JP2018046786A - Biochip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biochip that prevents a reaction liquid from adhering to the inner wall of a container and allows the reaction liquid to properly move inside the container.SOLUTION: The present invention provides a biochip in which a reaction liquid containing a surfactant moves in the longitudinal direction of a container. The biochip has: the container; and a liquid that is different from the reaction liquid in specific gravities and does not mix with the reaction liquid. The liquid has an amino modified silicone oil of 0.01 wt.% or more and 50 wt.% or less, the amino modified silicone oil having an amino group of 0.12 wt.% or more and 4.0 wt.% or less.SELECTED DRAWING: None

Description

  The present invention relates to a biochip.

  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 in the field of agriculture and livestock, many methods using genes for variety discrimination and variety improvement have been developed. Yes. As techniques for using genes, nucleic acid amplification techniques such as PCR (Polymerase Chain Reaction) are widely used. Today, the PCR method has become an indispensable technique for elucidating information on biological materials.

  In the PCR method, a method of performing a reaction using a container for performing a biochemical reaction, referred to as a tube or a chip (hereinafter referred to as a biochip) is common. However, in the conventional method, there are problems that a large amount of reagents and the like are required, and that the apparatus is complicated in order to realize a thermal cycle necessary for the reaction, and that the reaction takes time. Therefore, a biochip and a reaction apparatus for performing PCR in a short time using a very small amount of reagent or specimen are required.

  In order to solve such a problem, Patent Document 1 includes a liquid droplet (mineral oil or the like) that is not miscible with the reaction solution and has a specific gravity different from that of the reaction solution. A biochip and an apparatus for performing a reaction by applying a thermal cycle by reciprocating a reaction solution are disclosed.

  However, when the biochip disclosed in Patent Document 1 is used for an application in which fluorescence measurement is performed from the outside of the container to detect an amplification product, it is necessary to form the container with a transparent material. Transparent materials include resins and heat-resistant glass, but these materials are easily charged by friction or the like. It is possible to suppress charging by applying a hydrophilic treatment to the inner surface of the container, but the reaction solution, which is an aqueous solution, adheres to the container and hinders its movement. It was difficult.

  On the other hand, as a liquid that is not miscible with the reaction liquid and has a specific gravity different from that of the reaction liquid, silicone oil or mineral oil can be used from the viewpoint of heat and stability to the reaction liquid, but these oils are generally insulators. For this reason, the droplets of the reaction liquid introduced into the oil are easily polarized. For this reason, when a transparent material container is filled with oil and the reaction liquid is introduced, an electric field is generated between the reaction liquid and the container, and the reaction liquid is attracted to the inner wall of the container and adheres to it. Or may float. In such a state, as described in Patent Document 1, PCR of a method of moving a reaction solution in a biochip by gravity (hereinafter, this method is referred to as “elevating type” in this specification) is performed. In some cases, the reaction solution does not move properly, making it impossible to perform a desired heat cycle.

Therefore, in Patent Document 2, when using a container filled with a liquid that is immiscible with the reaction liquid in order to eliminate the charge generated in the biochip and to perform a stable thermal cycle on the reaction liquid, A biochip having a resistance greater than 0 Ω · cm and less than or equal to 5 × 10 ¹³ Ω · cm is disclosed.

JP 2009-136250 A JP2012-125169A

  However, even if charging is prevented as in Patent Document 2, there are cases where the droplets still adhere to the container and the droplets cannot move appropriately in the oil.

  One of the objects according to some embodiments of the present invention is to provide a biochip that can prevent the reaction solution from adhering to the inner wall of the container and can appropriately move the reaction solution through the container.

  SUMMARY An advantage of some aspects of the invention is to solve at least a part of the problems described above, and the invention can be implemented as the following aspects or application examples.

One aspect of the biochip according to the present invention is:
A biochip in which a reaction solution containing a surfactant moves in the longitudinal direction of the container,
The container;
A liquid having a specific gravity different from that of the reaction liquid and immiscible with the reaction liquid;
Including
The liquid contains 0.01 wt% or more and 50 wt% or less of amino-modified silicone oil containing 0.12 wt% or more and 4.0 wt% or less of amino groups.

  According to such a biochip, the reaction solution hardly adheres to the inner wall of the container, and the reaction solution can be easily moved in the container. That is, when the reaction solution is introduced into the biochip, amino-modified silicone oil is easily placed on the surface of the reaction solution (near the interface between the reaction solution and the liquid (oil, etc.)). Is formed, and the reaction solution is in a so-called soft capsule. Thereby, even when the interfacial tension of the reaction liquid is small, the reaction liquid is hardly wetted on the inner wall of the container, and the reaction liquid is less likely to adhere to the inner wall. Therefore, when the reaction solution is introduced into the biochip and, for example, PCR is performed, the reaction solution can be smoothly moved in the container.

In the reaction solution according to the present invention,
The reaction solution may contain a reagent for nucleic acid amplification reaction.

  According to such a biochip, the reaction solution can be introduced into the biochip, and the reaction solution can be smoothly moved in the container to perform PCR at a higher speed.

In the reaction solution according to the present invention,
The surfactant may be at least one of NP40, Triton-X100, and Tween20.

  According to such a biochip, for example, a surfactant effective in stabilizing the enzyme can be contained in the reaction solution, the enzyme can be stabilized, and the interfacial tension of the reaction solution is reduced accordingly. In addition, the reaction solution can move smoothly in the container, and PCR can be performed at a higher speed.

In the reaction solution according to the present invention,
The material of the container may include a resin.

  According to such a biochip, it is possible to make the reaction liquid hardly adhere to the inner wall even though the reaction liquid is more likely to adhere to the inner wall of the container. Thereby, for example, when PCR is performed, the reaction solution can be smoothly moved in the container.

In the reaction solution according to the present invention,
The material of the container may include polypropylene.

  According to such a biochip, the container can be formed more easily and at a lower cost, and the reaction solution is more easily adhered to the inner wall of the container, but the reaction solution is applied to the inner wall. It is possible to make it difficult to adhere.

The schematic diagram of the cross section of the chip | tip for nucleic acid amplification reaction which concerns on embodiment. In the figure, g represents the direction of gravity. The perspective view which shows typically the state which closed the cover of the thermal cycle apparatus which concerns on embodiment. The perspective view which shows typically the state which opened the cover of the thermal cycle apparatus which concerns on embodiment. The disassembled perspective view of the main body in the heat cycle apparatus which concerns on embodiment. It is a schematic diagram of the cross section in the AA line of FIG. 2A of the main body in the thermal cycle apparatus which concerns on embodiment, Comprising: 1st arrangement | positioning is shown. In the figure, g represents the direction of gravity. It is a schematic diagram of the cross section in the AA line of FIG. 2A of the main body in the heat cycle apparatus which concerns on embodiment, Comprising: 2nd arrangement | positioning is shown. In the figure, g represents the direction of gravity. The flowchart showing the process using the heat cycle apparatus which concerns on embodiment.

  Several embodiments of the present invention will be described below. Embodiment described below demonstrates an example of this invention. The present invention is not limited to the following embodiments, and includes various modified embodiments that are implemented within a range that does not change the gist of the present invention. Note that not all of the configurations described below are essential configurations of the present invention.

1. Biochip The biochip according to the present embodiment is a biochip in which a reaction liquid containing a surfactant moves in the longitudinal direction of the container. The biochip according to the present embodiment includes a container and a liquid having a specific gravity different from that of the reaction liquid and immiscible with the reaction liquid. In such a biochip, even when the droplet-like reaction liquid contains a surfactant, it is difficult for the reaction liquid to adhere to the inner wall of the container, and the reaction liquid droplets easily move in the longitudinal direction of the container. Below, an example of the structure is explained in full detail.

1.1. Container FIG. 1 is a cross-sectional view of a biochip 100. FIG. 1 shows a state in which the reaction solution 140 is put in the biochip 100.

  The biochip 100 according to the present embodiment includes a container 150 and a sealing unit 120. The size and shape of the biochip 100 are not particularly limited. For example, the amount of the liquid 130 that is not miscible with the reaction solution 140 (for example, oil or a mixture of oils, details will be described later), thermal conductivity, container 150, and The sealing portion 120 is designed in consideration of at least one of the shape and ease of handling.

  The container 150 of the biochip 100 can be formed from a transparent material. Thereby, the movement of the reaction solution 140 in the container 150 can be observed from the outside of the biochip 100, or the measurement can be used from the outside of the container 150, such as real-time PCR. In the present specification, when “transparent” is used, the visibility of the reaction liquid 140 in the container 150 from the outside of the container 150 can be secured, and the biochip 100 is not necessarily limited as long as this condition is satisfied. Is not necessarily transparent.

  Although the use of the biochip 100 is not particularly limited, for example, when used for an application involving fluorescence measurement, such as real-time PCR, the container 150 is preferably formed of a material with small spontaneous fluorescence. The container 150 is preferably made of a material that can withstand heating in PCR. The material of the container 150 is preferably a material that hardly adsorbs nucleic acids and proteins and does not inhibit an enzyme reaction by a polymerase or the like. The material satisfying these conditions is not particularly limited. For example, polypropylene, polyethylene, polyacrylic resin, resins such as cycloolefin polymer (for example, ZEONEX (registered trademark) 480R), heat resistant glass (for example, PYREX (registered trademark)) ) Glass) and the like and composite materials thereof. The material of the container 150 includes a resin or, more preferably, a polypropylene among the resins in that the container can be formed more easily and at a lower cost.

  In the biochip 100 shown in FIG. 1, the container 150 is formed in a cylindrical shape, and the central axis direction (vertical direction in FIG. 1) is the longitudinal direction. The container 150 used here is preferably a tube, and may be a small centrifuge tube or a tube designed for PCR, but has a longitudinal direction, that is, an elongated shape. When the temperature of the biochip 100 is controlled so that regions having different temperatures are formed in the liquid 130 in the container 150 using the elevating-type thermal cycler, it is easy to increase the distance between the regions having different temperatures. . Thereby, since it becomes easy to control the liquid 130 at different temperatures for each region in the container 150, a thermal cycle suitable for PCR can be realized. The elevating-type thermal cycler forms at least two temperature regions in the oil filled in the container 150, and the reaction solution 140 that is phase-separated from the liquid 130 is separated between these temperature regions by using gravity. It is a device that realizes a thermal cycle by reciprocating with a.

  The shape of the container 150 is not particularly limited as long as it has a longitudinal direction, but when used for elevating PCR, the ratio of the inner diameter D to the longitudinal length L is 1: 5 to 1.5. : The range of 20 is preferable. Furthermore, it is more preferable that the inner diameter D is 1.5 to 2 mm and the length L is 10 to 20 mm.

  The container 150 includes an opening and a sealing portion 120 that seals the opening. The container 150 includes a reaction liquid 140 and a liquid 130 that has a specific gravity different from that of the reaction liquid 140 and phase-separates. When the opening is sealed by the sealing part 120, it is preferable that no air remains in the container 150. This is because if bubbles remain in the container 150, the movement of the reaction solution 140 may be hindered. The sealing part 120 can be formed from the same material as the container 150. The structure of the sealing part 120 should just be a structure which can seal the container 150, for example, can be set as structures, such as a screw lid, a stopper, and fitting. In FIG. 1, the sealing part 120 has a stopper structure.

1.2. Liquid The biochip 100 of the present embodiment includes a liquid 130. The liquid 130 is not miscible with the reaction liquid 140. When the reaction liquid 140 is put in the container 150, the reaction liquid 140 and the liquid 130 are phase-separated, so that the reaction liquid 140 can be made into droplets in the liquid 130. Thus, the reaction liquid 140 is held in the liquid 130 in the form of droplets.

  The liquid 130 preferably has a specific gravity smaller than that of the reaction liquid 140. In this case, when the reaction liquid 140 is put in the liquid 130, the droplet of the reaction liquid 140 has a specific gravity greater than that of the liquid 130, and thus moves in the direction in which gravity acts due to the action of gravity. Further, the liquid 130 may be a liquid having a specific gravity greater than that of the reaction liquid 140. In this case, since the droplet of the reaction liquid 140 has a specific gravity smaller than that of the liquid 130, it moves in the direction opposite to the direction in which the gravity acts due to the action of gravity.

  The liquid 130 contains an amino-modified silicone oil. Here, the silicone means an oligomer or polymer having a siloxane bond as a main skeleton. In the present specification, silicone that is in a liquid state in a temperature range used for thermal cycle treatment is particularly referred to as silicone oil. In the present specification, amino-modified silicone oil refers to a kind of oil having a chemical structure in which an amino group is introduced into any part of the skeleton of silicone oil.

  In the present embodiment, the amino-modified silicone oil having an amino group content of 0.12 wt% or more and 4.0 wt% or less is used. The amino group content of the amino-modified silicone oil is preferably 0.23% by weight to 4.0% by weight, more preferably 0.5% by weight to 4.0% by weight, and even more preferably 0.82% by weight. The content is 4.0% by weight or less. When the amino group content of the amino-modified silicone oil is within this range, a sufficient effect can be obtained with the content of the amino-modified silicone oil in the liquid 130 described above. Examples of the amino group content of commercially available amino-modified silicone oils include KF393 (4%), KF-857 (1.77%), KF859 (0.23%), KF862 (0 .73%), KF867 (0.82%), TSF4703 (0.87%), TSF4708 (0.5%) manufactured by Momentive Performance Materials Japan GK, etc. Represents the group content (mass%).)

  Here, the amino group content refers to the amino group content per amino modified silicone oil, and the amount of nitrogen is determined by a general analysis method (atomic absorption, total nitrogen analysis, NMR, etc.). By multiplying the molecular weight (atomic weight) 16 to the weight of all amino groups and dividing this by the total weight of the amino-modified silicone oil, it can be obtained as the amino group content (% by weight) per amino-modified silicone oil. .

  The liquid 130 contains 0.01 wt% or more and 50 wt% or less of amino-modified silicone oil. The content of the amino-modified silicone oil in the liquid 130 is preferably 0.1% by weight to 50% by weight, more preferably 1% by weight to 50% by weight, further preferably 5% by weight to 50% by weight, Preferably they are 20 weight% or more and 50 weight% or less.

The liquid 130 contains 0.01% by weight or more and 50% by weight or less of the amino-modified silicone oil, but may contain an oil miscible with the amino-modified silicone oil (high affinity) as another component. Examples of such oil include silicone oil and mineral oil. In addition, in this specification, what is refine | purified from petroleum and is a liquid in the temperature range used for a heat cycle process is called mineral oil. These oils are highly stable against heat, and, for example, products having a viscosity of 5 × 10 ³ Nsm ⁻² or less are easily available, and thus are suitable for elevating PCR. Specific examples of such silicone oil include KF-96L-0.65cs, KF-96L-1cs, KF-96L-2cs, KF-96L-5cs manufactured by Shin-Etsu Silicone, SH200 manufactured by Toray Dow Corning. Examples thereof include dimethyl silicone oils such as C FLUID 5CS or TSF451-5A and TSF451-10 manufactured by Momentive Performance Materials Japan GK. Examples of the mineral oil include those containing alkanes having about 14 to 20 carbon atoms as the main component. That is, n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, n-octadecane, n-nonadecane, and n-tetracosane are exemplified.

  The liquid 130 may be used by mixing an amino-modified silicone oil other than an amino-modified silicone oil having an amino group content of 0.12 wt% or more and 4.0 wt% or less. X22-3820W (0.02%) manufactured by Shin-Etsu Silicone Co., Ltd. (in parentheses represent amino group content (mass%)).

  In addition, the liquid 130 may be a modified oil or modified resin other than amino-modified silicone oil (in this specification, a resin that is in a solid state alone in a temperature range to be used may be referred to as a resin. The resin is dissolved in oil. Such modified oil or modified resin may include carbinol-modified silicone oil or resin, carboxyl-modified silicone oil or resin, amino Examples thereof include a modified silicone resin, a polyether-modified silicone oil or a resin thereof, a silanol-modified silicone oil or a resin thereof, or a fluoro-modified silicone oil or a resin thereof. Specific examples of the modified oil or modified resin include silicone resins such as SR1000, SS4230, SS4267, YR3370 (above, Momentive), and fluoro-modified silicone resins such as XS66-C1191 (Momentive). .

1.3. Effects According to the biochip 100 of the present embodiment, the reaction solution 140 is less likely to adhere to the inner wall of the container 150, and the reaction solution 140 can be easily moved within the container 150. That is, when the reaction solution 140 is introduced into the biochip 100, amino-modified silicone oil is easily placed on the surface of the reaction solution 140 (near the interface between the reaction solution 140 and the liquid 130). As a result, a film is formed and the reaction solution 140 is in a so-called soft capsule. As a result, even when a surfactant is blended in the reaction solution 140 and the interfacial tension of the reaction solution 140 is reduced, the reaction solution 140 is less likely to get wet on the inner wall of the container 150 and the reaction solution 140 is less likely to adhere to the inner wall. . Therefore, when the reaction solution 140 is introduced into the biochip 100 and, for example, PCR is performed, the reaction solution 140 can be smoothly moved in the container 150.

1.4. Reaction Solution If the reaction solution 140 is introduced into the biochip 100 of the present embodiment, for example, PCR can be suitably performed using such a reaction solution. The reaction targeted by the reaction solution 140 is not particularly limited. For example, the reaction solution 140 may be used for a nucleic acid amplification reaction and may include a nucleic acid amplification reaction reagent or a target nucleic acid to be amplified.

Examples of the target nucleic acid include DNA prepared from specimens such as blood, urine, saliva, spinal fluid, and tissue, or cDNA obtained by reverse transcription of RNA prepared from these specimens. The reagent for nucleic acid amplification reaction may include a primer for amplifying the target nucleic acid, a buffer, a polymerase, dNTP, MgCl ₂ , a fluorescent probe for detecting the amplification product of the target nucleic acid, and the like.

  Although the DNA polymerase is not particularly limited, a thermostable enzyme or a PCR enzyme is preferable. For example, there are a large number of commercially available products such as Taq polymerase, Tfi polymerase, Tth polymerase, or improved versions thereof, but hot start is not possible. A DNA polymerase that can be used is preferred.

The concentration of dNTP or salt may be a concentration suitable for the enzyme to be used. Usually, dNTP is 10 to 1000 μM, preferably 100 to 500 μM, Mg ²⁺ is 1 to 100 mM, preferably 5 to 10 mM, and Cl − is 1 The total ion concentration is not particularly limited, but may be a concentration higher than 50 mM, a concentration higher than 100 mM is preferable, a concentration higher than 120 mM is more preferable, and 150 mM. Higher concentrations are more preferred, and concentrations greater than 200 mM are more preferred. The upper limit is preferably 500 mM or less, more preferably 300 mM or less, and even more preferably 200 mM or less. The primer oligonucleotide is 0.1 to 10 μM, preferably 0.1 to 1 μM.

  The reaction liquid 140 includes a surfactant. Although surfactant is not specifically limited, NP40, Triton-X100, Tween20 etc. can be illustrated and it is preferable that it is at least 1 type of these. By including the surfactant in the reaction solution 140, for example, the above-described enzyme can be stabilized, inactivation can be suppressed, and storage stability can be enhanced. The concentration of the surfactant in the reaction solution 140 is not particularly limited, but a concentration that does not inhibit the nucleic acid amplification reaction is preferable, and may be 0.001% to 0.1% or less, 0.002% to 0.02% It is preferable that it is 0.005%-0.01%. The surfactant may be brought in from the above-mentioned enzyme stock solution, but the surfactant solution may be added to the reaction solution 140 independently.

  In the present embodiment, since a surfactant is blended in the reaction liquid 140, the interfacial tension of the reaction liquid 140 is at least smaller than when the reaction liquid 140 is pure water. Therefore, particularly when the container 150 is formed of a material containing resin (polypropylene or the like), the reaction solution 140 is more likely to adhere to the inner wall. However, even in such a case, it is considered that the amino group contained in the liquid 130 described above adheres to the periphery of the reaction solution 140 and the amino-modified silicone oil forms a film around the reaction solution 140. Thereby, it is considered that the reaction liquid 140 hardly adheres to the inner wall of the container 150 (it is difficult to get wet). Therefore, when the reaction solution 140 is introduced into the biochip 100 and, for example, PCR is performed, the reaction solution 140 can be smoothly moved in the container 150.

2. Device Configuration of Thermal Cycle Device In this embodiment, an example in which a tube-type nucleic acid amplification reaction tube 100 is used as a biochip used for performing a nucleic acid amplification reaction will be described. Hereinafter, an example of an elevating nucleic acid amplification reaction (PCR) apparatus (hereinafter also referred to as a thermal cycle apparatus) suitable for the biochip 100 (hereinafter referred to as the nucleic acid amplification reaction tube 100) will be described in detail.

  2A and 2B show an example of the heat cycle apparatus 1. 2A shows a state in which the lid 50 of the heat cycle apparatus 1 is closed, and FIG. 2B shows a state in which the cover 50 of the heat cycle apparatus 1 is opened, and shows a state in which the nucleic acid amplification reaction tube 100 is attached to the attachment portion 11. FIG. 3 is an exploded perspective view of the main body 10 in the heat cycle apparatus 1 according to the embodiment. 4A and 4B are schematic views of a cross section of the main body 10 in the heat cycle apparatus 1 according to the embodiment, and are schematic views of a cross section taken along line AA in FIG. 2A.

  The heat cycle apparatus 1 includes a main body 10 and a drive mechanism 20 as shown in FIG. 2A. As shown in FIG. 3, the main body 10 includes a mounting part 11, a first heating part 12, 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 portion 11 has a structure for mounting a nucleic acid amplification reaction tube 100 described later. As shown in FIGS. 2B and 3, the mounting unit 11 of the present embodiment has a slot structure in which the nucleic acid amplification reaction tube 100 is inserted and mounted. The first heat block 12 b of the first heating unit 12, the spacer 14, The nucleic acid amplification reaction tube 100 is inserted into a hole penetrating the second heat block 13b of the second heating unit 13. The number of mounting parts 11 may be plural, and in the example of FIG. 2B, 20 mounting parts 11 are provided in the main body 10.

  The heat cycle apparatus 1 includes a structure that holds the nucleic acid amplification reaction tube 100 at a predetermined position with respect to the first heating unit 12 and the second heating unit 13. More specifically, as shown in FIG. 4A and FIG. 4B, the first region 111 of the flow path 110 constituting the later-described nucleic acid amplification reaction tube 100 is replaced by the first heating unit 12 and the second region 112 is replaced by the first region. Heating can be performed by the two heating unit 13. In this embodiment, the structure that determines the position of the nucleic acid amplification reaction tube 100 is the bottom plate 17. As shown in FIG. 4A, the first heating is performed by inserting the nucleic acid amplification reaction tube 100 to a position where it contacts the bottom plate 17. The nucleic acid amplification reaction tube 100 can be held at a predetermined position with respect to the unit 12 and the second heating unit 13.

  When the nucleic acid amplification reaction tube 100 is attached to the attachment unit 11, the first heating unit 12 heats the first region 111 of the nucleic acid amplification reaction tube 100 to the first temperature. In the example shown in FIG. 4A, the first heating unit 12 is disposed in the main body 10 at a position where the first region 111 of the nucleic acid amplification reaction tube 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 nucleic acid amplification reaction tube 100. In the example illustrated in FIG. 3, 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 12 b is a member that transfers heat generated from the first heater 12 a to the nucleic acid amplification reaction tube 100. In this embodiment, it is an aluminum block.

  When the nucleic acid amplification reaction tube 100 is attached to the attachment unit 11, the second heating unit 13 heats the second region 112 of the nucleic acid amplification reaction tube 100 to a second temperature different from the first temperature. In the example illustrated in FIG. 4A, the second heating unit 13 is disposed in the main body 10 at a position where the second region 112 of the nucleic acid amplification reaction tube 100 is heated. As shown in FIG. 3, 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 nucleic acid amplification reaction tube 100 to be heated and the heating temperature 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 nucleic acid amplification reaction tube 100 is heated to a desired temperature. In this embodiment, by controlling the first heating unit 12 to the first temperature and the second heating unit 13 to the second temperature, the first region 111 of the nucleic acid amplification reaction tube 100 is set to the first temperature. 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 rotates 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 heat 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 arranged in a predetermined manner. To control the time held in The control unit may be provided with a different mechanism for each item to be controlled, or may control all items at once. However, the control unit in the heat cycle apparatus 1 of the present embodiment is electronically controlled. Control all 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. 3 and 4A, 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. 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. 4A.

  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. 2B and 3, 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.

  The thermal cycle device 1 according to the present embodiment includes a lid 50. In the example of FIGS. 2A and 4A, the mounting portion 11 is covered with a lid 50. 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 FIGS. 2B and 3, a magnet is provided on the surface of the main body 10 that contacts the lid 50. Although not shown in FIG. 2B and FIG. 3, the lid 50 is also provided with a magnet at a position where the magnet of the main body 10 comes into contact. 10 is fixed. The fixing plate 19, the bottom plate 17, the lid 50, and the flange 16 are preferably formed using a heat insulating material.

3. Thermal Cycle Processing Using Thermal Cycle Device FIGS. 4A and 4B are cross-sectional views schematically showing a cross section of the thermal cycle device 1 taken along line AA in FIG. 2A. 4A and 4B show a state in which the nucleic acid amplification reaction tube 100 is attached to the heat cycle apparatus 1. 4A shows a first arrangement and FIG. 4B shows a second arrangement. FIG. 5 is a flowchart showing the procedure of the heat cycle process using the heat cycle apparatus 1 in the embodiment. Hereinafter, the nucleic acid amplification reaction tube 100 according to the embodiment will be described, and then, thermal cycle processing using the heat cycle apparatus 1 according to the embodiment when the nucleic acid amplification reaction tube 100 is used will be described.

  As shown in the example of FIG. 1, the nucleic acid amplification reaction tube 100 according to the embodiment includes a flow path 110 and a sealing portion 120. The flow path 110 is filled with the reaction solution 140 and a liquid 130 having a specific gravity smaller than that of the reaction solution 140 and immiscible with the reaction solution 140, and is sealed by the sealing portion 120. In the example of FIG. 1, the outer shape of the nucleic acid amplification reaction tube 100 is cylindrical, and a flow path 110 is formed in the central axis direction (vertical direction in FIG. 1). The shape of the flow path 110 is a cross section in a direction perpendicular to the longitudinal direction of the flow path 110, that is, a cross section perpendicular to the direction in which the reaction solution 140 moves in a region of the flow path 110 (this is the cross section of the flow path 110 “Cross section”) is a circular cylinder. Therefore, in the nucleic acid amplification reaction tube 100 of the present embodiment, the opposing inner wall of the flow channel 110 is a region including two points on the wall surface of the flow channel 110 constituting the diameter of the cross section of the flow channel 110, The reaction solution 140 moves in the longitudinal direction of the flow path 110 along the inner wall.

  The first region 111 of the nucleic acid amplification reaction tube 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 nucleic acid amplification reaction tube 100 of the present embodiment, the first region 111 is a region including one end in the longitudinal direction of the flow channel 110, and the second region 112 is the other in the longitudinal direction of the flow channel 110. It is an area | region including the edge part. In the example shown in FIGS. 4A and 4B, a region surrounded by a dotted line including the end portion on the sealing portion 120 side of the flow path 110 is the second region 112 and includes an end portion on the side far from the sealing portion 120. A region surrounded by a dotted line is a first region 111.

  As shown in FIG. 1, the flow path 110 includes a liquid 130 and reaction liquid droplets 140. The liquid 130 and the reaction liquid 140 are prepared according to the description of the biochip described above.

  Hereinafter, the thermal cycle process using the thermal cycle apparatus 1 according to the embodiment will be described with reference to FIGS. 4A and 4B. 4A and 4B, the direction of the arrow g (the 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. Occur.

  Generally, the high temperature in shuttle PCR is a temperature between 80 ° C. and 100 ° C., and the low temperature is a temperature between 50 ° C. 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.

  The appropriate time, temperature, and number of cycles (the number of repetitions of high temperature and low temperature) differ depending on the type and amount of reagent used, so an appropriate protocol was determined in consideration of the type of reagent and the amount of reaction solution 140. It is preferred to carry out the reaction above.

  First, the biochip 100 according to the present embodiment is mounted on the mounting unit 11 (step S101). In this embodiment, after introducing the reaction solution 140 into the flow path 110 filled with the gas 130, the biochip 100 sealed with the lid 120 is attached to the attachment portion 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 this embodiment, as shown in FIGS. 4A and 4B, 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. As shown in FIG. 4A, the first arrangement is an arrangement in which the first region 111 is vertically lower than the second region 112. In the present embodiment, the first region 111 of the biochip 100 is moved by the action of gravity. It is the arrangement that is positioned at the lowermost part of the flow path 110 in the direction to be. Therefore, the first region 111 is the flow channel 110 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. It is a part of the area. 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 gravity acts, the reaction liquid 140 having a specific gravity greater than that of the gas 130 is located in the first region 111. Yes. 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 a temperature suitable for the target reaction in the thermal cycle process. Of these, the temperature is relatively low. 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 treatment 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 heated to the first temperature. . Therefore, in step S102, the reaction solution 140 is reacted at the first temperature.

  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 following the mounting in step S101, that is, when the first step S103 is performed, the time since the heat cycle device 1 is operated reaches the first time. It is determined whether or not. In the first arrangement, since the reaction solution 140 is heated to the first temperature, the first time is preferably set to a time for causing the reaction solution 140 to react at the first temperature in the intended reaction. 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 drive 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 vertically lower than the first region 111. In the present embodiment, the second region 112 is positioned at the lowermost part of the flow path 110 in the direction in which gravity acts. Arrangement. In other words, the second region 112 includes the flow path 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 different from the first arrangement. This is the area located at the bottom.

  In step S104 of the present embodiment, the placement of the mounting unit 11, the first heating unit 12, and the second heating unit 13 is switched from the state of FIG. 4A to the state of FIG. 4B. 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. . In the example shown in FIGS. 4A and 4B, 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, since the positional relationship between the first region 111 and the second region 112 in the direction in which gravity acts is opposite to that in the first arrangement, the reaction solution 140 is moved from the first region 111 to the second region by the action of gravity. Move to region 112. 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 driving mechanism 20, the mounting unit 11, the first heating unit 12, and the second heating unit 13 are stopped. The arrangement of the two heating units 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, since the second region 112 is heated to the second temperature in step S102, the placement unit 11, the first heating unit 12, and the second heating unit 13 are arranged in step S105 of the present embodiment. It is determined whether or not the time since the second arrangement has reached the second arrangement has reached the second time. In the second arrangement, since the reaction liquid 140 is held in the second region 112, the reaction liquid 140 is heated to the second temperature for the time that the main body 10 is held in the second arrangement. Therefore, the second time is preferably a time for heating the reaction liquid 140 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, the reaction solution 140 is subjected to one thermal cycle. Therefore, the number of times Step S103 to Step S105 is performed can be set as 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 unit 11, the first heating unit 12, and the second heating unit 13 reaches the first placement, step S103 is started.

  When step S103 is performed subsequent to step S107, that is, in the second and subsequent steps S103, the time after the placement of the mounting portion 11, the first heating portion 12, and the second heating portion 13 reaches the first placement. It is determined whether or not the first time has been reached.

  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.

  If the biochip 100 of this embodiment is used for the PCR, the reaction solution 140 is less likely to adhere to the inner wall of the container 150, and the reaction solution 140 can be easily moved within the container 150, so that the reaction is very smooth. The liquid 140 can be moved in the channel 110, and high-speed PCR can be performed.

  Here, PCR is exemplified as an example of use of the biochip 100 of the present embodiment. However, the present invention has a configuration in which an aqueous solution containing a surfactant moves in a container, and includes a container and a liquid having a specific gravity different from that of the aqueous solution and immiscible with the aqueous solution, and the liquid is an amino-modified silicone oil. If the amino group content in the liquid is 0.164% or more, it is applicable not only to biochips and PCR, but also to various types of liquid containers and processing operation systems. That will be easily understood.

4). Experimental Examples Hereinafter, the present invention will be described more specifically with experimental examples, but the present invention is not limited to these experimental examples.

4.1. Preparation of Surfactant Solution A surfactant solution having a concentration of 0.02% by mass was prepared using pure water and a commercially available surfactant (NP40) (Roche).

4.2. Preparation of oil A polypropylene tube was filled with a liquid (a mixture of amino-modified silicone oil and silicone oil) (corresponding to the biochip 100). Table 1 shows the type of amino-modified silicone oil used, its amino group content, and its viscosity.

The amino-modified silicone oil in Table 1 is as follows.
・ KF8005, KF859, KF867, KF857, KF393, X-22-3820W: manufactured by Shin-Etsu Silicone ・ TSF4708, TSF4703, XF42-C5197, XF42-C5196: manufactured by Momentive Performance Materials Japan GK

4.3. Experiment contents In the experiment example, a surfactant solution having the above-mentioned concentration was prepared with reference to the concentration of the surfactant contained in a commercially available enzyme (DNA polymerase), and adhesion of the solution to the tube was observed. 1.6 μL of the prepared surfactant solution was introduced with a pipette into a polypropylene tube filled with the oil, and sealed with a stopper to prevent air from entering.

  In the experiment, immediately after introducing a droplet into the tube, the tube was moved vertically to move the droplet to the bottom of the tube. One minute after the droplet reached the bottom, the tube was turned upside down to Adhesion to the tube bottom was evaluated.

  A liquid was prepared by adding amino-modified silicone oils shown in Table 1 at different concentrations (% by weight) to KF-96L-2cs (manufactured by Shin-Etsu Chemical Co., Ltd.), which is a highly pure dimethyl silicone oil. In each experiment, the experiment was performed three times (n = 3), and the minimum concentration at which droplet adhesion did not occur was examined. In Table 1, “◯” indicates that the attachment of the droplet was not referred to, and “x” indicates that the attachment of the droplet occurred. In Table 1, “X” attached to X-22-3820W, XF42-C5197, and XF42-C5196 was not able to confirm the concentration that did not adhere because the viscosity of the liquid (mixed oil) increased. Indicates.

  From the above experiment, it was found that when the amino group content of the amino-modified silicone oil is 0.12 wt% or more and 4.0 wt% or less, there is a concentration that does not cause the surfactant solution to adhere to the container wall. did. Further, it was found that when the amino group content of the amino-modified silicone oil is 4% by weight, an excellent adhesion preventing effect can be obtained even at a low concentration of 0.01% by weight of the whole liquid. It was also found that if 50% by weight of the total liquid is blended, the adhesion preventing effect can be obtained even if the amino group content is 0.12% by weight. In general, when a surfactant is included in the droplet, the droplet adheres to the inner wall of the tube, and even if the tube is turned upside down, the droplet does not fall, but the amino group content of the amino-modified silicone oil In addition, it was confirmed that the adhesion was not caused by increasing the concentration to a certain level.

  The present invention is not limited to the above-described embodiments, and various modifications can be made. For example, the present invention includes substantially the same configuration (for example, a configuration having the same function, method and result, or a configuration having the same purpose and effect) as the configuration described in the embodiment. 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 ... Thermal cycle apparatus, 10 ... Main body, 11 ... Mounting part, 12 ... 1st heating part, 12a ... 1st heater, 12b ... 1st heat block, 13 ... 2nd heating part, 13a ... 2nd heater, 13b ... Second heat block, 14 ... spacer, 15 ... conductor, 16 ... flange, 17 ... bottom plate, 19 ... fixed plate, 20 ... drive mechanism, 50 ... lid, 51 ... fixed part, 100 ... biochip (in the example, nucleic acid (Amplification reaction tube), 110 ... channel, 111 ... first region, 112 ... second region, 120 ... sealed portion, 130 ... liquid (in the embodiment, oil), 140 ... reaction solution (in the embodiment, nucleic acid) Amplification reaction solution), 150 ... container

Claims (5)

A biochip in which a reaction solution containing a surfactant moves in the longitudinal direction of the container,
The container;
A liquid having a specific gravity different from that of the reaction liquid and immiscible with the reaction liquid;
Including
The biochip is a biochip, wherein the liquid contains 0.01% by weight or more and 50% by weight or less of an amino-modified silicone oil containing an amino group of 0.12% by weight to 4.0% by weight. In claim 1,
The said reaction liquid is a biochip containing the reagent for nucleic acid amplification reaction. In claim 1 or claim 2,
The biochip, wherein the surfactant is at least one of NP40, Triton-X100, and Tween20. In any one of Claims 1 to 3,
The material of the container is a biochip containing resin. In any one of Claims 1 thru | or 4,
The material of the container is a biochip including polypropylene.

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