the third substrate 10Bc, and the third suction passage 102C ( (See FIG. 14). Further, a groove 107 is formed on the lower surface of the third substrate 10Bc, and an introduction passage is constituted by the groove 107 and the upper surface of the lid body 13B (see FIG. 18).

The third substrate 10Bc is provided with a plurality of annular flow paths 14B at a predetermined angular interval around the center of the center hole 17B of the third substrate 10Bc (see FIG. 17). The center of the center hole 17B of the third substrate coincides with the axis AX of the shaft 41 of the motor 40 in a state where the thermal convection generating chip 10B is mounted on the thermal convection generating device 1.

Referring to FIG. 18, the detailed configuration of the introduction chamber 19B will be described. 18 is a view of the cross section of the thermal convection generating chip 10B taken along the line CC of FIG. The rectangular hole 106 B is trapezoidal in vertical section and is located above the groove 107. The rectangular hole 106B of the third substrate communicates with the groove 107 and the rectangular hole 106M of the second substrate, and the rectangular hole 106M communicates with the rectangular hole 106U of the first substrate. The rectangular hole 106U communicates with the outer space through the opening of the cover portion 25A.

Next, the cover portion 25A will be described with reference to FIG. FIG. 19 is a perspective view of the cover portion 25A. The cover portion 25A is a C-shaped band-like member in plan view, and has a plurality of concave portions 25a. The plurality of concave portions 25a are provided at predetermined intervals in the circumferential direction of the cover portion 25A.

《Positional relationship between heat convection chip and heat source》
In a state where the assembly is completed by attaching the thermal convection generation chip 10B to the thermal convection generation apparatus 1, the first heater 31 and the second heater 32 abut against the lower surface of the thermal convection generation chip 10B, as shown in FIG. In addition, the arrangement relationship is the same as that of the annular flow path 14 of the first embodiment and the first heater 31 and the second heater 32. This arrangement relationship may be changed as shown in FIG. Also in the third embodiment, the operation and effect described in the first embodiment can be obtained by the above configuration.

Next, a method of using the thermal convection generating chip 10B will be described with reference to FIG.

First, the sample liquid is injected into the first receiving portion 101A of the first liquid supply path 15Aa. The amount of the sample liquid injected into the first receiving portion 101A is larger than the amount of the sample liquid filled in the entire first suction passage 102A of the first liquid supply path, but the amount of the sample liquid needs to be accurately measured. There is no. The sample liquid injected into the first receiving portion 101A is filled into the entire first suction passage 102A by capillary action.

Similarly, the reaction reagent solution is injected into the second receiving portion 101B of the second liquid supply path 15Ba, and the reaction reagent solution is filled in the entire suction passage 102B. Further, mineral oil is injected into the third receiving part 101C of the third liquid supply path 15Ca to fill all of the suction path 102C by capillary action.

Next, the thermal convection generating chip 10B is mounted on the shaft 41 of the motor 40 of the thermal convection generating device 1 (see FIGS. 1 and 2), and the motor is driven to connect the thermal convection generating chip 10B to the axis AX of the shaft 41. When rotated around, centrifugal force is applied to the liquid in the suction passages 102A to 102C. As a result, in the suction passages 102A to 102C, the liquid in the first region T ′ and the liquid in the second region S ′ move away from each other, and the liquid in the first region T ′ moves to the introduction chamber 19B. The liquid flows in and the liquid in the second region S ′ returns to the receiving parts 101A to 101C.

Of the liquid (sample liquid, reaction reagent solution, and mineral oil) that has flowed into the introduction chamber 19B, the sample liquid and reaction reagent solution flow into the annular flow path 14B via the introduction passage 108 (see FIG. 17), and mineral oil. Stays at the position of the introduction passage 108. The sample liquid and the reaction reagent solution in the annular channel 14B are heated by the second heater 32 or the first heater 31 to cause thermal convection to mix the sample liquid and the reaction reagent solution. At that time, since the mineral oil blocks the introduction passage 108, the evaporation of the liquid in the annular flow path 14B and the backflow to the introduction chamber 19B are suppressed.

Hereinafter, the operation and effect of the thermal convection generation system 300 of the third embodiment will be described.

(16) The liquid supply path includes a first liquid supply path 15Aa for supplying the sample liquid to the annular flow path and a second liquid supply path 15Ba for supplying a reaction reagent solution for performing PCR to the annular flow path. ,
The volume of the first region T ′ of the first liquid supply channel 15Aa that supplies the sample liquid to the annular channel 14B and the first region of the second liquid supply channel 15Ba that supplies the reaction reagent solution to the annular channel 14B. Since the total volume of the solution of T ′ is equal to the volume of the annular channel 14B, each liquid of the PCR reaction solution can be simultaneously supplied to each of the annular channels 14B. As described above, the liquid is separated at the intermediate portion of the liquid supply path by centrifugation, and only a predetermined amount of liquid flows into the annular flow path, so that the user can save time for weighing each liquid.

As described above, according to the thermal convection generating chip 51, it is possible to simultaneously supply liquid to each of the plurality of thermal convection flow paths 11. Therefore, when performing the thermal convection PCR of a plurality of liquids of the same component at the same time, it is possible to reduce the time and effort for the user to weigh the liquids.

The specific embodiment of the present invention has been described above, but the present invention is not limited to this embodiment. Note that the drawings of the present embodiment schematically show each component mainly for easy understanding, and each illustrated component may be different from the actual for convenience of drawing. is there. The specific materials, shapes, and other configurations shown in the present embodiment are merely examples, and are not particularly limited, and various modifications are possible without departing from the effects of the present invention. It is.

For example, the number of annular channels of the thermal convection generating chip of the first to third embodiments is not limited and may be one. Further, the number of supply paths and substrates of the heat convection generating chips of the second and third embodiments is not limited, and may be one or four or more. By providing a plurality of supply paths for supplying the reaction reagent solution, the reagent components can be introduced separately.

In the third embodiment, the total capacity of the first region and the second region S of the first liquid supply path is equal to the annular flow path 14B. The volume of the convection flow path may be equal, or the volume of one first region communicating with the heat convection flow path may be equal to the volume of the heat convection flow path.

Further, according to the present invention, there is also provided a liquid weighing tool for weighing not only the liquid to be convected but also other liquids. 6 to 8, the liquid weighing instrument has a receiving portion 16A and a suction passage 17A that communicates with the receiving portion 16A and sucks the liquid in the receiving portion 16A by capillary action. Can be configured. As described above, by rotating the thermal convection generating chip 10A, the liquid in the first region T is separated from the liquid in the second region S and is discharged from the distal end portion of the suction passage 17A. Weighed into the annular channel 14A. In addition, various modifications can be made to the present embodiment without departing from the effects of the present invention.

Hereinafter, examples using the thermal convection generation system 100 of the first embodiment will be described.

[Example 1]
A temperature simulation was performed on the change of the liquid temperature by the thermal convection generation system 100 with the following contents.

<Dimensions of chip for generating heat convection>
Of the substrate 11 and the lid 13 constituting the thermal convection generating chip 10, the substrate 11 was set to a thickness of 2.0 mm and a diameter of 40 mm, and the lid 13 was set to a thickness of 0.19 mm and a diameter of 40 mm.

<Dimensions of annular channel>
The annular channel 14 was set to have a channel height (S): 0.3 mm, a channel width (W): 0.5 mm, and an annular channel diameter (outer diameter) (D): 6 mm (FIG. 2). (See (B)).

<Environmental temperature around the device>
The ambient temperature around the thermal convection generating device 1 was set to 10 ° C., and this temperature was maintained during thermal convection described later.

《Ratio of clearance》
The clearance CL between the first heater 31 and the second heater 32 was set to 0.5 mm. The area of the clearance CL (when viewed in plan: see FIG. 2A) is about 7% of the entire channel area of the annular channel 14.

<Determination of motor rotation speed>
20 and 21, the rotation speed of the shaft 41 of the motor 40 (the rotation speed of the thermal convection generating chip 10) is set to a voltage equivalent to 5G, that is, the shaft obtained when 4.0V is applied to the motor. The number of revolutions was determined (about 520 rpm). That is, it means that the thermal convection generating chip 10 is rotated at 520 rpm.

《Thermal convection treatment》
The temperature of the second heater 32 was set to 102 ° C., and the temperature of the first heater 31 was set to 60 ° C. When the motor 40 is driven to rotate the thermal convection generating chip 10 at a speed of 520 rpm and the liquid is subjected to centrifugal force, the liquid makes one round in the annular flow path 14 in about 8 seconds. As a result. FIG. 22A shows a temperature profile that changes when the liquid makes one round of the annular flow path in the annular flow path 14.

[Comparative Example 1]
In Comparative Example 1, a heat source as shown in FIG. 4 of Japanese Patent Laid-Open No. 2014-39498 (Patent Document 3) (the total area of the clearance between two heaters when viewed in plan as in FIG. 2A) The temperature simulation of the liquid moving in the annular channel was performed in the same manner as in Example 1 using about 38% of the channel area of the entire annular channel 14). The result is shown in FIG.

[Example 2]
In Example 2, the temperature simulation of the liquid moving in the annular flow path was performed in the same manner as in Example 1 except that the ambient temperature around the thermal convection generating device was changed to 25 ° C. The result is shown in FIG.

[Comparative Example 2]
In Comparative Example 2, the total area of the clearance between the two heaters when viewed in plan as shown in FIG. 2 (A) in FIG. 4 of JP 2014-39498 A (Patent Document 3) The temperature simulation of the liquid moving in the annular channel was performed in the same manner as in Example 2 using about 38% of the channel area of the entire annular channel 14). The result is shown in FIG.

[Example 3]
In Example 3, the temperature simulation of the liquid moving in the annular flow path was performed in the same manner as in Example 2 except that the ambient temperature around the thermal convection generating device was changed to 40 ° C. The result is shown in FIG.

[Comparative Example 3]
In Comparative Example 3, the heat source as shown in FIG. 4 of Japanese Patent Application Laid-Open No. 2014-39498 (Patent Document 3) (the total area of the clearance between the two heaters when viewed in plan as shown in FIG. 2A) The temperature simulation of the liquid moving in the annular channel was performed in the same manner as in Example 3 using about 38% of the channel area of the entire annular channel 14). The result is shown in FIG.

(Results and discussion)
The results of Comparative Examples 1 to 3 are shown in FIGS. 22 (B), (D), and (F). Here, the light gray band indicates that the liquid in the annular channel 14 passes through the channel area of the annular channel above the first heater 31. Further, the dark gray band portion means that the liquid in the annular flow path 14 passes through the flow path area of the annular flow path 14 above the second heater 32. Further, the white band means that the liquid in the annular channel 14 passes through the channel area of the annular channel 14 above the air where no heater or the like exists.

In Comparative Examples 1 to 3 (see FIGS. 22B, 22D and 22F), the first heater 31 and the second heater 32 are shown in FIG. 4 of Japanese Patent Laid-Open No. 2014-39498 (Patent Document 3). It arrange | positions as shown and the ratio of the clearance gap between 1st, 2nd heaters is set to 15% or more of annular flow paths. That is, as can be seen from the length of the horizontal axis of the white belt portion from FIGS. 22B, 22D and 22E, air cooling is performed in a flow path area corresponding to at least 2 radians. Since the annular flow path 14 is a perfect circle, the ratio of the portion where the liquid is air-cooled in the annular flow path 14 is calculated to be 31.8% or more with respect to the entire flow path area of the annular flow path 14. From the plot of the air-cooled liquid temperature, it is shown that the temperature of the liquid gradually decreases in the flow path area (see the mountain-shaped black line).

Further, as can be seen from a comparison between FIG. 22B (Comparative Example 1) and (F) (Comparative Example 3), when the outside air temperature is 10 ° C. and 40 ° C., the outside air temperature is naturally 10. The rate at which the temperature of the liquid is lowered is higher at 0 ° C., that is, the comparative example is easily affected by external factors, and it can be said that there is a problem in the reproducibility and robustness of thermal convection.

On the other hand, in Examples 1 to 3 (FIGS. 22A, 22C, and 22E), the first heater 31 and the second heater 32 are arranged as shown in FIG. There is no gap between the first heater and the second heater for air-cooling the annular flow path (a minimum necessary clearance is provided so that the temperature of the first heater and the second heater can be maintained). The clearance is set to about 7% with respect to the entire channel area of the annular channel (see FIG. 2A)).

Therefore, it is cooled at a speed faster than air cooling in the flow path area above the first heater, forms a steep gradient, and near the temperature (60 ° C.) of the first heater in a short time compared with Comparative Examples 1 to 3. You can see that it has reached. That is, it can be seen that the temperature of the liquid is adjusted efficiently and quickly in the example as compared with the comparative example. In addition, there is no difference in the cooling profile of the liquid in the annular channel between the case where the outside air temperature is 40 ° C. (FIG. 22E) and 10 ° C. (FIG. 22A), and the thermal convection is reproduced more than in the comparative example. It can be said that it is excellent in performance and robustness.

The present invention has been described through the first to third embodiments and examples. However, the present invention is not limited to these, and design changes are allowed without departing from the spirit of the invention described in the claims. The

DESCRIPTION OF SYMBOLS 1 ... Thermal convection production |

generation apparatus

10, 10A, 10B ... Chip 10Ba for thermal convection production | generation ... 1st board | substrate 10Bb ... 2nd board | substrate 10Bc ... 3rd board |

substrate

11, 11A ... board |

substrate

13 , 13A, 13B ...

lids

14, 14A, 14B ... annular channel 15 ... liquid supply channel 15a ... extension 15b ... liquid reservoir 15c ... communication unit 15A, 15Aa First liquid supply path 15B, 15Ba ... Second liquid supply path 15C, 15Ca ... Third liquid supply path 15D ... Liquid supply hole 16 ... Gas discharge path 16a ... Extender 16b ... Gas reservoir 16c ...

Communication parts

16A, 101A ... First receiving

part

16B, 101B ... Second receiving part 16C, 101C ... Third receiving part 17 ...

Center hole

17A, 102A:

first suction passages

17B, 102B,

Second suction passages

17C, 102C ...

third suction passages

18A, 18B, 18C, 103A ... air holes 19 ... introduction chamber 19a ... introduction passage 19A ... introduction chamber 20 ... stage 21 ... Heater mounting hole 22 ... first screw insertion hole 23 ... second screw insertion hole 24 ... center hole 25a ... concave portion 25b ... first inner wall surface 25c ... second Inner wall surface 25d ...

boundary portions

25, 25A ... cover portion 26 ... guide passage 26A ... guide passage forming portion 27 ... groove 28 ... hole 29 ... opening 30 ... heat source 31 ... 1st heater 31a ... Connection part 31b ... Heat source part 31c ... Hole 32 ... 2nd heater 32a ... Connection part 32b ... Heat source part 32c ... Through-hole 32a ... Ring-shaped connecting part 40 ... Motor 41 ... Shaft 50 ... Control means 51... Arithmetic control unit 52 .. display unit 53 .. input unit 60 .. heat sink 81 .. heat convection generator 90 .. optical detection system 91. ..Fluorescence detector 93... Detection light source 94... Detection light detector 95 .. detected portion 96 .. arithmetic control unit 97 .. control means 103 Bu, 103 Bm, 103 Cu, 103 Cm, 103 Cb .. Air conduction holes 104U, 104M ... holes 105U, 105M, 105B ...

holes

106U, 106B, 106M ... rectangular holes 107 ... grooves 108 ...

introduction passages

109U, 109M, 109B ... · Groove 110 ···

Hole

100, 200, 300 · · · Thermal convection generation system AX · · · axis L1 · · · light L2 · · · detection light P1 · · · detection point Sa · · · entrance S, S ' ... Second area T, T '··· first region

Claims

A rotating shaft capable of rotatably fixing a chip for generating heat convection having an annular flow path for circulating a liquid;
A first temperature adjustment unit having a first heat source unit for heating or cooling the liquid in the annular channel;
A second temperature adjustment unit having a second heat source unit for heating or cooling the liquid in the annular channel;
Rotation driving means for rotating the entire rotary channel around the axis of the rotary shaft by rotating the rotary shaft, and a direction (annular flow) perpendicular to the direction in which the liquid flows through the annular channel. When the annular flow path is viewed in plan on a plane that coincides with a circle presented by the road), the center of gravity of the annular flow path (center of the circular flow path) on the plane and the intersection of the axis and the plane A thermal convection generating device that does not coincide with the rotational center of the rotating shaft,
With respect to a straight line connecting the rotation center and the center of gravity (center of the annular channel), the center of gravity (center of the annular channel) is 30 ° to 150 ° or 210 ° to 330 °. In any one of the ranges, the at least one second heat source part is positioned and the flow passage area of the annular flow passage is heated or cooled in the range,
The first heat source unit heats or cools a channel area other than the channel area of the annular channel heated or cooled by the second heat source unit.
Having a clearance between the first heat source part and the second heat source part,
The thermal convection generating device according to claim 1, wherein an area of the clearance in the plan view is in a range of 0.1 to 15% with respect to an area of the entire flow path area.
The first heat source part of the first temperature control part and the second heat source part of the second temperature control part have a flat plate part,
3. The thermal convection generating device according to claim 2, wherein each heat source part is formed and arranged so that the clearance part surrounds the second heat source part in the state in plan view.
The flow path area of the annular flow path that overlaps the first heat source section of the first temperature control section is overlapped with the flow area of the annular flow path that overlaps the second heat source section of the second temperature control section when viewed in plan. The thermal convection generating device according to any one of claims 1 to 3, wherein the area is smaller than the area.
The thermal convection generating device according to any one of claims 1 to 4,
A chip for generating heat convection having an annular flow path for circulating a liquid,
The thermal convection generating chip has a liquid supply path communicating with the annular flow path, and the liquid in the liquid supply path is supplied to the annular flow path by centrifugal force applied to the thermal convection generating chip by the rotational drive. A thermal convection generation system.
The thermal convection generation system according to claim 5, wherein a gas discharge path communicating with the annular flow path is formed.
The thermal convection generation system according to claim 5 or 6, wherein a plurality of the annular flow paths are arranged so as to be symmetric with respect to the axis of the rotation axis.
The thermal convection generation system according to any one of claims 5 to 7, wherein a surface roughness Ra of the wall surface of the annular channel is 100 nm or less.
The thermal convection generation system according to any one of claims 5 to 8, wherein the annular flow path has a perfect circle shape when seen in a plan view.
The thermal convection generation system according to any one of claims 5 to 9, wherein a material of a wall surface of the annular flow path is any one of cyclic olefin, polypropylene, and polycarbonate.
An excitation light source that irradiates the liquid in the annular channel with excitation light that excites the fluorescent dye contained in the liquid in the annular channel;
A fluorescence detector that detects fluorescence emitted by the fluorescent dye by irradiating the fluorescent dye with the excitation light;
An arithmetic control unit that calculates the replication amount of the nucleic acid based on the fluorescence detected by the fluorescence detector;
The thermal convection generating system according to any one of claims 5 to 10, comprising:
The liquid supply path is
A receiving part for receiving the liquid;
A suction passage for communicating the receiving part and the annular flow path, and sucking the liquid in the receiving part by capillary action;
Have
The suction passage has a first region located between an intermediate portion of the suction passage and the annular flow path, and a second region located between the suction passage and the receiving portion,
The thermal convection according to any one of claims 5 to 11, wherein the liquid in the first region is separated from the liquid in the second region and supplied to the annular flow path by the rotation. Generation system.
The heat convection generating system according to claim 12, wherein the suction passage is bent at an acute angle at the intermediate portion.
The heat convection generating system according to claim 11 or 13, wherein the suction passage further has an air hole for introducing air into the intermediate portion.
A plurality of the liquid supply paths are provided,
The thermal convection generating system according to any one of claims 5 to 14, wherein the plurality of liquid supply paths supply different liquids to the annular flow path for each liquid supply path.
The plurality of liquid supply paths include a liquid supply path for supplying a sample liquid to the annular flow path, and a liquid supply path for supplying a reaction reagent solution for performing PCR to the annular flow path,
The volume of the first region of the liquid supply path for supplying the sample liquid to the annular channel and the volume of the first region of the liquid supply channel for supplying the reaction reagent solution to the annular channel. The thermal convection generation system of claim 15, wherein is equal to the volume of the annular flow path.
A convection PCR method using the thermal convection generating device or the thermal convection generating system according to any one of claims 1 to 16,
A liquid introduction step for introducing a sample solution, a reaction reagent solution, and other liquids necessary for a PCR reaction into a circular channel individually or integrally into a PCR reaction solution;
A PCR reaction step of performing convection PCR by refluxing the PCR reaction solution at a predetermined speed in a circular channel,
A convection PCR method in which the temperature difference between the heat source part of the first temperature control means and the heat source part of the second temperature control means is maintained at 10 ° C. or higher during the PCR reaction.