JP2006345814A - Method for reaction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To properly and easily carry out feeding and recovery of a reaction solution while readily suppressing an evaporation loss of the solution used for a reaction. <P>SOLUTION: A method for reaction is carried out as follows. The reaction solution is initially fed from an opening of a flow channel-like reaction part of a reaction chip toward the interior of the reaction part and then directed toward the interior of the reaction part for storing the reaction solution. A thermosetting resin is fed from the opening and superimposed on a liquid surface of the reaction solution exposed into the atmosphere in the interior of the reaction part. The temperature conditions of the reaction part are subsequently controlled to cause a polymerase chain reaction. The thermosetting resin having thermosetting properties and covering over the liquid surface of the reaction solution is simultaneously cured. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a reaction method.

Conventionally, as a reaction apparatus for processing a small amount of sample solution in, for example, a biochemical reaction, a plurality of recesses as reaction fields provided on the surface of a reaction chip base material, and the temperature state can be controlled for each recess There is known a reaction apparatus that includes a temperature control device that includes a Peltier element or the like and includes a transport device that transports a lid that can close each opening of a plurality of recesses (see, for example, Patent Document 1).
Conventionally, as a reactor used in, for example, a biochemical reaction, a plurality of concave portions as reaction fields provided on the surface of a base material of a reaction chip, and a plurality of concave portions that are thermally welded or pressure-bonded to the base material A reactor including a film capable of closing each opening is known (see, for example, Patent Document 2).
JP-A-5-317030 JP-A-9-99932

By the way, according to the reaction apparatus and the reactor according to the prior art, each opening of the plurality of recesses is closed by a lid or a film in order to suppress loss due to evaporation of a small amount of sample solution.
However, when a transport device that transports the lid is provided as in the reaction device according to the above-described prior art, there is a problem that the device configuration of the reaction device becomes complicated. In addition, when a common lid is used for reaction chips that are different depending on the transfer device, a cleaning device for cleaning the lid is required, which causes a problem that the device configuration is further complicated.
In addition, when each opening is closed with a film as in the reactor according to the above prior art, a troublesome work of peeling the film after completion of the reaction is required, and a series of processing steps can be efficiently performed. It becomes difficult.
In addition, in the method in which mineral oil having a relatively low specific gravity is layered on the sample solution, it is difficult to improve the effect of suppressing the evaporation of the sample solution, and when collecting the sample solution after the reaction is completed, There arises a problem that it is difficult to prevent oil from being mixed.
The present invention has been made in view of the above circumstances, and provides a reaction method capable of appropriately and easily supplying and recovering a reaction solution while easily suppressing evaporation loss of a solution used for the reaction. Objective.

  In order to solve the above problems and achieve the object, the reaction method of the present invention according to claim 1 is a container having at least one opening (for example, the opening 24 or 41b in the embodiment) ( For example, the reaction liquid supply step for supplying the reaction liquid to the reaction section 12 and the reaction member 41) in the embodiment, and the liquid thermosetting resin is overlaid on the liquid surface of the reaction liquid stored in the container It includes a thermosetting resin supply step and a reaction generation step for causing the reaction liquid to undergo a predetermined reaction that is in a temperature state exceeding the curing temperature of the thermosetting resin.

  Furthermore, the reaction method of the present invention described in claim 2 is characterized in that the thermosetting resin supply step supplies the thermosetting resin which is insoluble in the reaction solution.

  Furthermore, the reaction method of the present invention described in claim 3 is characterized in that the thermosetting resin supply step supplies the thermosetting resin having a specific gravity less than the specific gravity of the reaction solution.

  Furthermore, in the reaction method of the present invention according to claim 4, the thermosetting resin supply step supplies the thermosetting resin having a curing temperature lower than a reaction temperature of the reaction solution. .

  Furthermore, in the reaction method of the present invention according to claim 5, the thermosetting resin supply step supplies the thermosetting resin having a viscosity of 100 dPa · s to 1000 dPa · s at 23 ° C. It is said.

  Furthermore, the reaction method of the present invention according to claim 6 is characterized in that the reaction generation step generates an enzyme reaction.

  Furthermore, in the reaction method of the present invention according to claim 7, the enzyme reaction is a polymerase chain reaction.

  According to the reaction method of the present invention described in claim 1, the thermosetting resin is supplied so as to be superimposed on the reaction liquid stored in the container, and the liquid thermosetting is performed by a predetermined reaction in the reaction generation step. The resin hardens and becomes a cover that covers the entire liquid surface of the reaction liquid, and the evaporation loss of the reaction liquid can be suppressed.

  Furthermore, according to the reaction method of the present invention described in claim 2, the thermosetting resin layered on the reaction solution becomes insoluble in the reaction solution, so that the reaction solution and the thermosetting resin are not mixed. The evaporation loss of the reaction liquid can be suppressed by the thermosetting resin.

Furthermore, according to the reaction method of the present invention described in claim 3, the evaporation loss of the reaction liquid can be suppressed by making the specific gravity of the thermosetting resin layered on the reaction liquid less than the specific gravity of the reaction liquid. it can.
When the specific gravity of the thermosetting resin is larger than the specific gravity of the reaction liquid, the thermosetting resin cannot be overlaid on the liquid surface of the reaction liquid.

  Furthermore, according to the reaction method of the present invention as set forth in claim 4, since the curing temperature of the thermosetting resin is lower than the reaction temperature of the reaction liquid, the liquid thermosetting property is obtained by a predetermined reaction in the reaction generation step. The resin is properly cured and becomes a lid that covers the entire liquid surface of the reaction solution, and evaporation loss of the reaction solution can be suppressed.

Furthermore, according to the reaction method of the present invention as set forth in claim 5, the viscosity of the thermosetting resin layered on the reaction solution becomes 100 dPa · s to 1000 dPa · s at 23 ° C. Loss can be suppressed.
When the viscosity of the thermosetting resin is less than 100 dPa · s at 23 ° C., the thermosetting resin and the reaction liquid are mixed, and the thermosetting resin is layered on the liquid surface of the reaction liquid. On the other hand, when the viscosity of the thermosetting resin is greater than 1000 dPa · s at 23 ° C., the entire liquid surface of the reaction solution cannot be covered.

Furthermore, according to the reaction method of the present invention described in claim 6, it is possible to easily suppress the evaporation loss of the reaction solution used in the enzyme reaction.
Furthermore, according to the reaction method of the present invention described in claim 7, it is possible to easily suppress the evaporation loss of the reaction solution used in the polymerase chain reaction.

  Hereinafter, a reaction method according to an embodiment of the present invention will be described with reference to the accompanying drawings.

  The biochemical reaction device 1 according to the reaction method of the present embodiment includes, for example, a reagent storage device 2 that executes a reagent storage step of storing a reaction reagent in a reaction chip 10 and an enzyme reaction, for example, as shown in FIG. A reaction device 3 that executes a reaction step that causes a predetermined reaction such as a polymerase chain reaction (PCR), and a detection device 4 that executes a detection step that detects a sample such as DNA by optical analysis, for example. It is prepared for.

  The reagent storage device 2 of the biochemical reaction apparatus 1 includes, for example, sample reagents and other reagents used in various reaction processes such as polymerase chain reaction, various reagents used in the detection process, dilution liquid or buffer liquid, and the like. Is stored in the reagent storage portion 11 of the reaction chip 10.

And the reaction apparatus 3 of the biochemical reaction apparatus 1 is equipped with the temperature control apparatus 5 which comprises the Peltier device etc. which control the temperature state of the reaction liquid in the reaction process, for example.
For example, as shown in FIG. 1, the temperature control device 5 includes two Peltier elements arranged so as to sandwich a reaction portion 12 of a reaction chip 10 to be described later from both sides in the thickness direction (that is, the front surface side and the back surface side). The surface of each Peltier element part 5a, 5b provided with the parts 5a, 5b and abutting on the surface of the reaction chip 10 is shaped along the surface shape (for example, convex shape) of the reaction part 12 of the reaction chip 10 described later ( For example, a concave shape is formed.

  The detection device 4 of the biochemical reaction device 1 causes the sample adjusted by a predetermined reaction such as a polymerase chain reaction by the reaction device 3 to react with various detection reagents in the detection unit 13 of the reaction chip 10. Then, luminescence detection is performed to detect the presence or absence of a labeling substance (for example, a fluorescent substance) previously attached to the specimen or the nucleic acid probe, for example, from the back side of the detection unit 13 of the reaction chip 10 or the like.

For example, as shown in FIG. 2, the reaction chip 10 includes a reagent storage unit 11, a reaction unit 12, and a detection unit 13 provided on a single substantially rectangular plate-like base material 10 a. .
The base material 10a is preferably formed of, for example, each plastic such as PC (polycarbonate), PP (polypropylene), cycloolefin polymer, fluoropolymer, silicon resin, or an appropriate combination of a plurality of plastics, or glass. By doing so, it becomes excellent in heat resistance, chemical resistance, molding processability and the like.

  And the reagent storage part 11 is provided in one edge part along the longitudinal direction of the base material 10a, for example, and several concave hole-shaped reagent storage recessed parts 11a, ..., 11a provided on the surface of the base material 10a are provided. And contains, for example, sample reagents and other reagents used in various reaction processes such as polymerase chain reaction, various reagents used in the detection step, and a diluent or buffer solution.

And the reaction part 12 mentioned later is provided in the center part along the longitudinal direction of the base material 10a, for example.
And the detection part 13 is provided in the other edge part along the longitudinal direction of the base material 10a, for example, and is equipped with the several recessed hole-shaped detection recessed parts 13a, ..., 13a provided on the surface of the base material 10a. The reaction unit 12 accommodates a specimen adjusted by a predetermined reaction such as a polymerase chain reaction and various detection reagents.

The shape of each reagent storage recess 11a and each detection recess 13a is not particularly limited, and may be an appropriate well shape such as a truncated cone shape, a truncated pyramid shape, a cone shape, a truncated pyramid shape, or a curved bottom shape. It may be set appropriately depending on the processability, the solution injection property, and the like.
In addition, each reagent accommodation recessed part 11a and each detection recessed part 13a are formed by cutting, a shaping | molding process, etc., for example, when the base material 10a consists of plastics. Moreover, when the base material 10a consists of glass, it forms by cutting etc., for example.

In addition, the magnitude | size of each reagent accommodation recessed part 11a is set according to the quantity of the reagent to accommodate, for example, the hole diameter is 0.1-10 mm and the depth is 0.1-10 mm.
Since the amount of the reagent used for DNA analysis is very small, each detection recess 13a preferably has an opening diameter of 5 mm or less, particularly an opening diameter of 0.01 mm to 5 mm, and a depth of 5 mm or less. The depth is 0.01 mm to 5 mm.
Further, the inner surfaces of each reagent storage recess 11a and each detection recess 13a may be subjected to a surface treatment such as hydrophilicity or water repellency.

  Each reagent storage recess 11a and each detection recess 13a are, for example, PP (polypropylene), PC (polycarbonate), PS (polystyrene), PE (polyethylene), PET (polyethylene terephthalate), POM (polyacetal), PA (polyamide). , PAN (polyacrylonitrile), PMMA (polymethyl methacrylate), methylpentene films such as TPX film (Mitsui Chemicals), cycloolefin films such as ZEONOR (made by Nippon Zeon Co., Ltd.), silicon resin films, fluorine You may coat | cover with the coating film by each plastics, such as a polymer polymer film, or a suitable combination of several plastics.

  For example, as illustrated in FIGS. 3A to 3D, the reaction unit 12 covers the groove 21 provided on the back surface 10 </ b> B of the base material 10 a and the opening end 21 a of the groove 21 to open the groove 21. The film 23 that seals the portion and forms the flow path 22 and the base material 10a are penetrated in the thickness direction and connected to two openings 24 and 24 provided on the surface 10A of the base material 10a. In addition, two through holes 25 and 25 that open inside the groove portion 21 are provided. That is, the reaction part 12 has a flow path shape, and the solution supplied into the reaction part 12 from the one opening part 24 opened on the surface 10A of the base material 10a sequentially passes through the one through hole 25. The flow path 22 formed by the groove 21 and the film 23, the other through hole 25, and the other opening 24 can be circulated.

  The film 23 may be, for example, PP (polypropylene), PC (polycarbonate), PS (polystyrene), PE (polyethylene), PET (polyethylene terephthalate), POM (polyacetal), PA (polyamide), PAN (polyacrylonitrile), PMMA. (Polymethyl methacrylate), methylpentene films such as TPX films (manufactured by Mitsui Chemicals), cycloolefin films such as ZEONOR (manufactured by ZEON CORPORATION), plastics such as silicon resin films, fluorine polymer films, or A film of a single layer structure or a multilayer structure made of an appropriate combination of a plurality of plastics, or a single layer structure or a multilayer structure of an alloy of each metal such as aluminum, copper, gold, etc. Beam, further, a film such as a multilayer structure with a combination of plastic and metal.

And the thickness of the film 23 is 1-500 micrometers, for example, Preferably, it is 1-100 micrometers, Comprising: It becomes more preferable with becoming thin within this range.
If the thickness is less than 1 μm, the thermal deformation becomes excessively large and the desired strength cannot be ensured. On the other hand, if the thickness is greater than 500 μm, the thermal conductivity is excessively reduced, When controlling the temperature state of the solution in the reaction unit 12 from the outside, it becomes difficult to control the temperature state uniformly over the entire solution, and it becomes impossible to ensure the desired uniformity with respect to the reaction state. .
The film 23 made of metal preferably has a thickness of 1 to 50 μm.

The plastic film 23 preferably has a thermal conductivity of 0.1 kcal / mh ° C. or higher. For example, PP (polypropylene) has a thermal conductivity of about 0,119 kcal / mh ° C., and PC (polycarbonate). ) Has a thermal conductivity of about 0,166 kcal / mh ° C., and PE (polyethylene) has a thermal conductivity of about 0,252 kcal / mh ° C.
Further, the film 23 made of metal preferably has a thermal conductivity of 100 kcal / mh ° C. or more, for example, aluminum has a thermal conductivity of about 177 kcal / mh ° C., and copper has a thermal conductivity of 324 kcal / mh ° C. The thermal conductivity of gold is about 254 kcal / mh ° C.

The single-layer film 23 made of plastic preferably has a thickness of about 10 to 100 μm.
In addition, the film 23 having a single layer structure made of metal preferably has a thickness of about 5 to 80 μm in the case of soft aluminum, for example, and preferably has a thickness of about 5 to 50 μm in the case of hard aluminum.

Further, the multilayer film 23 made of plastic is formed of, for example, PET (polyethylene terephthalate) or OPP (stretched polypropylene), and preferably has a thickness of about 1 to 20 μm so that desired toughness can be obtained. And flexibility is ensured.
Further, the film 23 having a multilayer structure made of a combination of plastic and metal preferably has a thickness of about 7 to 50 μm, for example, in the case of aluminum. Further, the base material 10a of the reaction chip 10 is formed on the surface of the aluminum. A seal layer that can be attached to the surface by, for example, heat welding or pressure bonding is provided so as to be integrated with aluminum. This seal layer is formed, for example, by laminating a resin film-like sealant such as nylon on the surface of aluminum, or coating maleic acid-modified polypropylene or the like on the surface of aluminum. In this film 23, a film such as PET (polyethylene terephthalate) or OPP (stretched polypropylene) may be laminated on the aluminum layer side in order to further increase the strength.

  On the surface of the base material 10a to which the film 23 is attached, for example, a protrusion protruding from the surface around the groove 21 and the opening 24 of the reaction part 12 is provided, and the protrusion and the film 23 are in contact with each other. You may set so that it may touch.

  The biochemical reaction device 1 and the reaction chip 10 according to the reaction method of the present embodiment have the above-described configuration. Next, the operation of the biochemical reaction device 1 will be described.

  First, for example, in step S01 shown in FIG. 4, as a reagent storage process, the reagent storage device 2 performs, for example, sample reagents and other reagents used in various reaction processes such as polymerase chain reaction, and various reagents used in the detection process. Then, the diluted solution or the buffer solution is stored in the reagent storage unit 11 of the reaction chip 10.

  Next, in step S02, a predetermined reaction (for example, a polymerase chain reaction) is caused as a reaction process described later.

  Next, in step S03, as a detection process, the sample adjusted by the polymerase chain reaction in the reaction process and various reagents for detection (for example, nucleic acid probes) are detected in the detection unit 13 of the reaction chip 10. A series of processes is performed by detecting the presence or absence of a labeling substance (for example, a fluorescent substance) previously attached to a specimen or a nucleic acid probe by hybridization or the like, for example, from the back side of the detection unit 13 of the reaction chip 10 or the like. Exit.

Below, the reaction process in step S02 mentioned above is demonstrated.
First, for example, in step S <b> 11 shown in FIG. 5, as a reaction liquid supply process, the reaction liquid is supplied from the opening 24 of the reaction part 12 in the flow channel shape of the reaction chip 10 toward the inside of the reaction part 12.
The reaction solution for the polymerase chain reaction is, for example, DNA extracted from blood or the like, template DNA generated in advance, polymerase enzyme, dNTP (deoxynucleotide triphosphate) which is a material of each base, pH and concentration adjustment. And a diluting solution or buffer solution.

Next, in step S12, as the thermosetting resin supply step, the liquid thermosetting resin is supplied from the opening 24 toward the inside of the reaction section 12 storing the reaction liquid, for example, FIG. ), (B), a liquid thermosetting resin H is overlaid on the liquid surface of the reaction liquid R exposed to the atmosphere inside the reaction section 12.
This thermosetting resin is, for example, a liquid epoxy resin having a curing temperature lower than a temperature state (for example, about 96 ° C.) in a reaction generation step described later, and has a viscosity of 100 dPa · s to 1000 dPa at 23 ° C. S and the specific gravity is less than the specific gravity of the reaction solution and is insoluble in the reaction solution.

In addition, when the viscosity of the thermosetting resin is less than 100 dPa · s at 23 ° C., the thermosetting resin and the reaction liquid are mixed, and the thermosetting resin is layered on the liquid surface of the reaction liquid. On the other hand, when the viscosity of the thermosetting resin is greater than 1000 dPa · s at 23 ° C., the entire liquid surface of the reaction solution cannot be covered.
In addition, when the specific gravity of the thermosetting resin is larger than the specific gravity of the reaction solution, the thermosetting resin cannot be overlaid on the surface of the reaction solution.

In addition, when the specific gravity of a thermosetting resin is relatively heavy, you may mix the additive which can adjust specific gravity in this thermosetting resin. For example, the specific gravity can be adjusted by dispersing and arranging hollow particles. As the hollow particles, for example, inorganic particles such as silica and organic particles such as epoxy resin and acrylic resin can be used.
Further, if the thermosetting resin is insoluble in the reaction solution, mixing of the thermosetting resin and the reaction solution does not occur. In general, since the reaction solution is aqueous, the thermosetting resin may be a hydrophobic resin that is insoluble in water.
Moreover, as a thermosetting resin, a phenol resin, an epoxy resin, an unsaturated polyester resin, a polyurethane resin, a xylene resin, a melamine resin etc. can be used, for example.

  Next, in step S13, a polymerase chain reaction is caused as a reaction generation step to be described later, and a series of processes is terminated.

Below, the reaction production | generation process in step S13 mentioned above is demonstrated.
First, in step S21 shown in FIG. 7, for example, as a denaturing step, the temperature control device 5 changes the temperature state of the reaction unit 12 to a predetermined temperature (for example, 90 to 25 seconds) for a predetermined time (for example, 5 to 25 seconds). The reaction solution is denatured by heat.
In this denaturation step, the thermosetting resin layered on the liquid surface of the reaction liquid is cured to become a lid that covers the entire liquid surface of the reaction liquid, and the liquid surface of the reaction liquid is brought into the atmosphere inside the reaction unit 12. Exposure is further suppressed.

  Next, in step S22, as the annealing process, the temperature control device 5 changes the temperature state of the reaction unit 12 to a predetermined temperature (for example, about 50 to 60 ° C.) over a predetermined time (for example, 15 to 60 seconds). And various primers (that is, DNA fragments) are combined (annealed) with a desired gene sequence.

  Next, in step S23, as the extension reaction process, the temperature control device 5 changes the temperature state of the reaction unit 12 to a predetermined temperature (for example, 65 to 75 ° C.) for a predetermined time (for example, 1 to 5 minutes). The complementary strand synthesis by DNA polymerase is performed.

Next, in step S24, it is determined whether or not to continue a series of processes.
If this determination is “YES”, the flow returns to step S 21 described above.
On the other hand, when the determination result is “NO”, the series of processing ends.

Hereinafter, a method for manufacturing the reaction section 12 of the reaction chip 10 described above will be described.
First, a base material 10a made of an appropriate combination of plastics or a plurality of plastics such as PC (polycarbonate), PP (polypropylene), cycloolefin-based polymer, fluoropolymer, silicon resin, etc. The groove portion 21 is formed on the back surface 10B of (No. S31).

  Next, the substrate 10a is penetrated in the thickness direction by, for example, a cutting method, and is connected to the two openings 24 and 24 provided on the surface 10A of the substrate 10a and is opened inside the groove 21. Two through holes 25, 25 to be formed are formed (step S32).

Next, the film 23 covers the opening end 21a of the groove portion 21 and seals the opening portion of the groove portion 21, and the film 23 is thermally welded or pressed onto the back surface 10B of the base material 10a or, for example, polyacetic acid. Affixed via a thermoplastic resin adhesive such as vinyl and polyamide, and the flow path 22 is formed by the groove 21 and the film 23 (step S33).
Since the film 23 made of PE (polyethylene) or the like is heat-weldable, it can be bonded to the base material 10a without using an adhesive.

As described above, according to the reaction method according to the present embodiment, since the reaction part 12 of the reaction chip 10 is in a flow path shape, supply of the solution to the reaction part 12 and recovery of the solution from the reaction part 12 are prevented. It becomes easy. In addition to this, since the liquid thermosetting resin is supplied so as to be overlaid on the reaction liquid stored in the reaction section 12 of the reaction chip 10, it is possible to easily suppress the evaporation loss of the reaction liquid. it can. Moreover, in a predetermined reaction (for example, polymerase chain reaction) in the reaction generation step, the temperature of the thermosetting resin layered on the reaction liquid increases, so that the thermosetting resin is cured and the entire liquid surface of the reaction liquid is obtained. And the evaporation loss of the reaction solution can be further suppressed.
In addition, since the reaction chip 10 includes the reagent storage unit 11, the reaction unit 12, and the detection unit 13 on a single base material 10a, a series of reagent storage steps, reaction steps, and detection steps are performed. Can be executed continuously and efficiently.

In the above-described embodiment, the reaction chip 10 includes the reagent storage unit 11, the reaction unit 12, and the detection unit 13. However, the present invention is not limited to this, and for example, the type and number of reagents .., 11, a plurality of reaction units 12,..., And a plurality of detection units 13,. Also good.
In the above-described embodiment, in the reaction chip 10, the reagent storage unit 11, the reaction unit 12, and the detection unit 13 may be connected to each other by a flow path or the like. In this case, the inspection time can be shortened, various analyzes can be performed with a small amount of sample and reagent with high accuracy, and the cost required for the analysis can be reduced.

In the above-described embodiment, in the channel-shaped reaction part 12, the channel 22 is formed by the groove portion 21 provided on the back surface 10B of the base material 10a and the film 23 attached on the back surface 10B of the base material 10a. However, the present invention is not limited to this. For example, as in the first modified example shown in FIG. 8, the reaction portion 12 includes a groove portion 21 provided on the back surface 10B of the substrate 10a and the groove portion 21. A film 23 for sealing the opening of the groove 21 by covering the opening end 21a, and the two openings 24 provided on the surface 10A of the substrate 10a, penetrating the substrate 10a in the thickness direction, Are connected to the two through holes 25 and 25 that are open inside the groove 21 and on the surface 10A of the base material 10a at positions that do not interfere with the two openings 24 and 24 and are connected to the groove 21. Second groove portion 1 and, the second film 32 and may be configured with affixed on the bottom surface 31A of the second groove 31.
That is, in the first modification, an opening 31a connected to the groove 21 is formed on the bottom surface 31A of the second groove 31, and the second film 32 attached to the bottom 31A is opened. The channel 31 is formed by sealing the portion 31 a and covering the opening end 21 a of the groove portion 21 with the film 23.
In addition, this 2nd film 32 is a film equivalent to the film 23, for example.

Below, the manufacturing method of the reaction part 12 of the reaction chip 10 which concerns on this 1st modification is demonstrated.
First, a base material 10a made of an appropriate combination of plastics or a plurality of plastics such as PC (polycarbonate), PP (polypropylene), cycloolefin-based polymer, fluoropolymer, silicon resin, etc. The groove portion 21 is formed on the back surface 10B of (No. S41).

  Next, the substrate 10a is penetrated in the thickness direction by, for example, a cutting method, and is connected to the two openings 24 and 24 provided on the surface 10A of the substrate 10a and is opened inside the groove 21. Two through holes 25, 25 to be formed are formed (step S42).

  Next, the second groove portion 31 is formed on the surface 10A of the base material 10a at a position where it does not interfere with the two openings 24, 24 by, for example, a cutting method, and the second groove portion 31 on the bottom surface 31A. An opening 31a connected to the groove 21 is formed in the central part (step S43).

  Next, the film 23 covers the opening end 21a of the groove portion 21 and seals the opening portion of the groove portion 21, and the film 23 is thermally welded or pressed onto the back surface 10B of the base material 10a or, for example, polyacetic acid. Affixed via a thermoplastic resin adhesive such as vinyl and polyamide, and the flow path 22 is formed by the groove 21 and the film 23 (step S44).

Next, the opening 31a of the second groove 31 is sealed with the second film 32, and the second film 32 is thermally welded or pressed onto the bottom surface 31A of the second groove 31, or For example, it is pasted through a thermoplastic resin adhesive such as polyvinyl acetate and polyamide, and the flow path 22 is formed by the groove 21 and the film 23 and the second groove 31 and the second film 32 (step S45). .
In the first modification, the flow path 22 is formed by the second film 32 whose thermal conductivity is increased by reducing the thickness relative to the base material 10a forming the groove 21. Therefore, the temperature state of the entire reaction solution stored in the reaction unit 12 in the reaction generation step can be easily and uniformly controlled. Thereby, a predetermined reaction can be easily and uniformly generated with respect to the entire reaction liquid in the reaction unit 12.

  In the reaction chip 10 according to the first modified example, a lid that covers the entire liquid surface of the reaction liquid after the reaction generation step is performed, that is, the thermosetting resin layered on the liquid surface of the reaction liquid is cured. After that, when collecting the reaction solution, for example, as shown in FIG. 9, the reaction solution collecting probe P is inserted into the reaction solution so as to penetrate the second film 32.

In the above-described embodiment, the flow path-like reaction portion 12 is configured to include the film 23. However, the present invention is not limited to this, and for example, the second modification shown in FIGS. 10 (a) to 10 (d). Thus, the reaction part 12 is configured to include a hollow hole 35 that is hollow inside the base material 10a and is connected to each of the two openings 24, 24 provided on the surface 10A of the base material 10a. Also good.
In the method for manufacturing the reaction part 12 of the reaction chip 10 according to the second modification, for example, instead of the film 23 in the above-described embodiment, a substantially rectangular plate-like second substrate equivalent to the substrate 10a. 35a is pasted on the back surface 10B of the base material 10a through a thermoplastic resin adhesive such as polyvinyl acetate and polyamide, and the opening end 21a of the groove 21 is covered with the second base material 35a. The flow path 22 is formed by sealing the opening of the groove 21.
Moreover, in the manufacturing method of the reaction part 12 of the reaction chip 10 according to the second modified example, for example, PC (polycarbonate), PP (polypropylene), cycloolefin-based polymer, fluoropolymer, silicon resin, etc., by injection molding, for example. You may form the hollow hole 35 in the inside of the base material 10a which consists of an appropriate combination of each plastic or several plastics.

In the above-described embodiment, the reaction part 12 having the flow path shape is provided in the reaction chip 10. However, the present invention is not limited to this. For example, the third modification example shown in FIGS. In this way, the reaction part 12 is made of, for example, an appropriate one of each plastic or a plurality of plastics such as PC (polycarbonate), PP (polypropylene), cycloolefin polymer, fluoropolymer, silicon resin by an injection molding method or a cutting method. You may comprise and comprise the recessed part 36 of the recessed hole shape formed on the surface 10A of the base material 10a which consists of a combination.
In the reaction chip 10 according to the third modification, for example, as shown in FIG. 12, a recess 36 covers a through hole 36a that penetrates the base material 10a in the thickness direction and a back surface 10B of the base material 10a. 23, the thermosetting resin layered on the liquid surface of the reaction liquid is cured after the reaction generating step is performed, and the lid covers the entire liquid surface of the reaction liquid. Thereafter, when collecting the reaction solution, the reaction solution collecting probe P is inserted into the reaction solution so as to penetrate the film 23 from the back surface 10B side of the substrate 10a.
In the third modified example, the reaction portion 12 is configured to include a plurality of concave-hole-shaped concave portions 36, ..., 36 formed on the surface 10A of the substrate 10a. 36 may be connected by an appropriate flow path.

In the above-described embodiment, the reaction chip 10 including the flow channel-like reaction unit 12 is used in the reaction process of the reaction device 3. However, the present invention is not limited to this. For example, the fourth modification shown in FIG. As an example, a reaction member 41 formed of a tubular container having a closing part 41a and an opening part 41b may be used.
In the reaction member 41 according to the fourth modified example, when the reaction member 41 is formed of a relatively thin plastic or the like, the reaction member 41 is overlaid on the surface of the reaction liquid after the reaction generation step. After the thermosetting resin is cured and becomes a lid that covers the entire liquid surface of the reaction liquid, when collecting the reaction liquid, for example, as shown in FIG. The reaction member 41 is inserted into the reaction solution so as to penetrate.

In the above-described embodiment, for example, as shown in FIG. 4, the annealing process in step S12 and the extension reaction process in step S13 are sequentially performed. However, the present invention is not limited to this. For example, the annealing process And the extension reaction step may be performed simultaneously. In this case, the temperature state of the reaction unit 12 is controlled by the temperature control device 5 so as to reach a predetermined temperature (for example, about 50 to 70 ° C.) over a predetermined time (for example, 1 to 5 minutes). Thus, various primers (that is, DNA fragments) are combined (annealed) with a desired gene sequence, and complementary strand synthesis by DNA polymerase is performed.
In the embodiment described above, the polymerase chain reaction (PCR) may be multiplex PCR. In this multiplex PCR, a hot start method (that is, a method in which the extension reaction step is started after the reaction solution becomes relatively high temperature in order to suppress the occurrence of primer misannealing and oligomerization). It is preferable to apply.

The biochemical reaction apparatus 1 according to the embodiment of the present invention can be used for various biochemical reactions, and can be used, for example, for detection of antigen-antibody reaction and DNA reaction.
In the case of antigen detection by antigen-antibody reaction, for example, a sample containing an antigen is previously placed in each reaction part 12, a reagent containing an antibody is added later, and a labeling substance is attached to the antigen or antibody. The presence or absence of reaction can be detected. As the labeling substance, a luminescent substance such as fluorescence is generally used.

  In the case of DNA detection, for example, a nucleic acid probe is prepared in advance in each detection unit 13. Next, the sample DNA is supplied to the well-shaped detection unit 13, and the DNA can be detected by a hybridization reaction between the nucleic acid probe and the sample DNA. At this time, if a labeling substance is attached to the sample DNA, detection can be performed by detecting the presence or absence of the labeling substance. Further, as the sample DNA, DNA prepared by extracting DNA extracted from blood or the like by the PCR method, the LAMP method, or the like can be used. Further, by preparing a plurality of nucleic acids having different sequences as nucleic acid probes, it is possible to detect the sequence of the sample DNA.

  It can also be used to analyze single nucleotide gene polymorphisms (SNPs). In that case, there may be a plurality of probe nucleic acids and substances used for the detection, and one of these substances only needs to be labeled.

  In addition, as the labeling substance, a substance that specifically acts on the bound probe nucleic acid and the sample DNA can be added after the reaction. Such a thing includes an intercalator. Further, the labeling substance here includes indirect substances. That is, a substance that binds to a fluorescent substance or the like may be bound to the sample DNA as a labeling substance, and the fluorescent substance may be added later.

Alternatively, SNP or DNA may be detected by performing a multistep reaction.
For example, an invader assay method (Third Wave Technologies, Inc. (Madison, Wisconsin, USA)) may be used, thereby enabling realization of SNP analysis.

  In this case, a plurality of kinds of substances such as probe nucleic acids used for detection of detection DNA may be used. At least one kind of substance is put in advance in each reaction unit 12, and then the detection DNA and other substances are injected simultaneously or sequentially. However, a reaction may be performed.

In addition, a solution having a lighter specific gravity than the reaction solution such as mineral oil may be added to the reaction unit 12 for the purpose of preventing the reaction solution from drying.
The sample DNA or antigen may be fixed in the reaction unit 12 or may be held without being fixed.

It is a block diagram of the biochemical reaction apparatus which concerns on embodiment of this invention. It is a perspective view of the reaction chip concerning an embodiment of the invention. FIG. 3A is a perspective view of the reaction unit according to the embodiment of the present invention, and FIG. 3B is a plan view of the reaction unit according to the embodiment of the present invention as viewed from the surface side. 3 (c) is a plan view of the reaction part according to the embodiment of the present invention as seen from the back side, and FIG. 3 (d) is a cross-sectional view of the reaction part according to the embodiment of the present invention. is there. It is a flowchart which shows operation | movement of the biochemical reaction apparatus shown in FIG. It is a flowchart which shows the process of the reaction process in step S02 shown in FIG. FIG. 6 (a) is a perspective view of a reaction part according to an embodiment of the present invention, and shows a state in which a thermosetting resin is overlaid on the liquid surface of the reaction liquid inside the reaction part, FIG. 6B is a cross-sectional view of the reaction part according to the embodiment of the present invention, and shows a state in which a thermosetting resin is overlaid on the liquid surface of the reaction liquid inside the reaction part. It is a flowchart which shows the process of the reaction production | generation process in step S13 shown in FIG. FIG. 8A is a perspective view of a reaction unit according to a first modification of the embodiment of the present invention, and FIG. 8B is a reaction unit according to the first modification of the embodiment of the present invention. FIG. 8C is a plan view of the reaction unit according to the first modification of the embodiment of the present invention viewed from the back side, and FIG. 8D is a plan view of FIG. FIG. 5 is a cross-sectional view of a reaction unit according to a first modification of the embodiment of the present invention. It is a perspective view of the reaction part which concerns on the 1st modification of embodiment of this invention. FIG. 10 (a) is a perspective view of a reaction unit according to a second modification of the embodiment of the present invention, and FIG. 10 (b) is a reaction unit according to the second modification of the embodiment of the present invention. 10 (c) is a plan view of the reaction part according to the second modification of the embodiment of the present invention as seen from the back side, and FIG. 10 (d) is a plan view of FIG. FIG. 5 is a cross-sectional view of a reaction section according to a second modification of the embodiment of the present invention. FIG. 11 (a) is a perspective view of a reaction part according to a third modification of the embodiment of the present invention, and shows a state in which a thermosetting resin is overlaid on the liquid surface of the reaction liquid inside the reaction part. FIG. 11 (b) is a cross-sectional view of a reaction part according to a third modification of the embodiment of the present invention, and a thermosetting resin on the surface of the reaction liquid in the reaction part. It is a figure which shows the state which laminated | stacked. It is a perspective view of the reaction part which concerns on the 3rd modification of embodiment of this invention. It is a perspective view of the reaction member which concerns on the 4th modification of embodiment of this invention. It is a perspective view of the reaction member which concerns on the 4th modification of embodiment of this invention.

Explanation of symbols

10 reaction chip 12 reaction part (container)
24 Opening 41 Reaction member (container)
41b opening

Claims (7)

A reaction liquid supply step of supplying the reaction liquid to a container having at least one opening;
A thermosetting resin supply step of overlaying a liquid thermosetting resin on the liquid surface of the reaction solution stored in the container;
A reaction generating step of causing a predetermined reaction that causes a temperature state exceeding the curing temperature of the thermosetting resin to the reaction liquid. The reaction method according to claim 1, wherein the thermosetting resin supplying step supplies the thermosetting resin that is insoluble in the reaction solution. The reaction method according to claim 1, wherein the thermosetting resin supplying step supplies the thermosetting resin having a specific gravity less than a specific gravity of the reaction liquid. The reaction according to any one of claims 1 to 3, wherein the thermosetting resin supplying step supplies the thermosetting resin having a curing temperature lower than a reaction temperature of the reaction liquid. Method. The thermosetting resin supplying step supplies the thermosetting resin having a viscosity of 100 dPa · s to 1000 dPa · s at 23 ° C. 5. The reaction method described. The reaction method according to any one of claims 1 to 5, wherein the reaction generating step generates an enzyme reaction. The reaction method according to claim 6, wherein the enzyme reaction is a polymerase chain reaction.

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