JP2014135942A - Thermal cycler and thermal cycling method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a thermal cycler and a thermal cycling method that enable rapid thermal cycle.SOLUTION: A thermal cycler is provided, comprising: a loading part for loading a biochip 100 filled with a reaction liquid 140 mixed with a solid substance 9 having a greater specific gravity than the reaction liquid 140 and with a liquid 130 having a smaller specific gravity than the reaction liquid 140 and separable form the reaction liquid 140, the biochip 100 including a flow pass 110 in which the reaction liquid 140 travels; a heating part for heating a first region of the flow pass 110 when the biochip 100 is loaded in the loading part; and a driving mechanism for converting the disposition of the loading part and the heating part between a first disposition and a second disposition.

Description

  The present invention relates to a heat cycle apparatus and a heat cycle method.

  In recent years, gene-based medical care such as gene diagnosis and gene therapy has attracted attention due to the development of gene utilization technology, and many methods using genes for variety discrimination and variety improvement have been developed in the field of agriculture and livestock. . Techniques such as PCR (Polymerase Chain Reaction) are widely used as techniques for utilizing genes. Today, PCR has become an indispensable technique for elucidating information on biological materials.

  The PCR method is a technique for amplifying a target nucleic acid by subjecting a solution (reaction solution) containing a nucleic acid (target nucleic acid) to be amplified and a reagent to thermal cycling. The thermal cycle is a process in which two or more stages of temperature are periodically applied to the reaction solution. In the PCR method, a method of performing a two-stage or three-stage thermal cycle is common.

  In the PCR method, a container for performing a biochemical reaction, generally called a tube or a biological sample reaction chip (biochip), is used. However, the conventional methods have a problem that the amount of reagents and the like necessary for the reaction is large, the apparatus is complicated to realize a thermal cycle necessary for the reaction, and the reaction takes time. Therefore, a biochip and a reaction apparatus are required for performing the PCR method accurately and in a short time using a very small amount of reagent or specimen.

  In order to solve such problems, in the vertical PCR, the upper and lower parts of the rod-shaped reaction tube are heated by separate heaters, the reaction tube is filled with oil, and the PCR reaction droplets therein Apply two temperature cycles to move. Gravity is used as a means for moving the PCR droplet, and by utilizing the fact that the PCR droplet has a higher specific gravity than oil, the entire heater block in which the tube is installed is rotated halfway up and down, A biological sample reaction apparatus that performs thermal cycling by moving droplets by gravity has been disclosed (for example, see Patent Document 1).

JP 2012-115208 A

  In the biological sample reaction apparatus disclosed in Patent Document 1, the reaction solution is subjected to a thermal cycle by continuously rotating the biochip. However, since the reaction liquid moves in the biochip flow path as it rotates, it is necessary to speed up the movement of the reaction liquid in the biochip flow path in order to perform the PCR method accurately and in a short time. there were.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a thermal cycle apparatus and a thermal cycle method in which the heating time can be easily controlled.

  [Application Example 1] The thermal cycler according to this application example is a reaction liquid in which a solid substance having a specific gravity larger than that of the reaction liquid is mixed with the reaction liquid having a specific gravity smaller than that of the reaction liquid and phase-separating from the reaction liquid. A mounting portion for mounting a biochip that includes a flow path that is filled with a liquid and that moves close to the inner wall facing the reaction solution; and when the biochip is mounted on the mounting portion, A heating unit that heats one region, and a drive mechanism that switches the arrangement of the mounting unit and the heating unit between a first arrangement and a second arrangement.

  According to this application example, the reaction liquid is injected into the liquid in the biochip in order to perform the heating / cooling cycle by rotating the biochip. At this time, the reaction liquid in which a solid substance having a specific gravity larger than that of the reaction liquid is mixed. Form. Compared to the case where the same volume of reaction liquid is injected into the biochip in a single state, the reaction liquid moving speed (falling speed) is increased by mixing a solid substance with a specific gravity greater than the reaction liquid into the reaction liquid. Therefore, the moving speed becomes faster. This makes it possible to shorten the time required for raising or lowering the reaction liquid from high temperature to low temperature, or from low temperature to high temperature at each of the two levels, and to enable high-speed thermal cycling. I will provide a.

  Application Example 2 In the heat cycle apparatus according to this application example, the solid substance is fine particles.

  According to this application example, when the solid substance is made into fine particles, the movement of the reaction liquid becomes faster and the movement of the reaction liquid becomes smooth.

  Application Example 3 In the thermal cycle apparatus according to this application example, the surface of the solid substance is hydrophilic.

  According to this application example, the force to move the reaction liquid in the direction of gravity without the fine particles mixed in the reaction liquid in the liquid overcoming the surface tension of the reaction liquid and jumping out of the reaction liquid into the hydrophobic liquid. (Traction force) works.

  Application Example 4 In the heat cycle apparatus according to this application example, the solid substance is adsorbed with a nucleic acid to be detected.

  According to this application example, nucleic acid can be adsorbed on the surface of the solid material by nucleic acid extraction using the solid material from the sample, and the solid material after washing the impurities can be directly mixed into the reaction solution to perform the nucleic acid amplification reaction.

  [Application Example 5] In the thermal cycle method described in this application example, a solid substance having a specific gravity larger than that of the reaction liquid is mixed in the reaction liquid, the reaction liquid in which the solid substance is mixed, and the specific gravity is higher than that of the reaction liquid. Mounting a biochip including a channel that is small and filled with the liquid that is phase-separated from the reaction liquid, and the reaction liquid moves close to the opposing inner wall; and Holding the biochip in a first arrangement in which the first region of the path is located at the lowermost part of the flow path in the direction of gravity, heating the first region, Holding the biochip in a second arrangement in which the second region of the flow path, whose position in the moving direction is different from that of the first region, is located at the lowest part of the flow channel in the direction in which gravity acts; , Including.

  According to this application example, the reaction liquid is injected into the liquid in the biochip in order to perform the heating / cooling cycle by rotating the biochip. At this time, the reaction liquid in which a solid substance having a specific gravity larger than that of the reaction liquid is mixed. Form. Compared with injecting the same volume of reaction liquid into the biochip in a single state, the reaction liquid moving speed (falling speed) by gravity is increased by mixing the reaction liquid with fine particles having a specific gravity greater than that of the reaction liquid. Therefore, the moving speed becomes faster. This makes it possible to reduce the time required to raise or lower the reaction liquid from high temperature to low temperature, or from low temperature to high temperature at each of the two levels, and to enable high-speed thermal cycling. I will provide a.

The perspective view of the heat cycle apparatus which concerns on embodiment. (A) shows a state where the lid is closed, and (B) shows a state where the lid is opened. The disassembled perspective view of the main body in the heat cycle apparatus which concerns on embodiment. Sectional drawing which shows typically the cross section in the AA of FIG. 1 (A) of the main body in the thermal cycle apparatus which concerns on embodiment. (A) shows the first arrangement, and (B) shows the second arrangement. Sectional drawing of the biochip which concerns on embodiment. The figure which shows the method of measuring the time for a reaction liquid to move from an upper end to a lower end when the biochip which concerns on a present Example is half rotated. The graph which shows the result of having measured the time for a reaction liquid to move from an upper end to a lower end when the biochip which concerns on a present Example is half rotated. The table | surface which showed the conditions which implement PCR reaction in the state which mixed the microparticles | fine-particles with the reaction liquid which concerns on a present Example. The flowchart showing the procedure of the heat cycle process using the heat cycle apparatus which concerns on embodiment. The perspective view of the heat cycle apparatus which concerns on a modification. (A) shows a state where the lid is closed, and (B) shows a state where the lid is opened. Sectional drawing of the biochip which concerns on a modification. Sectional drawing which shows typically the cross section in the BB line of FIG. 9 (A) of the main body in the heat cycle apparatus which concerns on a modification.

DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments of the invention will be described with reference to the drawings in the following order. The embodiments described below do not unduly limit the contents of the present invention described in the claims. Also, not all of the configurations described below are essential constituent requirements of the present invention.
1. Embodiment 1-1. Configuration of Thermal Cycle Device in Embodiment 1-2. Thermal cycle process using thermal cycle apparatus in embodiment 1-3. 1. Effect of thermal cycle apparatus and thermal cycle process in the embodiment Modified example

1. Embodiment 1-1. Configuration of Thermal Cycle Device in Embodiment FIG. 1 is a perspective view of a thermal cycle device 1 according to the embodiment. (A) is the state which closed the lid | cover 50 of the thermal cycle apparatus 1, (B) is the state which opened the lid | cover 50 of the thermal cycle apparatus 1, and represents the state by which the biochip 100 was mounted | worn with the mounting part 11. FIG. FIG. 2 is an exploded perspective view of the main body 10 in the heat cycle apparatus 1 according to the embodiment. FIG. 3 is a cross-sectional view schematically showing a cross section taken along line AA of FIG. 1A of the main body 10 in the heat cycle apparatus 1 according to the embodiment. (A) shows the first arrangement, and (B) shows the second arrangement.

  The heat cycle apparatus 1 according to the embodiment includes a main body 10 and a drive mechanism 20 as shown in FIG. As shown in FIG. 2, the main body 10 includes a mounting part 11, a first heating part 12 (corresponding to a heating part), and a second heating part 13. A spacer 14 is provided between the first heating unit 12 and the second heating unit 13. In the main body 10 of the present embodiment, the first heating unit 12 is disposed on the bottom plate 17 side, and the second heating unit 13 is disposed on the lid 50 side. In the main body 10 of the present embodiment, the first heating unit 12, the second heating unit 13, and the spacer 14 are fixed to the flange 16, the bottom plate 17, and the fixing plate 19.

  The mounting part 11 has a structure for mounting a biochip 100 described later. As shown in FIGS. 1B and 2, the mounting unit 11 of this embodiment has a slot structure in which the biochip 100 is inserted and mounted, and the first heat block 12 b of the first heating unit 12 (heating unit). The biochip 100 is inserted into a hole penetrating the spacer 14 and the second heat block 13 b of the second heating unit 13. There may be a plurality of mounting portions 11, and in the example of FIG. 1B, 20 mounting portions 11 are provided in the main body 10.

  The thermal cycle device 1 of the present embodiment preferably includes a structure that holds the biochip 100 in a predetermined position with respect to the first heating unit 12 and the second heating unit 13. Accordingly, a predetermined region of the biochip 100 can be heated by the first heating unit 12 and the second heating unit 13. More specifically, as shown in FIG. 3, the first region 111 of the flow path 110 constituting the biochip 100 described later is formed by the first heating unit 12, and the second region 112 is formed by the second heating unit 13. Can be heated. In the present embodiment, the structure that determines the position of the biochip 100 is the bottom plate 17. As shown in FIG. 3A, the first heating unit 12 and the biochip 100 are inserted to a position where the biochip 100 contacts the bottom plate 17. The biochip 100 can be held at a predetermined position with respect to the second heating unit 13.

  The first heating unit 12 heats a first region 111 of the biochip 100 described later to a first temperature when the biochip 100 is mounted on the mounting unit 11. In the example illustrated in FIG. 3A, the first heating unit 12 is disposed in the main body 10 at a position where the first region 111 of the biochip 100 is heated.

  The first heating unit 12 may include a mechanism that generates heat and a member that transmits the generated heat to the biochip 100. In the example shown in FIG. 2, the first heating unit 12 includes a first heater 12a and a first heat block 12b. In the present embodiment, the first heater 12 a is a cartridge heater, and is connected to an external power source (not shown) by a conducting wire 15. The first heater 12a is inserted into the first heat block 12b, and the first heat block 12b is heated when the first heater 12a generates heat. The first heat block 12b is a member that transfers heat generated from the first heater 12a to the biochip 100. In this embodiment, it is an aluminum block.

  Since the temperature control of the cartridge heater is easy, the temperature of the first heating unit 12 can be easily stabilized by using the first heater 12a as a cartridge heater. Therefore, a more accurate thermal cycle can be realized. Since aluminum has high thermal conductivity, the biochip 100 can be efficiently heated by making the first heat block 12b aluminum. Moreover, since the heating unevenness hardly occurs in the first heat block 12b, a highly accurate thermal cycle can be realized. Further, since the processing is easy, the first heat block 12b can be accurately molded, and the heating accuracy can be improved. Therefore, a more accurate thermal cycle can be realized.

  The first heating unit 12 is preferably in contact with the biochip 100 when the biochip 100 is mounted on the mounting unit 11. Thereby, when the biochip 100 is heated by the first heating unit 12, the heat of the first heating unit 12 can be stably transmitted to the biochip 100, so that the temperature of the biochip 100 can be stabilized. . When the mounting unit 11 is formed as a part of the first heating unit 12 as in the present embodiment, the mounting unit 11 is preferably in contact with the biochip 100. Thereby, since the heat of the 1st heating part 12 can be stably transmitted to the biochip 100, the biochip 100 can be heated efficiently.

  When the biochip 100 is mounted on the mounting unit 11, the second heating unit 13 heats the second region 112 of the biochip 100 to a second temperature different from the first temperature. In the example shown in FIG. 3A, the second heating unit 13 is disposed in the main body 10 at a position where the second region 112 of the biochip 100 is heated. As shown in FIG. 2, the second heating unit 13 includes a second heater 13a and a second heat block 13b. The second heating unit 13 is the same as the first heating unit 12 except that the region of the biochip 100 to be heated and the temperature to be heated are different from those of the first heating unit 12.

  In this embodiment, the temperature of the 1st heating part 12 and the 2nd heating part 13 is controlled by the temperature sensor which is not shown in figure and the control part mentioned later. The temperatures of the first heating unit 12 and the second heating unit 13 are preferably set so that the biochip 100 is heated to a desired temperature. In this embodiment, the first region 111 of the biochip 100 is set to the first temperature by controlling the first heating unit 12 to the first temperature and the second heating unit 13 to the second temperature. The two regions 112 can be heated to a second temperature. The temperature sensor in this embodiment is a thermocouple.

  The drive mechanism 20 is a mechanism that drives the mounting unit 11, the first heating unit 12, and the second heating unit 13. In the present embodiment, the drive mechanism 20 includes a motor and a drive shaft (not shown), and the drive shaft and the flange 16 of the main body 10 are connected. The drive shaft in the present embodiment is provided perpendicular to the longitudinal direction of the mounting portion 11, and when the motor is operated, the main body 10 is rotated using the drive shaft as a rotation axis.

  The thermal cycle apparatus 1 of the present embodiment includes a control unit (not shown). The control unit controls at least one of a first temperature, a second temperature, a first time, a second time, and a thermal cycle number, which will be described later. When the control unit controls the first time or the second time, the control unit controls the operation of the drive mechanism 20 so that the mounting unit 11, the first heating unit 12, and the second heating unit 13 are controlled. Control the time held in a given arrangement. The control unit may provide a different mechanism for each item to be controlled, or may control all items at once.

  The control part in the heat cycle apparatus 1 of this embodiment is electronic control, and controls all the above items. The control unit of the present embodiment includes a processor such as a CPU (not shown) and a storage device such as a ROM (Read Only Memory) and a RAM (Random Access Memory). The storage device stores various programs and data for controlling the above items. The storage device also has a work area for temporarily storing in-process data and processing results of various processes.

  As shown in the example of FIGS. 2 and 3A, the main body 10 of the present embodiment is provided with a spacer 14 between the first heating unit 12 and the second heating unit 13. The spacer 14 of the present embodiment is a member that holds the first heating unit 12 or the second heating unit 13. By providing the spacer 14, the distance between the first heating unit 12 and the second heating unit 13 can be determined more accurately. That is, the position of the 1st heating part 12 and the 2nd heating part 13 with respect to the 1st field 111 and the 2nd field 112 of biochip 100 mentioned below can be defined more correctly.

  The material of the spacer 14 can be appropriately selected as necessary, but is preferably a heat insulating material. Thereby, since the influence which the heat of the 1st heating part 12 and the 2nd heating part 13 mutually has can be decreased, temperature control of the 1st heating part 12 and the 2nd heating part 13 becomes easy. In the case where the spacer 14 is a heat insulating material, when the biochip 100 is mounted on the mounting portion 11, the spacer 14 surrounds the biochip 100 in a region between the first heating unit 12 and the second heating unit 13. Is preferably arranged. Thereby, since the heat radiation from the area | region between the 1st heating part 12 of the biochip 100 and the 2nd heating part 13 can be suppressed, the temperature of the biochip 100 becomes more stable. In the present embodiment, the spacer 14 is a heat insulating material, and the mounting portion 11 penetrates the spacer 14 in the example of FIG. Accordingly, when the biochip 100 is heated by the first heating unit 12 and the second heating unit 13, the heat of the biochip 100 is difficult to escape, so that the temperatures of the first region 111 and the second region 112 are further stabilized. be able to.

  The main body 10 of the present embodiment includes a fixing plate 19. The fixed plate 19 is a member that holds the mounting unit 11, the first heating unit 12, and the second heating unit 13. In the example shown in FIGS. 1B and 2, two fixing plates 19 are fitted to the flange 16, and the first heating unit 12, the second heating unit 13, and the bottom plate 17 are fixed. Since the structure of the main body 10 is further strengthened by the fixing plate 19, the main body 10 is hardly damaged.

  The thermal cycle device 1 according to the present embodiment includes a lid 50. In the example of FIGS. 1A and 3A, the mounting portion 11 is covered with a lid 50. By covering the mounting portion 11 with the lid 50, when the first heating unit 12 is heated, heat radiation from the main body 10 to the outside can be suppressed, so that the temperature inside the main body 10 can be stabilized. The lid 50 may be fixed to the main body 10 by the fixing portion 51. In the present embodiment, the fixing part 51 is a magnet. As shown in the example of FIG. 1B and FIG. 2, a magnet is provided on the surface of the main body 10 that contacts the lid 50. Although not shown in FIG. 1B and FIG. 2, the lid 50 is also provided with a magnet at a position where the magnet of the main body 10 comes into contact. 50 is fixed to the main body 10. Thereby, when the main body 10 is driven by the drive mechanism 20, the lid 50 can be prevented from being removed or moved. Accordingly, it is possible to prevent the temperature in the heat cycle apparatus 1 from changing due to the removal of the lid 50, so that a more accurate heat cycle is applied to the reaction liquid 140 in which the solid substance 9 having a higher specific gravity than the reaction liquid 140 described later is mixed. be able to.

  The main body 10 preferably has a highly airtight structure. If the main body 10 has a highly airtight structure, the air inside the main body 10 is difficult to escape to the outside of the main body 10, so that the temperature inside the main body 10 becomes more stable. In the present embodiment, as shown in FIG. 2, the space inside the main body 10 is sealed by the two flanges 16, the bottom plate 17, the two fixing plates 19, and the lid 50.

  The fixing plate 19, the bottom plate 17, the lid 50, and the flange 16 are preferably formed using a heat insulating material. Thereby, since the heat radiation from the main body 10 to the outside can be further suppressed, the temperature in the main body 10 can be further stabilized.

1-2. FIG. 4 is a sectional view of a biochip 100 according to the embodiment. Below, the biochip 100 which concerns on embodiment is demonstrated first.

  As shown in the example of FIG. 4, the biochip 100 according to the present embodiment includes a flow path 110 and a sealing unit 120. In the channel 110, the reaction liquid 140 in which the solid substance 9 having a specific gravity larger than that of the reaction liquid 140 is mixed, and the liquid having a specific gravity smaller than that of the reaction liquid 140 and phase-separated from the reaction liquid 140 (hereinafter “liquid”). 130) and is sealed by the sealing portion 120.

  The solid substance 9 may be fine particles. According to this, by making the solid substance 9 into fine particles, the movement of the reaction liquid 140 becomes faster and the movement of the reaction liquid 140 becomes smooth. Moreover, the solid substance 9 will not be specifically limited if it is a substance which does not inhibit PCR or RT-PCR reaction. Further, the shape, particle size, etc. are not particularly limited. Moreover, the one where the density of the solid substance 9 is larger is more preferable by increasing the reaction liquid moving speed.

  The surface of the fine particles (solid substance) 9 may be hydrophilic. According to this, the fine particles 9 mixed in the reaction liquid 140 in the liquid 130 overcome the surface tension of the reaction liquid 140 and do not jump out of the reaction liquid 140 into the hydrophobic liquid 130, so that the reaction liquid 140 is moved in the gravity direction. The force to move (traction force) works.

  The fine particles 9 may be adsorbed with a nucleic acid to be detected. According to this, nucleic acid is adsorbed on the surface of the fine particle 9 by nucleic acid extraction using the fine particle 9 from the sample, and the fine particle 9 having been washed with impurities is directly mixed in the reaction solution 140, whereby the nucleic acid amplification reaction can be performed.

  The flow path 110 is formed so that the reaction liquid 140 mixed with the fine particles 9 having a specific gravity larger than that of the reaction liquid 140 moves near the opposing inner walls. Here, the “opposite inner walls” of the flow channel 110 mean two regions of the wall surface of the flow channel 110 that are in a positional relationship facing each other. “Proximity” means that the distance between the reaction solution 140 and the wall surface of the channel 110 is short, and includes the case where the reaction solution 140 contacts the wall surface of the channel 110. Therefore, “the reaction solution 140 moves close to the opposing inner wall” means “the reaction solution 140 is in a state where the distance is close to both of the two regions on the wall surface of the flow channel 110 that are in a facing positional relationship”. Means that the reaction liquid 140 moves along the opposing inner walls. In other words, the distance between the two opposing inner walls of the flow path 110 is such a distance that the reaction solution 140 moves close to the inner wall.

  When the flow path 110 of the biochip 100 has such a shape, the direction in which the reaction liquid 140 moves in the flow path 110 can be regulated. Therefore, a first area 111 and a first area 111 of the flow path 110 described later The path through which the reaction solution 140 moves between different second regions 112 can be defined to some extent. Thereby, the time required for the reaction solution 140 to move between the first region 111 and the second region 112 can be limited to a certain range. Therefore, the degree of “proximity” is such that fluctuations in the time during which the reaction solution 140 moves between the first region 111 and the second region 112 do not affect the heating time of the reaction solution 140 in both regions, That is, it is preferable that it is a grade which does not affect the result of reaction. More specifically, it is desirable that the distance in the direction perpendicular to the direction in which the reaction solution 140 moves between the inner walls facing each other is such that two or more droplets of the reaction solution 140 do not enter.

  In the example of FIG. 4, the outer shape of the biochip 100 is a columnar shape, and the flow path 110 is formed in the central axis direction (vertical direction in FIG. 4). The shape of the flow path 110 is a cross section perpendicular to (crossing) the longitudinal direction of the flow path 110, that is, a cross section perpendicular to the direction in which the reaction liquid 140 moves in a region of the flow path 110 ( The “cross section” of the flow path 110 is a circular cylinder. Therefore, in the biochip 100 of the present embodiment, the opposed inner wall of the flow channel 110 is a region including two points on the wall surface of the flow channel 110 that constitutes the diameter of the cross section of the flow channel 110, and The reaction solution 140 moves along the longitudinal direction of the flow path 110 along the path.

  The first region 111 of the biochip 100 is a partial region of the flow path 110 that is heated to the first temperature by the first heating unit 12. The second region 112 is a partial region of the flow path 110 that is heated to the second temperature by the second heating unit 13 and is different from the first region 111. In the biochip 100 of the present embodiment, the first region 111 is a region including one end in the longitudinal direction of the flow path 110, and the second region 112 is the other end in the longitudinal direction of the flow path 110. It is an area including In the example illustrated in FIGS. 3A and 3B, the region surrounded by the dotted line including the end portion on the sealing portion 120 side of the flow path 110 is the second region 112 and is far from the sealing portion 120. A region surrounded by a dotted line including the end on the side is a first region 111.

  The flow path 110 is filled with a liquid 130 and a reaction liquid 140 in which fine particles 9 having a specific gravity larger than that of the reaction liquid 140 are mixed. The liquid 130 is phase-separated from the reaction liquid 140. In other words, since it does not mix, the reaction liquid 140 is held in a state where fine particles 9 having a specific gravity greater than that of the reaction liquid 140 are mixed in the liquid 130 as shown in FIG. Since the specific gravity of the reaction liquid 140 is larger than that of the liquid 130, the reaction liquid 140 is located in the lowermost region in the gravity direction of the flow path 110. As the liquid 130, for example, dimethyl silicone oil or paraffin oil can be used. The reaction liquid 140 is a liquid containing components necessary for the reaction. When the reaction is PCR, DNA (target nucleic acid) amplified by PCR, DNA polymerase necessary for amplifying DNA, primers, and the like are included. For example, when PCR is performed using oil as the liquid 130, the reaction solution 140 is preferably an aqueous solution containing the above components.

Example 1
(Change in reaction solution moving time when fine particles 9 are mixed in the reaction solution 140)
FIG. 5 is a diagram illustrating a method of measuring the time for the reaction solution 140 to move from the upper end to the lower end when the biochip 100 according to the present embodiment is rotated halfway. FIG. 6 is a graph showing the results of measuring the time for the reaction solution 140 to move from the upper end to the lower end when the biochip 100 according to this example is rotated halfway. The upper side of the drawing is called “upper end” and the lower side is called “lower end”.

  In clinical practice, it is desired to shorten the time required for PCR in order to know the test results as soon as possible. It was confirmed that the moving speed of the reaction solution was increased by mixing the fine particles 9 in the reaction solution 140 in the vertical PCR. The implementation method is shown below.

  As the fine particles 9, MagExtractor-Viral RNA- (manufactured by Toyobo Co., Ltd.) was used (average particle size: 2.7 μm). The channel 110 of the biochip 100 has an average inner diameter of 2.3 mm and a length in the longitudinal direction of 24 mm. The flow path 110 is filled with a liquid 130 containing 80% (v / v) of silicon oil having a kinematic viscosity of 2 cSt (25 ° C.) and 20% (v / v) of silicon oil having a kinematic viscosity of 6 cSt (25 ° C.). The reaction solution 140 containing 6 μL of the fine particles 9 or the reaction solution 140 not containing the fine particles 9 was added and sealed with the sealing portion 120. Thereafter, as shown in FIG. 5, when the biochip 100 was rotated halfway, the time for the reaction solution 140 to move from the upper end to the lower end was measured (n = 3).

  In this example, a PCR system having a denaturation temperature of 95 ° C. and an annealing / elongation reaction temperature of 55 ° C. is assumed as a model case, and the whole biochip 100 is evaluated at an average value of 75 ° C. The results are shown in FIG. In addition, 2 * standard deviation is shown with a beard.

  For example, when a 50-cycle PCR reaction is performed with a high temperature holding (DNA denaturation) time of 3 seconds and a low temperature holding (annealing / DNA extension) time of 8 seconds, the reaction solution 140 containing no fine particles 9 is Since the average value of the movement time is 3.9 seconds, assuming that the half rotation time of the biochip 100 is 0.5 seconds, the high temperature holding time: 3 seconds → half rotation time: 0.5 seconds → droplet movement time: 3. 9 seconds → low temperature holding time: 8 seconds → half rotation time: 0.5 seconds → droplet moving time: 3.9 seconds or more is one cycle, and the time for one cycle is 19.8 seconds. In the case of 50 cycles, 19.8 × 50 = 990 (seconds). However, since the last cycle does not require a step for shifting to the next cycle of “rotation 0.5 seconds → droplet movement 3.9 seconds”, 990−0.5−3.9 = 985.6 ( Seconds) = 16.4 (minutes), and the PCR reaction is completed in a total of 16.4 minutes.

  On the other hand, in the reaction solution 140 containing 400 mg / mL fine particles 9, the average value of the reaction solution moving time is 0.95 seconds, so the high temperature holding time: 3 seconds → half rotation time: 0.5 seconds → droplet moving time: 0.95 seconds → low temperature holding time: 8 seconds → half rotation time: 0.5 seconds → droplet moving time: 0.95 seconds or more is one cycle, and the time for one cycle is 13.9 seconds. In the case of 50 cycles, 13.9 × 50 = 695 (seconds). However, in the last cycle, a step for shifting to the next cycle of “rotation 0.5 seconds → droplet movement 0.95 seconds” is not necessary, so 695−0.5−0.95 = 693.55 ( Seconds) = 11.6 (minutes), and the PCR reaction is completed in a total of 11.6 minutes. That is, the time can be shortened by about 5 minutes. In addition, if finer particles or denser particles are used, a further time reduction effect can be considered.

(Example 2)
(PCR reaction with fine particles 9 mixed in the reaction solution 140)
FIG. 7 is a table showing conditions for carrying out the PCR reaction in a state where the microparticles 9 are mixed in the reaction solution 140 according to this example.

  First, nucleic acid extraction was performed from an influenza A positive specimen (nasal swab) using MagExtractor-Viral RNA (manufactured by Toyobo Co., Ltd.). Using the eluate containing this nucleic acid, 1 step RT-PCR was carried out in a state where fine particles 9 were mixed in the reaction vessel.

  RT-PCR (reverse transcription-polymerase chain reaction) is a technique for detecting or quantifying RNA. Using reverse transcriptase, reverse transcription to DNA is performed at 50 ° C. using RNA as a template, and cDNA synthesized by reverse transcription is amplified by PCR. In general RT-PCR, a reverse transcription reaction step and a PCR step are independent, and a container is exchanged and a reagent is added between the reverse transcription step and the PCR step. On the other hand, 1step RT-PCR performs reverse transcription and PCR reaction continuously by using a dedicated reagent. RT-PCR was performed according to the CDC protocol of realtime RTPCR for swine influenza A (H1N1).

  That is, 8 μL of a solution containing 500 mg / mL microparticles, 1 μM forward primer, 1 μM reverse primer, 0.25 μM probe, 1.25 × SuperScriptIII RT / Platinum Taq Mix, 1.25 × PCR Master Mix, 0.25 (wt)% bovine serum albumin And 2 μL of eluate was added thereto. After stirring, 1.6 μL of the solution was placed in the biochip 100 shown in Example 1 and sealed with the sealing part 120, and then RT-PCR was performed under the conditions shown in FIG.

  As a result of measuring the fluorescence of the reaction solution 140 after the reaction, it was confirmed that the nucleic acid was amplified and that RT-PCR could be carried out without any problems even if the microparticles 9 existed.

  According to Example 1 and Example 2, mixing the microparticles 9 into the RT-PCR reaction solution 140 increases the reaction solution dropping speed due to gravity, and as a result, shortens the total RT-PCR reaction time. it can.

(Example 3)
(Silica magnetic fine particles used for nucleic acid extraction are directly brought into PCR)
When extracting nucleic acid from samples such as blood, soil, food, etc., an extraction method using magnetic fine particles on a silica surface is used. In general, at the end of the extraction operation, pure water or an eluent such as a low-concentration buffer is added to the magnetic fine particles to which the nucleic acid has been adsorbed, the nucleic acid is eluted in the eluate, and the eluate containing the nucleic acid and the magnetic fine particles are separated. An operation of separating and collecting only the supernatant is necessary. In this example, it is shown that RT-PCR can be performed in a magnetic particle-mixed reaction solution. Therefore, magnetic particles adsorbed with nucleic acid are added directly to the RT-PCR reaction solution 140, and the RT-PCR reaction is in progress. It can be expected that the elution operation can be omitted and the time required for nucleic acid extraction and RT-PCR can be shortened. In order to test this hypothesis, magnetic fine particles (fine particles 9) adsorbed with nucleic acids were directly mixed in the RT-PCR reaction solution 140. RT-PCR reaction was performed. The details of the experiment will be described below.

  Extraction operation was performed from the influenza A positive specimen (nasal swab) using MagExtractor-Viral RNA- (manufactured by Toyobo Co., Ltd.). However, after washing the fine particles 9 to which the nucleic acid was attached, the elution operation was not performed, and the fine particles 9 were directly mixed in the RT-PCR reaction solution 140.

  Specifically, 10 μL of a solution containing 0.8 μM forward primer, 0.8 μM reverse primer, 0.2 μM probe, 1 × SuperScriptIII RT / Platinum Taq Mix, 1 × PCR Master Mix, 0.2 (wt)% bovine serum albumin was prepared, and 4 mg of nucleic acid adsorbed fine particles 9 was added. After stirring, 1.6 μL of the solution was placed in the biochip 100 shown in Example 1 and sealed with the sealing part 120, and then RT-PCR was performed under the conditions shown in FIG. As a result, amplification of the nucleic acid was confirmed, and it was shown that pretreatment and RT-PCR can be performed without the elution operation. This makes it possible to shorten the inspection time including pretreatment and RT-PCR.

  According to this example, the elution operation can be omitted by adding the nucleic acid-adsorbed magnetic fine particles (fine particles 9) washed in the nucleic acid extraction operation directly to the RT-PCR reaction solution 140. As a result, the number of operation steps is reduced, the complexity of the operator's operation is reduced, and the total inspection time can be shortened.

Next, the thermal cycle process using the thermal cycle apparatus 1 according to the embodiment when the biochip 100 is used will be described.
FIG. 8 is a flowchart showing the procedure of the heat cycle process using the heat cycle apparatus 1 in the present embodiment.

  Hereinafter, thermal cycle processing using the thermal cycle apparatus 1 according to the embodiment will be described with reference to FIGS. 3 (A), 3 (B), and 8. In FIGS. 3A and 3B, the direction of arrow g (downward direction in the figure) is the direction in which gravity acts. In the present embodiment, a case where shuttle PCR (two-stage temperature PCR) is performed will be described as an example of thermal cycle processing. In addition, each process demonstrated below shows an example of a heat cycle process. If necessary, the order of processes may be changed, two or more processes may be performed continuously or in parallel, or processes may be added.

  Shuttle PCR is a technique for amplifying nucleic acids in a reaction solution by repeatedly applying a two-step temperature treatment of high temperature and low temperature to the reaction solution. Dissociation of double-stranded DNA occurs during high-temperature treatment, and annealing (reaction where the primer binds to single-stranded DNA) and extension reaction (reaction in which a complementary strand of DNA is formed starting from the primer) are performed during low-temperature treatment. Done.

  Generally, the high temperature in shuttle PCR is a temperature between 80 and 100 ° C, and the low temperature is a temperature between 50 and 70 ° C. The treatment at each temperature is performed for a predetermined time, and the time for keeping at a high temperature is generally shorter than the time for keeping at a low temperature. For example, the high temperature may be about 1 to 10 seconds, and the low temperature may be about 10 to 60 seconds. Depending on the reaction conditions, the time may be longer.

  Note that the appropriate time, temperature, and number of cycles (the number of repetitions of high temperature and low temperature) vary depending on the type and amount of reagent used, so an appropriate protocol is determined in consideration of the type of reagent and the amount of reaction solution 140. It is preferable to carry out the reaction.

  First, in step S <b> 101, the biochip 100 according to the present embodiment is mounted on the mounting unit 11. In this embodiment, after introducing the reaction solution 140 into the flow path 110 filled with the liquid 130, the biochip 100 sealed by the sealing unit 120 is mounted on the mounting unit 11. The reaction solution 140 can be introduced using a micropipette, an ink jet type dispensing device, or the like. In a state where the biochip 100 is mounted on the mounting unit 11, the first heating unit 12 is in contact with the biochip 100 at a position including the first region 111 and the second heating unit 13 includes the second region 112. In the present embodiment, as shown in FIG. 3A, the biochip 100 is attached to the first heating unit 12 and the second heating unit 13 by attaching the biochip 100 so as to contact the bottom plate 17. Can be held in the position.

  In this embodiment, arrangement | positioning of the mounting part 11, the 1st heating part 12, and the 2nd heating part 13 in step S101 is a 1st arrangement | positioning. As shown in FIG. 3A, the first arrangement is an arrangement in which the first region 111 of the biochip 100 is positioned at the lowest part of the flow path 110 in the direction in which gravity acts. Therefore, the first region 111 is a flow channel located at the lowermost portion of the flow channel 110 in the direction in which gravity acts when the mounting unit 11, the first heating unit 12, and the second heating unit 13 are in a predetermined arrangement. 110 is a partial region. In the first arrangement, since the first region 111 is located at the lowermost part of the flow path 110 in the direction in which the gravity acts, the reaction liquid 140 in which the fine particles 9 having a specific gravity greater than that of the reaction liquid 140 are mixed is It is located in region 111. In the present embodiment, when the biochip 100 is mounted on the mounting unit 11, the mounting unit 11 is covered with the lid 50, and the heat cycle apparatus 1 is operated. In the present embodiment, when the heat cycle apparatus 1 is operated, step S102 and step S103 are started.

  In step S <b> 102, the biochip 100 is heated by the first heating unit 12 and the second heating unit 13. The first heating unit 12 and the second heating unit 13 heat different regions of the biochip 100 to different temperatures. That is, the first heating unit 12 heats the first region 111 to the first temperature, and the second heating unit 13 heats the second region 112 to the second temperature. Thereby, a temperature gradient in which the temperature gradually changes between the first temperature and the second temperature is formed between the first region 111 and the second region 112 of the flow path 110. In the present embodiment, the first temperature is a relatively high temperature among the temperatures suitable for the target reaction in the thermal cycle process, and the second temperature is suitable for the target reaction in the thermal cycle process. Of these temperatures, it is a relatively low temperature. Therefore, in step S102 of the present embodiment, a temperature gradient is formed in which the temperature decreases from the first region 111 toward the second region 112. Since the thermal cycle process of this embodiment is shuttle PCR, it is preferable that the first temperature is a temperature suitable for dissociation of double-stranded DNA, and the second temperature is a temperature suitable for annealing and extension reaction.

  Since the placement unit 11, the first heating unit 12, and the second heating unit 13 are arranged in the first arrangement in step S102, when the biochip 100 is heated in step S102, the reaction solution 140 is brought to the first temperature. Heated. Therefore, in step S102, the reaction at the first temperature is performed on the reaction liquid 140 in which the fine particles 9 having a specific gravity greater than that of the liquid 130 are mixed.

  In step S103, it is determined whether or not the first time has elapsed in the first arrangement. In this embodiment, the determination is performed by a control unit (not shown). The first time is a time for holding the mounting unit 11, the first heating unit 12, and the second heating unit 13 in the first arrangement. In the present embodiment, when step S103 is performed subsequent to the mounting in step S101, that is, when the first step S103 is performed, the time since the heat cycle device 1 is operated is the first time. It is determined whether or not it has been reached. In the first arrangement, since the reaction solution 140 is heated to the first temperature, the reaction solution 140 in which fine particles 9 having a specific gravity greater than that of the reaction solution 140 are mixed in the target reaction is the first time. It is preferable to make it time to react at the temperature of. In this embodiment, it is preferable to set the time required for dissociation of double-stranded DNA.

  If it is determined in step S103 that the first time has elapsed (yes), the process proceeds to step S104. If it is determined that the first time has not elapsed (no), step S103 is repeated.

  In step S104, the main body 10 is driven by the driving mechanism 20, and the arrangement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 is switched from the first arrangement to the second arrangement. The second arrangement is an arrangement in which the second region 112 is positioned at the lowest part of the flow path 110 in the direction in which gravity acts. In other words, the second region 112 is a flow path in the direction in which gravity acts when the mounting unit 11, the first heating unit 12, and the second heating unit 13 are in a predetermined arrangement different from the first arrangement. This is an area located at the bottom of 110.

  In step S104 of the present embodiment, the arrangement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 is switched from the state of FIG. 3A to the state of FIG. In the heat cycle apparatus 1 of the present embodiment, the drive mechanism 20 rotationally drives the main body 10 under the control of the control unit. When the flange 16 is rotationally driven by a motor using the drive shaft as a rotation axis, the mounting portion 11, the first heating portion 12, and the second heating portion 13 fixed to the flange 16 are rotated. Since the drive shaft is an axis perpendicular to the longitudinal direction of the mounting portion 11, when the drive shaft is rotated by the operation of the motor, the mounting portion 11, the first heating portion 12, and the second heating portion 13 are rotated. The In the example shown in FIGS. 3A and 3B, the main body 10 is rotated 180 degrees. Thereby, arrangement | positioning of the mounting part 11, the 1st heating part 12, and the 2nd heating part 13 is switched from 1st arrangement | positioning to 2nd arrangement | positioning.

  In step S104, the positional relationship in the direction in which gravity acts between the first region 111 and the second region 112 is opposite to that in the first arrangement, so that the reaction liquid in which fine particles 9 having a higher specific gravity than the reaction liquid 140 are mixed. 140 moves from the first region 111 to the second region 112 by the action of gravity. When the placement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 reaches the second configuration, when the control unit stops the operation of the drive mechanism 20, the mounting unit 11, the first heating unit 12. In addition, the arrangement of the second heating unit 13 is held in the second arrangement. When the placement of the mounting portion 11, the first heating portion 12, and the second heating portion 13 reaches the second placement, Step S105 is started.

  In step S105, it is determined whether the second time has elapsed in the second arrangement. The second time is a time for holding the mounting unit 11, the first heating unit 12, and the second heating unit 13 in the second arrangement. In the present embodiment, the second region 112 is heated to the second temperature in step S102. Therefore, in step S105 of the present embodiment, the mounting unit 11, the first heating unit 12, and the second heating unit 13 are used. It is determined whether or not the time since the arrangement of the second arrangement has reached the second arrangement has reached the second time. In the second arrangement, the reaction liquid 140 in which the fine particles 9 having a specific gravity larger than that of the reaction liquid 140 are mixed is held in the second region 112. Therefore, the reaction liquid 140 is maintained for a time during which the main body 10 is held in the second arrangement. The reaction liquid 140 in which the fine particles 9 having a higher specific gravity are mixed is heated to the second temperature. Therefore, the second time is preferably set to a time for heating the reaction liquid 140 in which the fine particles 9 having a specific gravity larger than that of the reaction liquid 140 are mixed to the second temperature in the target reaction. In the present embodiment, it is preferable to set the time required for annealing and extension reaction.

  If it is determined in step S105 that the second time has elapsed (yes), the process proceeds to step S106. If it is determined that the second time has not elapsed (no), step S105 is repeated.

  In step S106, it is determined whether the number of thermal cycles has reached a predetermined number of cycles. Specifically, it is determined whether or not the procedure from step S103 to step S105 has been completed a predetermined number of times. In the present embodiment, the number of times step S103 and step S105 are completed is determined by the number of times determined as “yes”. When Step S103 to Step S105 are performed once, a thermal cycle is performed for the reaction liquid 140 in which the fine particles 9 having a specific gravity larger than that of the reaction liquid 140 are mixed. Therefore, the number of times Step S103 to Step S105 is performed is calculated. , The number of thermal cycles. Therefore, it can be determined by step S106 whether or not the number of thermal cycles necessary for the target reaction has been performed.

  If it is determined in step S106 that the thermal cycle has been performed for the predetermined number of cycles (yes), the processing is completed (END). When it is determined that the thermal cycle is not performed for the predetermined number of cycles (no), the process proceeds to step S107.

  In step S107, the arrangement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 is switched from the second arrangement to the first arrangement. By driving the main body 10 by the drive mechanism 20, the placement of the mounting portion 11, the first heating portion 12, and the second heating portion 13 can be set to the first placement. When the placement of the mounting part 11, the first heating part 12, and the second heating part 13 reaches the first placement, step S103 is started.

  When step S103 is performed following step S107, that is, in the second and subsequent steps S103, the placement of the mounting portion 11, the first heating portion 12, and the second heating portion 13 has reached the first placement. It is determined whether the time has reached a first time.

  The direction in which the mounting unit 11, the first heating unit 12, and the second heating unit 13 are rotated by the drive mechanism 20 is preferably opposite to the rotation in step S104 and the rotation in step S107. Thereby, since the twist produced in wiring, such as the conducting wire 15, by rotation, can be eliminated, deterioration of wiring can be suppressed. The direction of rotation is preferably reversed every time the drive mechanism 20 operates. Thereby, compared with the case where rotation in the same direction is continuously performed a plurality of times, the degree of twisting of the wiring can be reduced.

1-3. Effects of Thermal Cycle Device and Thermal Cycle Process in Embodiment According to the thermal cycle device and the thermal cycle method according to the present embodiment, the following effects can be obtained.
In the present embodiment, the reaction liquid 140 is injected into the liquid 130 in the biochip 100 in order to perform the heating / cooling cycle by rotating the biochip 100, and at that time, fine particles 9 having a specific gravity larger than that of the reaction liquid 140 are mixed. A reaction solution 140 is formed. Compared to the case where the same volume of the reaction solution 140 is injected into the biochip 100 in a single state, the reaction solution 140 is mixed with fine particles 9 having a specific gravity greater than that of the reaction solution 140, so that the reaction solution moving speed due to gravity (falling) (Speed) increases, so movement speed increases. As a result, the time required for raising or lowering the reaction liquid 140 from a high temperature to a low temperature, or from a low temperature to a high temperature at each of the two levels can be shortened, and a high-speed thermal cycle is possible.

2. Modified Examples Hereinafter, modified examples will be described based on the embodiment.
FIG. 9 is a perspective view of a heat cycle device 2 according to a modification. FIG. 9A shows a state where the lid 50 is closed, and FIG. 9B shows a state where the lid 50 is opened. FIG. 10 is a cross-sectional view of a biochip 100a according to Modification 5. FIG. 11 is a cross-sectional view schematically showing a cross section taken along line BB in FIG. 9A of the main body 10a of the heat cycle apparatus 2 according to the modification. The following modifications can be arbitrarily combined as long as the configurations are not contradictory to each other, and the heat cycle apparatus 2 shown in FIGS. 9A, 9B, and 11 has modifications 2, 5 and , 17 and 18 are combined. A corresponding modification will be described with reference to FIGS. 9 to 11. In the following, a configuration different from that of the embodiment will be described in detail, and the same configuration as that of the embodiment will be denoted by the same reference numeral and description thereof will be omitted.

(Modification 1)
In the embodiment, an example in which the thermal cycle device 1 does not include a detection device has been shown. However, as shown in FIGS. 9A and 9B, the thermal cycle device 2 according to this modification includes a fluorescence detector. 40 may be included. Thereby, for example, the thermal cycle apparatus 2 can be used for applications involving fluorescence detection such as real-time PCR. The number of the fluorescence detectors 40 is arbitrary as long as detection can be performed without any problem. In this modification, the fluorescence detection is performed by moving one fluorescence detector 40 along the slide 22. When performing fluorescence detection, it is preferable to provide a measurement window 18 (see FIG. 11) on the second heating unit 13 side of the main body 10a. Thereby, since the member which exists between the fluorescence detector 40 and the reaction liquid 140 can be decreased, more appropriate fluorescence measurement can be performed.

  In the present modification, in the heat cycle apparatus 2 shown in FIGS. 9A, 9B, and 11, the first heating unit 12 is provided on the lid 50 side, and on the side far from the lid 50. A second heating unit 13 is provided. That is, the positional relationship between the first heating unit 12 and the second heating unit 13 and other members included in the main body 10 is different from that of the heat cycle apparatus 1. The functions of the first heating unit 12 and the second heating unit 13 are the same as those in the first embodiment except that the positional relationship is different. In this modification, as shown in FIG. 11, a measurement window 18 is provided in the second heating unit 13. Thereby, appropriate fluorescence measurement can be performed in real-time PCR in which fluorescence measurement is performed on the low temperature side (temperature at which annealing and extension reaction are performed). When fluorescence measurement is performed from the lid 50 side, it is preferable that the sealing portion 120 and the lid 50 be designed so as not to affect the measurement.

(Modification 2)
In the embodiment, the first temperature and the second temperature are constant from the start to the end of the thermal cycle process. However, even if at least one of the first temperature and the second temperature is changed during the process. Good. The first temperature and the second temperature can be changed by the control of the control unit. The reaction solution 140 can be heated to the changed temperature by moving the reaction solution 140 by switching the arrangement of the first heating unit 12 and the mounting unit 11. Therefore, a reaction that requires a combination of two or more types of temperatures, such as reverse transcription PCR, can be performed without increasing the number of heating units or complicating the structure of the apparatus.

(Modification 3)
In the embodiment, an example in which the mounting unit 11 has a slot structure has been described, but the mounting unit 11 may have a structure that can hold the biochip 100. For example, a structure in which the biochip 100 is fitted in a recess that matches the shape of the biochip 100 or a structure in which the biochip 100 is held therebetween may be employed.

(Modification 4)
In the embodiment, the structure for determining the position of the biochip 100 is the bottom plate 17. However, the structure for determining the position may be any structure that can hold the biochip 100 in a desired position. The structure for determining the position may be a structure provided in the heat cycle apparatus 1, a structure provided in the biochip 100, or a combination of both. For example, a screw, a plug-type rod, a structure in which a protruding portion is provided on the biochip 100, or a structure in which the mounting portion 11 and the biochip 100 are fitted with each other can be adopted. When using a screw or a rod, the holding position can be adjusted in accordance with the reaction conditions of the thermal cycle, the size of the biochip 100, etc. by changing the length of the screw, the length to be screwed in, or the position to insert the rod. It may be.

  For example, as shown in FIGS. 9, 10, and 11, the structure in which the biochip 100 and the mounting portion 11 are fitted with each other is a structure in which the protruding portion 113 provided in the biochip 100 is fitted into the recess 60 provided in the mounting portion 11. Can be adopted. Thereby, the direction of the biochip 100 with respect to the 1st heating part 12 or the 2nd heating part 13 can be kept constant. Therefore, since it can suppress that the direction of the biochip 100 changes in the middle of a thermal cycle, heating can be controlled more precisely. Therefore, a more accurate thermal cycle can be applied to the reaction solution.

(Modification 5)
In the embodiment, the first heating unit 12 and the second heating unit 13 are both cartridge heaters. However, the first heating unit 12 can heat the first region 111 to the first temperature. That's fine. The 2nd heating part 13 should just be what can heat the 2nd field 112 to the 2nd temperature. For example, as the 1st heating part 12 and the 2nd heating part 13, a carbon heater, a sheet heater, IH (electromagnetic induction heating), a Peltier device, heating liquid, and heating gas can be used. In addition, different heating mechanisms may be employed for the first heating unit 12 and the second heating unit 13.

(Modification 6)
In the embodiment, an example in which the biochip 100 is heated by the first heating unit 12 and the second heating unit 13 is shown, but a cooling unit that cools the second region 112 may be provided instead of the second heating unit 13. Good. As the cooling unit, for example, a Peltier element can be used. Thereby, for example, even when the temperature of the second region 112 is not easily lowered by the heat from the first region 111 of the biochip 100, a desired temperature gradient can be formed in the flow path 110. Further, for example, the reaction liquid 140 can be subjected to a heat cycle in which heating and cooling are repeated.

(Modification 7)
In the embodiment, the example in which the material of the first heat block 12b and the second heat block 13b is aluminum is shown. However, the material of the heat block takes into account conditions such as thermal conductivity, heat retention, and ease of processing. Can be selected. For example, a copper alloy may be used and a plurality of materials may be combined. Further, the first heat block 12b and the second heat block 13b may be made of different materials.

(Modification 8)
As exemplified in the embodiment, when the mounting unit 11 is formed as a part of the first heating unit 12, a mechanism for bringing the mounting unit 11 into close contact with the biochip 100 may be provided. The mechanism for making it adhere | attaches should just be able to make at least one part of the biochip 100 adhere to the mounting part 11. FIG. For example, the biochip 100 may be pressed against one wall surface of the mounting portion 11 by a spring provided on the main body 10 or the lid 50. Thereby, since the heat of the 1st heating part 12 can be more stably transmitted to the biochip 100, the temperature of the biochip 100 can be further stabilized.

(Modification 9)
In the embodiment, the example in which the temperatures of the first heating unit 12 and the second heating unit 13 are controlled to be substantially equal to the temperature for heating the biochip 100 has been described. The temperature control of the two heating unit 13 is not limited to the embodiment. The temperature of the 1st heating part 12 and the 2nd heating part 13 should just be controlled so that the 1st field 111 and 2nd field 112 of biochip 100 may be heated to desired temperature. For example, considering the material and size of the biochip 100, the temperature of the first region 111 and the second region 112 can be more accurately heated to a desired temperature.

(Modification 10)
In the embodiment, an example in which the drive mechanism 20 is a motor has been described. However, the drive mechanism 20 may be any mechanism that can drive the mounting unit 11, the first heating unit 12, and the second heating unit 13. When the drive mechanism 20 is a mechanism that rotates the mounting unit 11, the first heating unit 12, and the second heating unit 13, the drive mechanism 20 can be controlled to a rotation speed that does not disturb the temperature gradient of the liquid 130 by centrifugal force. It is preferable that In addition, it is preferable that the direction of rotation can be reversed in order to eliminate the twist generated in the wiring. As such a mechanism, for example, a handle, a mainspring or the like can be adopted.

(Modification 11)
In the embodiment, the example in which the mounting unit 11 is a part of the first heating unit 12 has been described. However, when the drive mechanism 20 is operated, the mounting unit 11 and the first heating unit are not changed unless the positional relationship between them is changed. A member different from the part 12 may be used. When the mounting part 11 and the 1st heating part 12 are another members, it is preferable that both are being fixed directly or via another member. Moreover, although the mounting part 11 and the 1st heating part 12 may be driven by the same mechanism or may be driven by separate mechanisms, it is preferable to operate so as to keep the positional relationship between them constant. Thereby, when the drive mechanism 20 is operated, the positional relationship between the mounting unit 11 and the first heating unit 12 can be maintained constant, so that a predetermined region of the biochip 100 can be heated to a predetermined temperature. In addition, when the mechanism which drives the mounting part 11, the 1st heating part 12, and the 2nd heating part 13 is a separate mechanism, they are set as the drive mechanism 20 together.

(Modification 12)
In the embodiment, the temperature sensor is a thermocouple. However, for example, a resistance temperature detector or a thermistor may be used.

(Modification 13)
In the embodiment, an example in which the fixing portion 51 is a magnet has been described, but the fixing portion 51 may be anything that can fix the lid 50 and the main body 10. For example, a hinge or catch clip may be employed.

(Modification 14)
In the embodiment, the direction of the drive shaft is perpendicular to the longitudinal direction of the mounting unit 11, but the direction of the drive shaft is the arrangement of the mounting unit 11, the first heating unit 12, and the second heating unit 13. As long as it can be switched between the first and second arrangements. When the drive mechanism 20 is a mechanism that rotationally drives the mounting unit 11, the first heating unit 12, and the second heating unit 13, a straight line that is not parallel to the longitudinal direction of the mounting unit 11 is used as the axis of rotation. The arrangement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 can be switched.

(Modification 15)
In the embodiment, an example in which the control unit is electronic control has been described. However, the control unit (time control unit) that controls the first time or the second time controls the first time or the second time. Anything is possible. In other words, any device that can control the timing of operation or stop of the drive mechanism 20 may be used. Moreover, the control part (cycle number control part) which controls the cycle number of a thermal cycle should just be what can control the cycle number. As the time control unit and the cycle number control unit, for example, a physical mechanism, an electronic control mechanism, and a combination thereof can be adopted.

(Modification 16)
The heat cycle apparatus may include a setting unit 25 as illustrated in FIGS. 9A and 9B. The setting unit 25 is a UI (user interface), and is a device that sets conditions for thermal cycling. By operating the setting unit 25, at least one of the first temperature, the second temperature, the first time, the second time, and the number of cycles of the thermal cycle can be set. The setting unit 25 is mechanically or electronically linked with the control unit, and the setting in the setting unit 25 is reflected in the control of the control unit. Thereby, since the conditions of reaction can be changed, a desired thermal cycle can be applied to the reaction solution 140. Even if the setting unit 25 can individually set any of the above items, for example, if one is selected from a plurality of reaction conditions registered in advance, the necessary items are automatically set. It may be a thing. In the example of FIG. 9, the setting unit 25 is a button type, and reaction conditions can be set by pressing a button for each item.

(Modification 17)
The heat cycle apparatus may include a display unit 24 as illustrated in FIGS. 9A and 9B. The display unit 24 is a display device, and displays various information related to the heat cycle device. The display unit 24 may display the conditions set by the setting unit 25 and the actual time and temperature during the heat cycle process. For example, when setting, the input conditions are displayed, or during the thermal cycle process, the temperature measured by the temperature sensor, the time elapsed in the first arrangement or the second arrangement, and the thermal cycle were applied. The number of cycles may be displayed. Further, when the heat cycle process is completed or when some abnormality occurs in the apparatus, the fact may be displayed. Furthermore, notification by voice may be performed. By performing notification by display or voice, the user of the apparatus can easily grasp the progress or termination of the thermal cycle process.

(Modification 18)
In the embodiment, the biochip 100 having a circular cross section of the flow path 110 is illustrated, but the shape of the flow path 110 is arbitrary as long as the reaction solution 140 can move in the vicinity of the opposed inner walls. That is, as long as the fluctuation of the time during which the reaction solution moves between the first region 111 and the second region 112 does not affect the heating time of the reaction solution 140 in both regions, it is arbitrary. In addition, when the cross section of the flow path 110 of the biochip 100 is a polygon, the “opposite inner wall” is opposed to the flow path when the cross section inscribed in the flow path 110 is assumed to be a circular flow path. It shall be an inner wall. That is, the flow path 110 may be formed so that the reaction solution 140 moves in the vicinity of the opposing inner wall of the virtual flow path that is inscribed in the flow path 110 and has a circular cross section. Thereby, even when the cross section of the flow path 110 is a polygon, the path | route for the reaction liquid 140 to move between the 1st area | region 111 and the 2nd area | region 112 can be prescribed | regulated to some extent. Therefore, the time required for the reaction solution 140 to move between the first region 111 and the second region 112 can be limited to a certain range.

(Modification 19)
In the embodiment, the rotation direction in step S104 and the rotation direction in step S107 are opposite directions. However, after the rotation in the same direction is performed a plurality of times, the rotation may be performed the same number of times in the opposite direction. Thereby, since the twist which arose in wiring can be eliminated, compared with the case where the rotation to an opposite direction is not performed, deterioration of wiring can be suppressed.

(Modification 20)
Although the heat cycle apparatus 1 in the embodiment includes the first heating unit 12 and the second heating unit 13, the second heating unit 13 may be omitted. That is, the heating unit may be only the first heating unit 12. Thereby, since the number of members to be used can be reduced, manufacturing cost can be reduced.

  In the present modification, the first region 111 of the biochip 100 is heated by the first heating unit 12, thereby forming a temperature gradient in the biochip 100 whose temperature decreases as the distance from the first region 111 increases. Since the second region 112 is a region different from the first region 111, the second region 112 is maintained at a second temperature lower than that of the first region 111. In this modification, the second temperature is controlled by, for example, the design of the biochip 100, the properties of the liquid 130, the temperature setting of the first heating unit 12, and the like.

  In this modification, the drive mechanism 20 switches the arrangement of the mounting unit 11 and the first heating unit 12 between the first arrangement and the second arrangement, so that the reaction solution 140 is changed between the first region 111 and the second region. It can be moved between the areas 112. Since the first region 111 and the second region 112 are maintained at different temperatures, the reaction solution 140 can be subjected to a heat cycle.

  When there is no second heating unit 13, the spacer 14 holds the first heating unit 12. Thereby, since the position of the 1st heating part 12 in the main body 10 can be determined more correctly, the 1st area | region 111 can be heated more reliably. In the case where the spacer 14 is a heat insulating material, the spacer 14 is arranged so as to surround the region of the biochip 100 other than the region heated by the first heating unit 12, so that the first region 111 and the second region 112 The temperature can be made more stable.

  The heat cycle device of this modification may have a mechanism for keeping the temperature of the main body 10 constant. Thereby, since the temperature of the 2nd field 112 of biochip 100 becomes more stable, a more exact thermal cycle can be given to reaction liquid 140. As a mechanism for keeping the main body 10 warm, for example, a thermostatic bath can be used.

(Modification 21)
In the embodiment, the example in which the heat cycle apparatus 1 includes the lid 50 has been described, but the lid 50 may be omitted. Thereby, since the number of members to be used can be reduced, manufacturing cost can be reduced.

(Modification 22)
In the embodiment, the example in which the thermal cycle device 1 includes the spacer 14 has been described, but the spacer 14 may not be provided. Thereby, since the number of members to be used can be reduced, manufacturing cost can be reduced.

(Modification 23)
In the embodiment, the example in which the heat cycle apparatus 1 includes the bottom plate 17 has been described, but the bottom plate 17 may be omitted as illustrated in FIG. 11. Thereby, since the number of members to be used can be reduced, manufacturing cost can be reduced.

(Modification 24)
In the embodiment, the example in which the heat cycle apparatus 1 includes the fixed plate 19 is shown, but the fixed plate 19 may be omitted. Thereby, since the number of members to be used can be reduced, manufacturing cost can be reduced.

(Modification 25)
In the embodiment, the example in which the spacer 14 and the fixing plate 19 are separate members has been described, but the spacer 14 and the fixing plate 19 may be integrally formed as shown in FIG. Further, the bottom plate 17 and the spacer 14 or the bottom plate 17 and the fixed plate 19 may be formed integrally.

(Modification 26)
The spacer 14 and the fixing plate 19 may be transparent. Thereby, when the transparent biochip 100 is used for thermal cycle processing, it is possible to observe how the reaction liquid 140 moves from the outside of the apparatus. Therefore, it can be visually confirmed whether the heat cycle process is performed appropriately. Accordingly, the degree of “transparency” here may be such that the movement of the reaction liquid 140 can be visually recognized when these members are employed in the heat cycle apparatus 1 and the heat cycle process is performed.

(Modification 27)
In order to observe the inside of the thermal cycler 1, both the spacer 14 and the fixing plate 19 can be used, even if the spacer 14 is transparent and the fixing plate 19 is omitted, or the fixing plate 19 is transparent and the spacer 14 is omitted. May be eliminated. The smaller the number of members existing between the observer and the biochip 100 to be observed, the less the influence of light refraction by the object, and the easier the internal observation becomes. Moreover, if there are few members, manufacturing cost can be reduced.

(Modification 28)
In order to observe the inside of the heat cycle apparatus 1, an observation window 23 may be provided in the main body 10a as illustrated in FIGS. The observation window 23 may be, for example, a hole or a slit formed in the spacer 14 or the fixing plate 19. In the example of FIG. 11, the observation window 23 is a recess provided in the transparent spacer 14 formed integrally with the fixing plate 19. By providing the observation window 23, the thickness of a member existing between the observer and the biochip 100 to be observed can be reduced, so that the inside can be easily observed.

(Modification 29)
In the embodiment, the example in which the first heating unit 12 is arranged on the bottom plate 17 side of the main body 10 and the second heating unit 13 is arranged on the lid 50 side is shown. However, as shown in FIG. The first heating unit 12 may be disposed on the side. When the first heating unit 12 is disposed on the lid 50 side, the mounting unit 11, the first heating unit 12, and the second heating unit 13 when the biochip 100 is mounted in step S101 of the embodiment. The arrangement is the second arrangement. That is, the second region 112 is disposed at the lowermost portion of the flow path 110 in the direction in which gravity acts. Therefore, when the heat cycle device 2 of the present modification is applied to the heat cycle process according to the embodiment, when the biochip 100 is mounted on the mounting portion 11, switching to the first arrangement is performed. Specifically, the process of step S107 is performed before moving from step S101 to step S102 and step S103.

(Modification 30)
In the embodiment, the step of heating the biochip 100 by the first heating unit 12 and the second heating unit 13 (step S102), the step of determining whether or not the first time has passed (step S103), However, although the example which starts when the biochip 100 is mounted on the mounting unit 11 (step S101) has been shown, the timing of starting step S102 is not limited to the embodiment. As long as the first region 111 is heated to the first temperature by the time point when the timing is started in step S103, step S102 may be started at an arbitrary timing. The timing for performing step S102 is determined in consideration of the size and material of the biochip 100 to be used, the time required for heating the first heat block 12b, and the like. For example, it may be any of before step S101, simultaneously with step S101, and after step S101 and before step S103.

(Modification 31)
In the embodiment, the first temperature, the second temperature, the first time, the second time, the number of cycles of the thermal cycle, and the operation of the drive mechanism 20 are controlled by the control unit. It is also possible for the user to control at least one of the items. When the user controls the first temperature or the second temperature, for example, the temperature measured by the temperature sensor is displayed on the display unit 24, and the user operates the setting unit 25 to adjust the temperature. Good. When the user controls the number of cycles of the heat cycle, the user stops the heat cycle apparatus 1 when the predetermined number of times is reached. The number of cycles may be counted by the user or the thermal cycle device 1 may count and display the number of cycles on the display unit 24.

  When the user controls the first time or the second time, it is determined whether or not the user has reached a predetermined time, and the mounting unit 11, the first heating unit 12, In addition, the arrangement of the second heating unit 13 is switched. That is, the user performs at least a part of steps S103 and S105 and steps S104 and S107 in FIG. The time may be measured using a timer that is not linked to the heat cycle apparatus 2, or the elapsed time may be displayed on the display unit 24 of the heat cycle apparatus 2. The switching of the arrangement may be performed by operating the setting unit 25 (UI), or may be performed manually by using a handle for the drive mechanism 20.

(Modification 32)
In the embodiment, an example in which the rotation angle when the placement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 is switched by the rotation of the driving mechanism 20 is 180 ° is shown. Any angle that changes the positional relationship between the first region 111 and the second region 112 in the gravity direction may be used. For example, if the rotation angle is less than 180 °, the moving speed of the reaction solution 140 becomes slow. Therefore, by adjusting the rotation angle, it is possible to adjust the time during which the reaction solution 140 moves between the first temperature and the second temperature. That is, the time for the temperature of the reaction solution 140 to change between the first temperature and the second temperature can be adjusted.

  The present invention is not limited to the above-described embodiments, and various modifications can be made. For example, the present invention includes configurations that are substantially the same as the configurations described in the embodiments (for example, configurations that have the same functions, methods, and results, or configurations that have the same objects and effects). In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiment or a configuration that can achieve the same object. In addition, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

  DESCRIPTION OF SYMBOLS 1, 2 ... Thermal cycle apparatus 9 ... Fine particle (solid substance) 10, 10a ... Main body 11 ... Mounting part 12 ... 1st heating part (heating part) 12a ... 1st heater 12b ... 1st heat block 13 ... 2nd heating part 13a ... second heater 13b ... second heat block 14 ... spacer 15 ... conductor 16 ... flange 17 ... bottom plate 18 ... measurement window 19 ... fixing plate 20 ... drive mechanism 22 ... slide 23 ... observation window 24 ... display unit 25 ... setting unit DESCRIPTION OF SYMBOLS 40 ... Fluorescence detector 50 ... Cover 51 ... Fixed part 60 ... Recessed part 100, 100a ... Biochip 110 ... Channel 111 ... 1st area | region 112 ... 2nd area | region 113 ... Protrusion part 120 ... Sealing part 130 ... Liquid 140 ... Reaction liquid.

Claims (5)

The reaction liquid in which a solid substance having a specific gravity larger than that of the reaction liquid is mixed and the liquid whose specific gravity is smaller than that of the reaction liquid and phase-separated from the reaction liquid are filled, and the reaction liquid is close to the opposing inner wall. A mounting part for mounting a biochip including a flow path that moves,
When the biochip is attached to the attachment part, a heating part that heats the first region of the flow path;
A drive mechanism for switching the arrangement of the mounting part and the heating part between the first arrangement and the second arrangement;
A thermal cycle apparatus comprising: The thermal cycle apparatus according to claim 1, wherein
The heat cycle apparatus, wherein the solid substance is fine particles. The thermal cycle apparatus according to claim 1 or 2,
The heat cycle apparatus, wherein the surface of the solid substance is hydrophilic. In the heat cycle apparatus according to any one of claims 1 to 3,
A thermal cycle apparatus, wherein the solid substance is adsorbed with a nucleic acid to be detected. Mixing a solid substance having a specific gravity greater than that of the reaction solution into the reaction solution;
The reaction liquid in which the solid substance is mixed and a liquid having a specific gravity smaller than that of the reaction liquid and phase-separated from the reaction liquid are filled, and the reaction liquid moves close to the opposing inner walls. Attaching a biochip including a flow path to the attachment part;
Holding the biochip in a first arrangement in which the first region of the flow path is located at the lowest part of the flow path in the direction in which gravity acts;
Heating the first region;
The biochip is placed in a second arrangement in which the second region of the flow path, which is different from the first region in the direction in which the reaction solution moves, is located at the lowest part of the flow channel in the direction in which gravity acts. Holding,
A thermal cycle method comprising:

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