JP4804090B2 - Reaction vessel - Google Patents

Description

  The present invention relates to a reaction vessel used for, for example, a biochemical reaction.

Conventionally, as a reaction vessel for processing a small amount of sample solution in, for example, a biochemical reaction, one having a plurality of recesses or flow paths as a reaction field has been provided (for example, see Patent Document 1). In such a reaction vessel, the reaction solution supplied to each recess or flow path is heated by a reaction apparatus including a temperature control device including a Peltier element that can control the temperature state of each recess or flow path.
Here, in the case of a reaction vessel having a flow channel-like reaction section, the reaction solution supplied to the flow path is exposed to the outside only from the opening of the flow path, thereby reducing the evaporation of the reaction solution during the reaction. be able to. However, due to the expansion of mixed bubbles, the surface condition of the flow path-like reaction part and the surface condition such as processing stripes, the reaction liquid moves due to the pinching condition of the temperature control device, etc. Furthermore, the reaction solution may reach the opening and evaporate. At this time, in order to further prevent evaporation of the reaction solution, for example, a sealing liquid such as mineral oil is further supplied to the flow path to which the reaction solution is supplied, so that the flow path is sealed with the sealing liquid. The reaction solution may be blocked by the flow path and the sealing liquid.
Japanese Patent No. 2759071

  However, the following problems remain in the conventional reaction vessel. That is, when a reaction solution or a sealing liquid is supplied to the flow path, air may be mixed in the flow path as bubbles. The bubbles expand when the reaction solution is heated, which may cause the sealing liquid to be pushed out of the opening and out of the flow path. For this reason, there exists a problem that a reaction solution evaporates and the loss of a reaction solution generate | occur | produces.

  The present invention has been made in view of the above-described problems, and an object of the present invention is to provide a reaction vessel that can avoid loss of a reaction solution.

The present invention employs the following configuration in order to solve the above problems. That is, the reaction container of the present invention includes a reaction part having a flow path provided in a base material, and the flow path can supply the sample solution from the reaction main part storing the sample solution and the outside of the base material. such a liquid feeding portion, possess a bent portion communicating at one end and one end of the liquid feed portion of the reaction headquarters, the flow path is a groove formed on one surface of the base material, the open end of the groove portion It is formed by the film which has the heat conductivity which covers at least one part of this.

In this invention, the sample solution is stored in the reaction headquarters, and the sample solution is sent over the bent portion by supplying sealing liquid for sealing the sample solution in the flow path to the liquid feeding portion and the bent portion, respectively. It can prevent reaching the liquid part. That is, even when the sample solution is heated to a predetermined temperature in a state where bubbles are mixed in the flow path when supplying the sample solution or the sealing liquid, and the bubbles expand with heating, the reaction headquarters and the liquid supply unit Since the bent portion is provided between the sample head and the sample head, the sample solution is prevented from being pushed out from the reaction head portion over the bent portion to the liquid feeding portion. Therefore, loss of the sample solution can be avoided.
Moreover, since the reaction part has a flow path, supply of the solution to the flow path and recovery of the supplied sample solution and sealing liquid are facilitated.
Furthermore, in this invention, since the flow path is formed by a film having a thermal conductivity larger than that of the base material because it is relatively thin with respect to the base material forming the groove, the entire solution stored in the flow path is formed. The temperature state can be easily and uniformly controlled.

Further, the reaction vessel of the present invention, the cross-sectional area of the flow path is preferably at 0.1 mm ² or more 10 mm ² or less.
In this invention, the components of the purified sample solution can be made uniform by setting the cross-sectional area of the flow channel to 0.1 mm ² or more and 10 mm ² or less.

Further, in the reaction container of the present invention, the flow path has another liquid feeding part capable of supplying the sample solution, and one end of the other liquid feeding part is the other end of the reaction main part and another bent part. It is good also as being connected via.
In this invention, after storing the sample solution in the reaction headquarters, the sample solution is sealed with the sealing liquid by storing the sealing liquid in the two liquid feeding sections, respectively.

In the reaction container of the present invention, the thickness of the film is preferably 1 μm or more and 500 μm or less.
In the reaction vessel of the present invention, the film preferably has a thermal conductivity of 0.1 kcal / mh ° C. or higher .

Moreover, it is preferable that the reaction container of this invention is equipped with the detection part which can be optically analyzed on the surface of the said base material.
According to the present invention, it is possible to continuously and efficiently execute at least a process for causing a desired reaction and a detection process on a single substrate.

Moreover, it is preferable that the reaction container of this invention is provided with the reagent storage part which accommodates the reaction reagent on the surface of the said base material.
In this invention, the process which accommodates at least a reaction reagent and the process which produces a desired reaction with respect to a single base material can be performed continuously and efficiently.

In the reaction container of the present invention, the reagent container is preferably concave.
In this invention, the reagent storage part can be easily formed on the surface of the substrate.

Moreover, the reaction container of this invention is good also as the said reaction part being for enzyme reactions.
In this invention, an enzyme reaction can be easily and uniformly generated with respect to the entire solution in the reaction part.

In the reaction container of the present invention, the enzyme reaction may be a polymerase chain reaction.
In this invention, the polymerase chain reaction can be easily and uniformly generated with respect to the entire solution in the reaction part.

  According to the reaction container of the present invention, the sample solution is stored in the reaction head portion and the sealing solution is supplied to the liquid feeding portion and the bending portion, respectively, so that the sample solution reaches the liquid feeding portion beyond the bending portion. Can be prevented. Moreover, since the reaction part has a flow path, supply of the solution to the flow path and recovery of the supplied solution are facilitated.

Hereinafter, an embodiment of a reaction container according to the present invention will be described with reference to the drawings.
The reaction container 1 according to the present embodiment includes a reagent storage unit 3, a reaction unit 4, and a detection unit 5 provided on a single substantially rectangular plate-like base material 2, for example, as shown in FIG. Yes.

  The substrate 2 is made of, for example, each plastic such as PC (polycarbonate), PP (polypropylene), cycloolefin polymer, fluorine polymer, silicon resin, or an appropriate combination of these plastics, glass, etc. Excellent in chemical properties, chemical resistance, and moldability.

The reagent storage unit 3 is provided, for example, at one end along the longitudinal direction of the base material 2, and is configured by a plurality of concave hole-shaped reagent storage recesses 11 formed on the surface 2 </ b> A of the base material 2. ing.
In the plurality of reagent storage recesses 11, for example, various reagents such as specimen reagents used in various reaction processes such as polymerase chain reaction (PCR), dilution liquid, buffer liquid, and the like are stored. Here, the magnitude | size of the reagent accommodation recessed part 11 is suitably set according to the quantity of the reagent to accommodate, for example, the opening diameter is 0.1 mm-10 mm, and the depth is 0.1 mm-10 mm.

The shape of the reagent containing recess 11 is not particularly limited, and may be any 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 is set appropriately depending on the injection property of the solution and the like. Further, the inner surface of the reagent containing recess 11 may be subjected to a surface treatment such as hydrophilicity or water repellency.
Further, the inner surface of the reagent containing recess 11 is made of, for example, PC, PP, PS (polystyrene), PE (polyethylene), PET (polyethylene terephthalate), POM (polyacetal), PA (polyamide), PAN (polyacrylonitrile), PMMA (polyethylene). Methyl pentene films such as methyl methacrylate) and TPX films (manufactured by Mitsui Chemicals), cycloolefin films such as ZEONOR (manufactured by ZEON CORPORATION), silicon resin films, plastics such as fluoropolymer films, or a plurality of these You may coat | cover with the coating film which combined these plastics suitably.

The reaction part 4 is provided, for example, in the central part along the longitudinal direction of the base material 2 and is formed on the back surface (one surface) 2B of the base material 2 as shown in FIGS. The groove 14 and the flow path 14 which is a space formed by the film 13 covering the opening end 12A of the groove 12 and 2 provided on the surface 2A of the substrate 2 through the thickness direction of the substrate 2 The liquid injection parts 17 and 18 which are the through-holes which connect each of the opening parts 15 and 16 and the groove part 12 are provided.
That is, the reaction unit 4 has a flow path shape, and the solution supplied into the reaction unit 4 from the one opening 15 opened on the surface 2A of the base 2 is sequentially supplied to the one injection unit 17. The flow path 14 formed by the groove part 12 and the film 13 and the other liquid injection part 18 can be circulated.

The flow path 14 includes a reaction head 21, a pair of bent portions 22 and 23 that communicate with both ends of the reaction head portion 21, and a pair of liquid feeding portions 24 and 25 that respectively communicate with the pair of bent portions 22 and 23. Has been. That is, the flow path 14 is connected so as to bend at least two places between the liquid injection parts 17 and 18. Here, the angle θ1 formed between the axis L1 of the reaction head unit 21 and the axis L2 of the liquid feeding unit 24 is 5 ° to 100 °, and the angle θ2 formed between the axis L1 and the axis L3 of the liquid feeding unit 25 is It is 5 ° or more and 100 ° or less. That is, the direction of the path of the flow path 14 is changed by the angle θ1 by the bent portion 22 and changed by the angle θ2 by the bent portion 23. Moreover, the cross-sectional area of the flow path 14 is 0.1 mm ² or more and 10 mm ² or less.
Note that a recess may be formed by cutting or forming a mold from the surface 2A side of the base material 2 toward the groove portion 12, and the wall thickness on the surface 2A side of the flow path 14 may be reduced. In this way, when the heat source for reaction is arranged at a position facing the surface 2A (for example, a position above the surface 2A), heat is quickly and uniformly transferred into the flow path 14.

  The film 13 is made of a methylpentene film such as PC, PP, PS, PE, PET, POM, PA, PAN, PMMA, TPX film (manufactured by Mitsui Chemicals), Zeonore (manufactured by Nippon Zeon Co., Ltd.), or the like. Each plastic such as cycloolefin film, silicon resin film, fluorine polymer film, etc., or a film having a single layer structure or multilayer structure appropriately combining these plastics, or each metal such as aluminum, copper, gold, etc. It consists of a film having a single layer structure or a multilayer structure in which a plurality of metals are appropriately combined, and a film having a multilayer structure by a combination of plastic and metal.

  And the thickness of the film 13 is 1-500 micrometers, for example, Preferably it is 1-100 micrometers, Comprising: It becomes more preferable as it becomes thin within this range. When the thickness is less than 1 μm, thermal deformation becomes excessively large and desired strength cannot be ensured. On the other hand, when the thickness of the film 13 is greater than 500 μm, the thermal conductivity is excessively reduced, and the temperature state of the solution in the reaction unit 4 is controlled uniformly from the outside when the temperature state of the solution in the reaction unit 4 is controlled from the outside. It becomes difficult to control, and the desired uniformity with respect to the reaction state cannot be ensured. Moreover, the film 13 made of metal preferably has a thickness of 1 to 50 μm.

The plastic film 13 preferably has a thermal conductivity of 0.1 kcal / mh ° C. or higher. For example, PP has a thermal conductivity of about 0.119 kcal / mh ° C., and PC has a thermal conductivity of about 0.1. It is about 166 kcal / mh ° C., and the thermal conductivity of PE is about 0.252 kcal / mh ° C.
The film 13 made of metal preferably has a thermal conductivity of 100 kcal / mh ° C. or higher, 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 13 made of plastic preferably has a thickness of about 10 μm to 100 μm.
In addition, the film 13 having a single layer structure made of metal preferably has a thickness of about 5 μm to 80 μm in the case of soft aluminum, for example, and preferably has a thickness of about 5 μm to 50 μm in the case of hard aluminum.

The multilayer film 13 made of plastic is formed of, for example, PET or OPP (stretched polypropylene), and preferably has a desired toughness and flexibility by setting the thickness to about 1 μm to 20 μm. Secured.
Further, the film 13 having a multilayer structure made of a combination of plastic and metal, for example, in the case of aluminum, preferably has a thickness of about 7 μm to 50 μm. Further, the base material 2 of the reaction vessel 1 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 by laminating a sealant in the form of a resin film such as nylon on the surface of aluminum, or by coating maleic acid-modified polypropylene or the like on the surface of aluminum. In the film 13, a film such as PET or OPP may be laminated on the aluminum layer side in order to further increase the strength.

The detection unit 5 is provided, for example, at the other end portion along the longitudinal direction of the base material 2, and is configured by a plurality of detection recesses 26 having a concave shape formed on the surface of the base material 2.
Here, the detection recess 26 is appropriately set according to the amount of reagent used for DNA analysis. However, since the amount of the reagent is very small, for example, the opening diameter is 0.01 mm to 5 mm and the depth is 0. .01 mm or more and 5 mm or less.
The shape of the detection recess 26 is not particularly limited, as is the case with the reagent storage recess 11, and may be an appropriate well shape as described above, and may be appropriately set depending on processability formation, solution injection property, and the like. The Further, the inner surface of the detection recess 26 may be subjected to a surface treatment such as hydrophilicity or water repellency.
Further, the inner surface of the detection recess 26 may be covered with a covering film in which each plastic or a plurality of these plastics is appropriately combined, as described above.

The reaction vessel 1 configured as described above is used for performing a biochemical reaction test using a biochemical reaction apparatus 30 as shown in FIG.
The biochemical reaction device 30 includes a reagent storage device 31 that stores a reaction reagent in the reaction vessel 1 and a reaction device 32 that generates a predetermined reaction such as a polymerase chain reaction (PCR) that is an enzyme reaction. And a detection device 33 that detects a sample such as DNA (deoxyribonucleic acid) by optical analysis or the like.

The reagent storage device 31 is configured to store in the reaction container 1 specimen reagents used in various reaction processes such as PCR, other reagents, diluents, buffer solutions, and the like.
The reaction device 32 includes a temperature control device 34 including a Peltier element that controls a temperature state of a reaction solution described later. For example, as shown in FIG. 3, the temperature control device 34 is arranged so as to sandwich the reaction section 4 of the reaction vessel 1 from both sides in the thickness direction (that is, the front surface side and the back surface side of the reaction vessel 1). Two Peltier elements 35 and 36 are provided. Here, the Peltier elements 35 and 36 that contact the surface of the reaction vessel 1 have a shape (for example, a concave shape) along the surface shape (for example, a convex shape) of the reaction portion 4 of the reaction vessel 1. It is configured.
The detection device 33 reacts a sample adjusted by a predetermined reaction such as PCR by the reaction device 32 with various detection reagents in the detection unit 5 of the reaction vessel 1 and labels the sample or nucleic acid probe previously attached thereto. Luminescence detection is performed to detect the presence or absence of a substance (for example, a fluorescent substance) from, for example, the back side of the detection unit 5 of the reaction vessel 1.

Next, operation | movement of the biochemical reaction apparatus 30 using the reaction container 1 is demonstrated, referring FIG.
First, the reagent storage device 31 performs a reagent storage process in which various reagents and the like are stored in the reaction container 1 (step ST1 shown in FIG. 4). This includes, for example, specimen reagents and other reagents used in various reaction processes such as PCR, various reagents used in detection, a diluent or a buffer solution, etc., in the reagent storage unit 3 of the reaction container 1. To do.

Next, for example, a reaction step that causes PCR or the like is performed. This consists of a reaction solution supply step, a sealing step, and a reaction generation step.
First, a reaction solution supply process for supplying a reaction solution (sample solution) is performed (step ST2 shown in FIG. 4). For example, as shown in FIGS. 5A and 5B, the reaction solution R is supplied from the opening 15 of the reaction unit 4 to the inside of the flow path 14. Here, the reaction solution R is supplied so as to be stored in the reaction headquarters 21. As a reaction solution for PCR, for example, DNA extracted from blood or pre-purified template DNA, polymerase enzyme, dNTP (deoxynucleotide triphosphate) which is a material of each base, and pH and concentration adjustment Dilute solution or buffer solution.

  And the sealing process which supplies mineral oil as sealing liquid is performed (step ST3 shown in FIG. 4). This is because the mineral oil is supplied from the openings 15 and 16 so as to go to the inside of the flow path 14 storing the reaction solution. For example, as shown in FIGS. The mineral oil M is layered on the liquid surface of the reaction solution R exposed to the atmosphere in the interior of 14 to seal the interior of the flow path 14. Here, the mineral oil M is supplied so as to fill the liquid feeding parts 24 and 25 and the bent parts 22 and 23. Therefore, the reaction solution R stays in the reaction headquarters 21 and is not stored in the bent portions 22 and 23. In addition, when supplying the reaction solution R and the mineral oil M, bubbles may be mixed in the flow path 14.

Then, the reaction production | generation process which produces PCR is performed. This consists of a denaturation step, an annealing step and an extension reaction step.
In this step, a denaturation step is first performed in which the DNA in the reaction solution is thermally denatured (step ST4 shown in FIG. 4). This is because the temperature control device 34 controls the temperature state of the reaction unit 4 to be a predetermined temperature (for example, about 90 ° C. to 100 ° C.) over a predetermined time (for example, 5 seconds to 25 seconds). DNA is heat denatured.

  Next, an annealing process for binding (annealing) DNA is performed (step ST5 shown in FIG. 4). This is because the temperature controller 34 controls the temperature state of the reaction unit 4 to be a predetermined temperature (for example, about 50 ° C. to 60 ° C.) over a predetermined time (for example, 15 seconds to 60 seconds). The DNA fragment is bound to the desired gene sequence.

Then, an extension reaction step of performing complementary strand synthesis by DNA polymerase is performed (step ST6 shown in FIG. 4). This is because the temperature control device 34 controls the temperature state of the reaction unit 4 to be a predetermined temperature (for example, about 65 ° C. to 75 ° C.) over a predetermined time (for example, 1 minute to 5 minutes). Perform complementary strand synthesis with polymerase. Here, even if the bubbles mixed in the flow path 14 are expanded by heating by heating the reaction section 4 in the denaturation process, annealing process, and extension reaction process, the flow path 14 is bent at the bent portions 22 and 23. Therefore, the reaction solution is prevented from being extruded beyond the bent portions 22 and 23 and the liquid feeding portions 24 and 25. As a result, since the closed state of the reaction solution is maintained, loss due to evaporation of the reaction solution is avoided.
Thereafter, it is determined whether or not to continue the series of processes (step ST7 shown in FIG. 4). If the process is continued, the process returns to step ST4, and if the process is ended, the process proceeds to the next detection process.

Next, a detection process using a specimen and various reagents for detection is performed (step ST8 shown in FIG. 4). This is because a sample prepared by PCR in the reaction generation step and various detection reagents (for example, a nucleic acid probe) are reacted in the detection unit 5 of the reaction vessel 1 by hybridization or the like, and the sample or nucleic acid is previously obtained. Luminescence detection is performed to detect the presence or absence of a labeling substance (for example, a fluorescent substance) attached to the probe from, for example, the back side of the detection unit 5 of the reaction container 1.
As described above, the operation of the biochemical reaction apparatus 30 using the reaction vessel 1 is performed.

Here, the manufacturing method of the reaction part 4 of the reaction container 1 mentioned above is demonstrated.
First, the back surface of the base material 2 made of, for example, each plastic such as PC, PP, cycloolefin polymer, fluorine polymer, silicon resin, or an appropriate combination of a plurality of plastics, for example, by an injection molding method or a cutting method. A groove 12 is formed on 2B.
Next, there are a pair of through-holes that communicate with each of the openings 15 and 16 provided on the surface 2A of the base material 2 through the thickness direction of the base material 2 and the groove portion 12 by, for example, a cutting method. Liquid injection parts 17 and 18 are formed.
Then, the film 13 covers the opening end 12A of the groove 12 and seals the groove 12, so that the film 13 is thermally welded or pressure-bonded on the back surface 2B of the substrate 2, or a polyvinyl acetate type or a polyamide type is used. The channel 14 is formed by the groove 12 and the film 13 by sticking using a thermoplastic resin adhesive.
In addition, when the film 13 consists of PE etc., since it is heat-weldability, it can bond together with the base material 2 without using an adhesive agent. The film 13 may be formed by laminating an adhesive layer or applying a sealant to a resin film, a metal film, or a film in which these films are laminated.

According to the reaction container 1 configured as described above, even when the bubbles are expanded by heating to a predetermined temperature in a state where bubbles are mixed in the flow path 14, the reaction head unit 21 and the liquid feeding unit 24 are used. , 25 are provided with the bent portions 22, 23, so that the reaction solution is prevented from being pushed out from the reaction head portion 21 over the bent portions 22, 23 to the liquid feeding portions 24, 25 during heating. For this reason, it is prevented that mineral oil flows out from the opening parts 15 and 16, and the obstruction | occlusion state of a reaction solution is maintained. Therefore, loss of the reaction solution can be avoided. Here, since the direction of the path of the flow path 14 in the bent portions 22 and 23 is changed by 5 ° or more and 100 ° or less, the reaction solution can be more reliably prevented from being pushed out to the liquid feeding portions 24 and 25. .
Moreover, since the reaction part 4 has the flow path 14, supply of the solution to the flow path 14 and collection | recovery of the supplied solution become easy.
Further, the cross-sectional area of the passage 14 by a 0.1 mm ² or more 10 mm ² or less, it is possible to equalize the components of the purified reaction solution.
Furthermore, since the film 13 is formed of a heat conductive film, the temperature state of the entire solution stored in the reaction unit 4 can be more easily and uniformly controlled.
In addition, since the reagent storage unit 3, the reaction unit 4, and the detection unit 5 are provided for a single base material 2, a series of reagent storage steps, reaction steps, and detection steps are executed continuously and efficiently. be able to.

Next, the reaction container according to the present invention will be specifically described with reference to examples.
First, as an example, a base 2 of a resin plate (PP made by Novatec) made of PP of 26 mm × 19 mm × 3 mm was formed by an injection molding method. And the reagent storage part 3 which has the eight reagent storage recessed parts 11, the reaction part 4, and the detection part 5 which has 24 detection recessed parts 26 were formed in this base material 2 by cutting.

  Here, two of the eight reagent containing recesses 11 have an opening diameter (diameter) of 8 mm and a depth of 5 mm, and the other two have an opening diameter (diameter) of 6 mm and a depth of 4 mm. The remaining four have an opening diameter (diameter) of 3.4 mm and a depth of 4 mm. Moreover, the bottom part of the reagent storage recess 11 has a conical columnar shape, and each inner surface is subjected to a hydrophilic treatment. Reagent storage recesses 11 store reagents such as specimen reagents used in PCR reactions, diluents, reagents used in the invader method, enzymes, and mineral oil of sealing liquid.

The flow path 14 of the reaction unit 4 has a rectangular shape with a width of 1 mm, a height of 1 mm, and a cross-sectional area of 1 mm ^2. The reaction head unit 21 has a length of 20 mm and the liquid feeding units 24 and 25 have a length. Are each 3 mm. The angles θ1 and θ2 formed by the axis of the reaction head unit 21 and the axes of the liquid feeding units 24 and 25 are 45 °. Furthermore, in order to make it easy to transfer heat to the reaction solution, the vicinity of the central portion (including the portion where the reaction solution exists) of the base material 2 in the flow path 14 does not penetrate from the upper part of the base material 2 to the flow path 14. It is dug down. That is, the base material 2 is dug down by 13 mm × 19 mm and a depth of 1.7 mm in a region including the vicinity of the central portion of the flow path 14 in plan view.
The film 13 is formed by superposing a resin film made of PP having a thickness of 70 μm and a metal film made of aluminum having a thickness of 30 μm, and has a thickness of 100 μm.

The detection recess 26 has a truncated cone shape with an opening diameter (diameter) of 3 mm and a depth of 1.7 mm. The inner surface of each detection recess 26 is subjected to a hydrophilic treatment.
The reaction device 32 includes a temperature control device 34 having Peltier elements 35 and 36 arranged so as to sandwich the upper and lower sides of the reaction unit 4. In the detection device 33, a nucleic acid probe to which a labeling substance is attached in advance is arranged in the detection unit 5, and a sample and various reagents adjusted by the PCR reaction are reacted to detect fluorescence from the back side of the detection unit 5. .

And 5 microliters of mineral oil, 5 microliters of PCR reaction solutions, and 5 microliters of mineral oil were put into the flow path 14 from the opening parts 15 and 16 in order. Here, each solution is supplied so that the PCR reaction solution is located in the center of the flow path 14.
Thereafter, heaters were arranged on the upper and lower surfaces in the thickness direction of the reaction unit 4 via aluminum blocks for improving thermal conductivity, and a PCR reaction as a reaction generation step was performed under the following conditions.
In this reaction generation step, heating is first performed at 94 ° C. for 2 minutes, followed by 45 cycles of heating at 95 ° C. for 1 second, 60 ° C. for 1 second, 72 ° C. for 1 second, and further heating at 15 ° C. Yes.
Thereafter, reaction and detection were performed using an invader reagent.

  Similarly, as another example, a reaction container in which the angles θ1 and θ2 formed by the axis of the reaction main unit 21 and the axes of the liquid feeding units 24 and 25 are 90 ° was prepared, and the PCR reaction similar to the above was performed. . And reaction and detection were performed using the invader reagent.

Subsequently, as a comparative example, a flow channel and a liquid injection part were formed using the same resin plate as in the example, and a reaction vessel having a hollow flow channel was created by attaching a film. Here, the flow path has a reaction head section and a liquid feeding section communicating with both ends of the reaction head section, and is not provided with a bent portion unlike the reaction container of the embodiment. Therefore, the angles formed by the axis of the reaction headquarters and the axis of the liquid feed unit are 180 °.
In the same manner as in the example, mineral oil and a PCR reaction solution were put into the flow path from the opening, and the same PCR reaction was performed. Thereafter, reaction and detection were performed using an invader reagent.

As a result, in the reaction container of the example, almost the entire amount of the PCR reaction solution was recovered without evaporation, and it was confirmed that the reaction proceeded well. On the other hand, in the reaction container of the comparative example, the mixed bubbles are heated and expanded and the PCR reaction solution is removed from the heating area by the heater, so that the sufficient PCR reaction is not performed, or the PCR reaction solution is pushed out by the bubbles and evaporated. As a result, it was confirmed that the reaction efficiency was lowered or the solution could not be recovered. Further, in the reaction container of the comparative example, even when there is a slight imbalance in the cross-sectional area of the flow path and a slight unbalance in the heat transfer of the heating control unit, the PCR reaction solution moves in the same manner from the heating region. It was confirmed that sufficient PCR reaction may not be performed due to detachment.
As a result, even when bubbles mixed in the flow path 14 expand, the bent portions 22 and 23 are provided between the reaction head portion 21 and the liquid feeding portions 24 and 25, so that the reaction solution is heated during heating. Was suppressed from being pushed out from the reaction head part 21 to the liquid feeding parts 24 and 25 beyond the bent parts 22 and 23, and it was confirmed that the closed state of the reaction solution was maintained.

In addition, this invention is not limited to the said embodiment, A various change can be added in the range which does not deviate from the meaning of this invention.
For example, in the above-described embodiment, the two openings 15 and 16 are provided on the surface 2A of the base material 2 in the reaction unit 4, but only the opening 15 is provided as shown in FIGS. It is good also as a structure currently formed in the surface 2A. The reaction part 40 is formed with an air hole 41 that is a through hole that communicates the one end side of the liquid feeding part 25 of the flow path 14 that is separated from the reaction main part 21 and the surface 2A of the substrate 2.
Here, when the liquid feeding part 25 communicates with the surface 2A of the substrate 2 through the air holes 41 in this way, the bent part 23 is not provided between the liquid feeding part 25 and the reaction head part 21, and the liquid feeding part 25 is fed. It is good also as a structure which is connected in the state in which the axis line of the liquid part 25 and the axis line of the reaction head part 21 correspond. Moreover, you may provide a bending part in three or more places.

  Moreover, in the said embodiment, although the flow path 14 has a meandering shape by planar view, it is good also as the reaction part 50 which has the flow path 51 as shown to Fig.7 (a)-(d). The flow channel 51 extends so that the liquid feeding parts 52 and 53 are separated from the openings 54 and 55 in plan view, respectively, and one end of the liquid feeding parts 52 and 53 reacts via the bent parts 56 and 57. The headquarters 58 communicates.

  Moreover, in the said embodiment, although the axis line of the reaction headquarters 21 is a straight line, as shown to Fig.8 (a)-(d), it is good also as the reaction headquarter 61 whose axis line is a meandering shape. The flow path 62 of the reaction part 60 includes a reaction main part 61 whose axis is meandering, and liquid feeding parts 24 and 25 that are communicated with both ends of the reaction main part 61 via bent parts 22 and 23, respectively. ing.

Moreover, in the said embodiment, although the flow path 14 is formed with the groove part 12 formed on the back surface 2B of the base material 2, and the film 13 stuck on the back surface 2B of the base material 2, FIG. As shown in (d), the flow path 71 may be constituted by a hollow hole formed in the substrate 2. The flow path 71 is hollow inside the base material 2 and is formed to communicate with the openings 15 and 16 formed on the surface 2 </ b> A of the base material 2.
Here, the manufacturing method of the reaction part 70 which has this flow path 71 is the base material 2 instead of the film 13 in the said embodiment, the 2nd base material 72 of the substantially rectangular plate shape equivalent to the base material 2, for example. For example, a thermoplastic resin adhesive such as polyvinyl acetate and polyamide is applied to the back surface 2B, and the opening end of the groove portion is sealed by covering the opening end of the groove portion with the second base material 72. A flow path 71 is formed. Moreover, in the manufacturing method of this reaction part 70, the base material which consists of what combined each plastics or several plastics, such as PC, PP, a cycloolefin type polymer, a fluorine-type polymer, a silicon resin, for example by the injection molding method suitably The flow path 71 may be configured by forming a hollow hole in the interior of 2.

Moreover, although the cross-sectional area of the flow path is 0.1 mm ² or more and 10 mm ² or less, it may be narrower than 0.1 mm ² or larger than 10 mm ² .
In addition, the angle formed between the axis of the reaction head and the axis of the liquid feeding part communicated via the bent part is less than 5 ° or 100 ° if the reaction solution is kept closed even by expansion of bubbles during heating. It may be an angle exceeding.
Moreover, although the reaction container is provided with the reagent storage part, the reaction part, and the detection part, the reaction container should just be equipped with the reaction part at least.
In addition, the reaction container may be configured to include a plurality of reagent storage units, a plurality of reaction units, and a plurality of detection units according to, for example, the type and number of reagents, the type and number of samples, and the like.
In the reaction container, the reagent storage unit, the reaction unit, and the detection unit may be connected to each other by a flow path or the like. In this case, the inspection time can be shortened, and 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.
Moreover, although mineral oil is added to the reaction part as a sealing liquid, other solutions may be added as long as the specific gravity is lighter than the reaction solution.
In addition, the sample DNA or antigen may be fixed in the reaction part or may be held without being fixed.

Further, although the annealing step and the extension reaction step are sequentially executed, the annealing step and the extension reaction step may be executed simultaneously. At this time, the temperature state of the reaction part is controlled by the temperature control device so as to be a predetermined temperature (for example, about 50 ° C. to 70 ° C.) over a predetermined time (for example, 1 minute to 5 minutes). A primer (that is, a DNA fragment) is combined with a desired gene sequence, and complementary strand synthesis is performed with a DNA polymerase.
The PCR may be multiplex PCR. In this multiplex PCR, it is preferable to apply a hot start method in which the execution of the extension reaction step is started after the reaction solution is at a relatively high temperature in order to suppress the occurrence of primer misannealing and oligomerization.

The biochemical reaction device can be used for various biochemical reactions such as detection of antigen-antibody reaction and DNA reaction.
In the case of antigen detection by antigen-antibody reaction, for example, the presence or absence of a reaction can be detected by adding a reagent containing an antigen in the reaction part in advance and attaching a labeling substance to the antigen or antibody. Here, as the labeling substance, a luminescent substance such as fluorescence is generally used.

In the case of detection of DNA, for example, a nucleic acid probe is prepared in advance in the detection unit, and then the sample DNA is supplied to the well-shaped detection unit and a DNA reaction is performed by a hybridization reaction between the nucleic acid probe and the sample DNA. Can be detected. Further, as the sample DNA, a DNA extracted from blood or the like prepared 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.
Further, the biochemical reaction apparatus can be used for analysis of single nucleotide polymorphism (SNP). At this time, there may be a plurality of probe nucleic acids and substances used for detection thereof, and one of those substances only needs to be labeled.

  Further, 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, Wis., USA)) may be used. This makes it possible to realize SNP analysis.
In this case, there may be a plurality of types of substances such as probe nucleic acid used for detection of detection DNA, and at least one type of substance is put in the reaction part in advance, and then the detection DNA and other substances are injected simultaneously or sequentially, A reaction may be performed.

It is a perspective view which shows the reaction container in one Embodiment of this invention. 1A and 1B show a reaction part in FIG. 1, in which FIG. 1A is a perspective view, FIG. 1B is a plan view, FIG. 1C is a back view, and FIG. It is a block diagram which shows the structure of a biochemical reaction apparatus. It is a flowchart which shows operation | movement of the biochemical reaction apparatus of FIG. The reaction part shows the state which overlaid the mineral oil on the liquid level of the reaction solution, (a) is a perspective view, (b) is sectional drawing. The other reaction part which can apply this invention is shown, (a) is a perspective view, (b) is a top view, (c) is a back view, (d) is a BB arrow cross section of (c). FIG. Similarly, the other reaction part which can apply this invention is shown, (a) is a perspective view, (b) is a top view, (c) is a back view, (d) is CC arrow of (c). FIG. Similarly, the other reaction part which can apply this invention is shown, (a) is a perspective view, (b) is a top view, (c) is a back view, (d) is DD arrow of (c). FIG. Similarly, the other reaction part which can apply this invention is shown, (a) is a perspective view, (b) is a top view, (c) is a back view, (d) is the EE arrow of (c). FIG.

Explanation of symbols

1 reaction vessel 2 base material 2B back side (one side)
3 Reagent storage part 4, 40, 50, 60, 70 Reaction part 5 Detection part 12 Groove part 12A Open end 13 Film 14, 51, 62, 71 Flow path 21, 58, 61 Reaction head part 22, 23, 56, 57 Bending part 24, 25, 52, 53 Liquid feeding part R Reaction solution (sample solution)

Claims (10)

Comprising a reaction part having a flow path provided in a substrate;
The flow path includes a reaction head for storing a sample solution, a liquid feeding portion capable of supplying the sample solution from the outside of the base material, a bent portion communicating with one end of the reaction head and one end of the liquid feeding portion. I have a,
The reaction characterized in that the flow path is formed by a groove formed on one surface of the base material and a film having thermal conductivity covering at least a part of the open end of the groove. container. ^2. The reaction container according to claim 1, wherein a cross-sectional area of the flow path is 0.1 mm ² or more and 10 mm ² or less.   The flow path has another liquid feeding part capable of supplying the sample solution, and one end of the other liquid feeding part is communicated with the other end of the reaction head via another bent part. The reaction container according to claim 1 or 2, characterized in that The thickness of the said film is 1 micrometer or more and 500 micrometers or less, The reaction container of any one of Claim 1 to 3 characterized by the above-mentioned. The reaction container according to any one of claims 1 to 4, wherein the film has a thermal conductivity of 0.1 kcal / mh ° C or more . The reaction container according to any one of claims 1 to 5 , further comprising a detection unit capable of optical analysis on a surface of the base material. The reaction container according to any one of claims 1 to 6 , wherein a reagent storage section for storing a reaction reagent is provided on the surface of the base material. The reaction container according to claim 7 , wherein the reagent container is concave. The reaction container according to any one of claims 1 to 8 , wherein the reaction section is for enzyme reaction. The reaction container according to claim 9 , wherein the enzyme reaction is a polymerase chain reaction.

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