JP4804091B2 - Reaction vessel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reaction vessel that can avoid the loss of reaction solution. <P>SOLUTION: The reaction vessel is equipped with the reaction part that is formed on the base material 2 as a flow channel 14 for storing the reaction solution in which the flow channel 14 has a cross-section of 0.5 mm<SP>2</SP>to 10 mm<SP>2</SP>. The one end of the flow channel 14 is connected through the openings 15, 16 formed on the surface 2A of the base material 2 and the liquid-injecting parts 17, 18, and the areas of the opening parts 15, 16 are each 1 mm<SP>2</SP>to 50 mm<SP>2</SP>and are 1.5 fold to 10 fold larger than the cross-section of the flow channel 14. <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

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. 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 supplying the reaction solution or the sealing liquid to the flow path, air may be mixed as bubbles in the flow path. Usually, the thickness of a reaction vessel used for biochemical reaction is several centimeters or less, and when an opening or a channel is formed in such a reaction vessel, the length of a communication hole that communicates the opening and the channel Is limited by the thickness of the reaction vessel. For this reason, when the bubbles expand when the reaction solution is heated, the sealing liquid is pushed out from the opening and flows 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 is a reaction container in which a reaction part for performing a biochemical reaction is formed on a substrate, the reaction part including a flow path to which a sample solution is supplied, and the sample solution. A liquid injection part for injecting into the flow path, and the liquid injection part has an opening opened on the outer surface of the base material and has a cross-sectional area larger than at least the cross-sectional area of the flow path. The liquid reservoir and a communication having a cross-sectional area smaller than the cross-sectional area of the liquid reservoir and a cross-sectional area larger than the cross-sectional area of the flow channel, and communicating one end of the flow channel with the liquid reservoir. And a portion .

In this invention, by making the area of the opening larger than the cross-sectional area of the flow path, the sample solution is pushed out from the opening through the communication hole when the sample solution stored in the flow path is heated. Can be prevented. That is, since the area of the opening is larger than the cross-sectional area of the flow path, a liquid reservoir having a cross-sectional area wider than the cross-sectional area of the flow path is formed in at least a portion including the vicinity of the opening of the communication hole. . Even when air bubbles are mixed in the flow path when the sample solution or the sealing liquid is supplied to the flow path and the bubbles expand with heating, the sealing liquid pushed out of the flow path is not liquid. It is suppressed that it is stored in the reservoir and flows out of the opening. Therefore, loss of the sample solution can be avoided.
Moreover, since the cross-sectional area of the opening is wider than the cross-sectional area of the flow path as described above, it is easy to supply the solution from the opening to the flow path.
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.

In the reaction container of the present invention, the area of the opening is preferably 1 mm ² or more and 50 mm ² or less.
In this invention, it can avoid that it becomes difficult to supply the solution from the opening to the flow path by setting the area of the opening to 1 mm ² or more and 50 mm ² or less.

Further, the reaction vessel of the present invention, the cross-sectional area of the flow path is preferably at 0.5 mm ² or more 10 mm ² or less.
In the present invention, by setting the cross-sectional area of the flow path to 0.5 mm ² or more and 10 mm ² or less, the sample solution can be uniformly heated, and the components of the purified sample solution can be made uniform.

In the reaction container of the present invention, the area of the opening is preferably 1.5 to 10 times the cross-sectional area of the flow path.
In this invention, by making the area of the opening 1.5 times or more and 10 times or less with respect to the cross-sectional area of the flow path, it is possible to avoid difficulty in supplying the solution and to uniformly heat the solution. The components of the sample solution purified by performing the above can be made uniform.

Moreover, the reaction container of this invention is good also as the other end of the said flow path communicating with the other opening part formed in the one surface of the said base material via the other communicating hole.
In this invention, after storing the sample solution in the channel, the sample solution is sealed with the sealing solution by supplying the sealing solution to the channel from the two openings.

In the reaction container of the present invention, it is preferable that the flow path is formed by a groove formed on one surface of the substrate and a film covering at least a part of the opening end of the groove.
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 temperature of the entire solution stored in the flow path The state can be easily and uniformly controlled.

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, when the area of the opening is made larger than the cross-sectional area of the flow path, the sample solution is opened through the communication hole when the sample solution stored in the flow path is heated. It can prevent being pushed out from the part. In addition, the solution can be easily supplied from the opening to the channel. Furthermore, 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.

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 a plurality of concave hole-shaped reagent storage recesses 11 formed on the surface (one surface) 2 </ b> A of the base material 2. It is constituted by.
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 (other surface) 2B of the base material 2 as shown in FIGS. Provided on the surface 2A of the base 2 through the flow path 14 which is a space formed by the groove 12 formed and the film 13 covering the open end 12A of the groove 12 and the thickness of the base 2 Liquid injection parts (communication holes) 17 and 18 which are through-holes communicating the two openings 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.
Here, the cross-sectional area of the flow path 14 is 0.5 mm ² or more and 10 mm ² or less, and the areas of the openings 15 and 16 are 1 mm ² or more and 50 mm ² or less. However, the areas of the openings 15 and 16 are configured to be 1.5 times to 10 times the cross-sectional area of the flow path 14.

The liquid injection parts 17 and 18 include liquid reservoirs 21 and 22 that communicate with the openings 15 and 16, respectively, and communication parts 23 and 24 that communicate the liquid reservoirs 21 and 22 with both ends of the flow path 14, respectively. Each has.
The liquid reservoirs 21 and 22 are formed so that the cross-sectional area is equal to the area of the openings 15 and 16. Further, the communication portions 23 and 24 are configured so that the cross-sectional area thereof is narrower than the cross-sectional area of the liquid reservoir portions 21 and 22.

  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 channel 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 the 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 0.00. It is 01 mm to 5 mm.
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. 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. Here, the area of the openings 15 and 16 is 1 mm ² or more and 50 mm ² or less, and the area of the openings 15 and 16 is 1.5 times or more and 10 times or less of the cross-sectional area of the flow path 14. Therefore, the reaction solution can be easily supplied to the flow path 14.

  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. 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 bubble mixed in the flow path 14 expands by heating by heating the reaction part 4 in these denaturation process, annealing process, and extension reaction process, the area of the openings 15 and 16 becomes the flow path 14. Since the liquid injection parts 17 and 18 having a wider cross-sectional area than the flow path 14 are formed in the liquid injection parts 17 and 18 respectively, the mineral oil pushed out from the flow path 14 is stored in the liquid storage parts 21 and 22. It is stored in. As a result, the mineral oil is prevented from being pushed out from the openings 15 and 16, so that the reaction solution is kept closed, and loss due to evaporation of the reaction solution is avoided. Moreover, since the cross-sectional area of the flow path 14 is 0.5 mm ² or more and 10 mm ² or less, the sample solution is uniformly heated and the components of the purified sample solution are made uniform.
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 expand by heating to a predetermined temperature in a state where the bubbles are mixed in the flow path 14, the areas of the openings 15 and 16 are reduced. Since the liquid reservoirs 21 and 22 having a larger cross-sectional area than the flow path 14 are formed in the liquid injection parts 17 and 18, respectively, which are larger than the cross-sectional area of the flow path 14, the mineral oil pushed out from the flow path 14 The liquid is stored in the liquid reservoirs 21 and 22. 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, the area of the openings 15 and 16 is 1 mm ² or more and 50 mm ² or less, and the area of the openings 15 and 16 is 1.5 times or more and 10 times or less of the cross-sectional area of the flow path 14. Therefore, the reaction solution can be easily supplied to the flow path 14. Moreover, since the cross-sectional area of the flow path 14 is 0.5 mm ² or more and 10 mm ² or less, the sample solution is uniformly heated, and the components of the purified sample solution can be made uniform.
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.
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, the groove portion 12 and the liquid injection portions 17 and 18 were formed on the base material 2 of a resin plate made of PP (PP made by Novatec Co., 3 mm thick) by cutting. ABI PRISM Optical Cover (manufactured by ABI, 100 μm thickness) was attached as the film 13 so as to seal the opening end 12 </ b> A of the groove portion 12, thereby producing a reaction vessel 1 having a hollow channel 14. Here, the openings 15 and 16 have a circular shape in plan view, and the diameter thereof is 3 mm. Further, the length of the liquid reservoirs 21 and 23 in the thickness direction of the base material 2 is 1.7 mm. Furthermore, the flow path 14 has a circular cross section, and its diameter is 1 mm.

And 12 microliters of mineral oil, 4 microliters of PCR reaction solutions, and 12 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, first heating is performed at 94 ° C. for 2 minutes, followed by 35 cycles of heating at 95 ° C. for 1 second, 60 ° C. for 1 second, and 75 ° C. for 1 second, and further heating at 15 ° C. Yes.

Subsequently, as a comparative example, a groove portion and a liquid injection portion were respectively formed using the same resin plate as in the example, and a reaction vessel having a hollow flow path was created by attaching a film. Here, the opening has a circular shape in plan view, and its diameter is 1 mm. Moreover, the flow path has a circular cross section, and its diameter is 1 mm.
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.

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, it was confirmed that evaporation of the PCR reaction solution occurred in the middle of the PCR reaction, and the reaction efficiency was lowered or the solution could not be recovered.
As a result, even when the bubbles mixed in the flow path 14 expand, the liquid reservoirs 21 and 22 are formed because the areas of the openings 15 and 16 are larger than the cross-sectional area of the flow path 14. Therefore, it is confirmed that the mineral oil pushed out from the flow path 14 is stored in the liquid reservoirs 21 and 22 and the mineral oil is prevented from flowing out from the openings 15 and 16 and the blocked state of the reaction solution is maintained. It was done.

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 portion 40 is formed with an air hole 41 that is a through hole that communicates one end side of the flow path 14 away from the opening 15 and the surface 2A of the substrate 2.

  Moreover, in the said embodiment, although the liquid injection parts 17 and 18 are each provided with the liquid storage parts 21 and 22 and the communication parts 23 and 24, as shown to Fig.7 (a)-(d), the liquid injection part 51 is shown. , 52 may have the same cross-sectional area as that of the openings 15 and 16 in the whole. That is, the cross-sectional areas of the liquid injection parts 51 and 52 are the same as the cross-sectional areas of the liquid reservoirs 21 and 22 in the above embodiment over the thickness direction of the base material 2. Also in the reaction part 50 having such a configuration, the mineral oil pushed out from the flow path 14 when the reaction solution is heated is stored in the liquid injection parts 51 and 52 in the same manner as in the above embodiment. The state is maintained and loss of the reaction solution can be avoided.

Moreover, in the said embodiment, although the flow path 14 is formed with the groove | channel 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 61 may be constituted by hollow holes formed in the substrate 2. The flow path 61 is hollow inside the base material 2 and is formed so as 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 60 which has this flow path 61 is the base material 2 instead of the film 13 in the said embodiment, the 2nd base material 62 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 attached 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 62. A flow path 61 is formed. Moreover, in the manufacturing method of this reaction part 60, 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 silicone resin, for example by injection molding, for example suitably The flow path 61 may be configured by forming a hollow hole in the interior of 2.

Moreover, although the area of the opening is 1 mm ² or more and 50 mm ² or less, the reaction solution is not pushed out from the opening due to the expansion of bubbles when the reaction solution is heated, and the area of the opening is wider than the cross-sectional area of the flow path. The area of the opening may be narrower than 1 mm ² or larger than 50 mm ² .
Similarly, although the cross-sectional area of the flow path is 0.5 mm ² or more and 10 mm ² or less, the reaction solution is not pushed out from the opening due to the expansion of bubbles when the reaction solution is heated, and the area of the opening is the cross-sectional area of the flow path. The cross-sectional area of the flow path can be made smaller than 0.5 mm ² or larger than 10 mm ² if the components of the purified reaction solution can be made uniform by heating uniformly to the reaction solution. You may do it.
Furthermore, although the area of the opening is 1.5 times or more and 10 times or less with respect to the cross-sectional area of the flow path, it is possible to avoid the difficulty of supplying the solution and to uniformly heat the solution. If the components of the purified reaction solution can be made uniform, it may be less than 1.5 times or more than 10 times.

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.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Reaction container, 2 base material, 2A surface (one side), 2B back surface (other side), 3 reagent accommodating part 4, 40, 50, 60 reaction part, 5 detection part, 12 groove part, 12A opening end 13 film, 14, 61 Channel, 17, 18, 51, 52 Injection section (communication hole)
R reaction solution (sample solution)

Claims (11)

A reaction vessel in which a reaction part for performing a biochemical reaction is formed on a substrate,
The reaction part is
A channel through which a sample solution is supplied;
A liquid injection part for injecting the sample solution into the flow path;
Have
The liquid injection part is
A liquid reservoir having an opening opened on the outer surface of the substrate and having a cross-sectional area larger than at least the cross-sectional area of the flow path;
A communication section having a cross-sectional area smaller than the cross-sectional area of the liquid reservoir and having a cross-sectional area larger than the cross-sectional area of the flow path, and communicating one end of the flow path with the liquid reservoir;
A reaction vessel characterized by comprising: ^2. The reaction container according to claim 1, wherein an area of the opening is 1 mm ² or more and 50 mm ² or less. The reaction container according to claim 1 or 2, wherein a cross-sectional area of the flow path is 0.5 mm ² or more and 10 mm ² or less.   The reaction container according to any one of claims 1 to 3, wherein an area of the opening is 1.5 to 10 times a cross-sectional area of the flow path.   The other end of the flow path is communicated with another opening formed on one surface of the base material via another communication hole. Reaction vessel.   The said flow path is formed of the groove part formed in the other surface of the said base material, and the film which covers at least one part of the opening end of this groove part, The any one of Claim 1 to 5 characterized by the above-mentioned. The reaction container according to Item.   The reaction container according to claim 1, 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 7, 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 8, wherein the reagent container is concave.   The reaction container according to any one of claims 1 to 9, wherein the reaction section is for enzyme reaction. The reaction container according to claim 10, wherein the enzyme reaction is a polymerase chain reaction.

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