JP5000861B2 - Reaction chip - Google Patents

Description

  The present invention relates to a reaction chip.

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 above prior art, the concave portion provided on the surface of the base material or the base material is used as the reaction field, and when the temperature state of the sample solution stored in the concave portion is controlled. The temperature state of the entire sample solution may be difficult to control uniformly. That is, since the base material or base material of the reaction chip is formed of a material having relatively low thermal conductivity, such as silicon or polypropylene, the sample solution stored in the recess of the base material or base material is used. Thus, for example, when heating or cooling is performed by a temperature control device or the like disposed outside the reaction chip, the sample solution located in a region closer to the base material or the base material can suppress the temperature change. Thus, it may be difficult to uniformly generate a desired reaction for the entire sample solution.
This invention is made | formed in view of the said situation, and it aims at providing the reaction chip which can generate | occur | produce a desired reaction uniformly in a reaction part.

In order to solve the above problems and achieve the object, the reaction chip of the present invention according to claim 1 (for example, the reaction chip 10 in the embodiment) is a reaction chip used for a biochemical reaction. The groove (for example, the groove 21 in the embodiment) provided on the surface of the substrate (for example, the substrate 10a in the embodiment) and the opening end of the groove (for example, the opening in the embodiment) A channel-like reaction section (for example, the reaction section 12 in the embodiment) including a heat conductive film (for example, the film 23 in the embodiment) covering at least a part of the end 21a), and the base material A second groove portion (for example, an embodiment of the present invention) that is provided at a position that does not interfere with the opening portion of the groove portion on the surface opposite to the surface on which the thermal conductive film is disposed and connected to the groove portion. Second groove 31) in the form and the second Second film of a thermally conductive film attached on the bottom surface of the groove (e.g., the second film 32 in the embodiment) and, wherein the second groove portion and the second film, the When performing a biochemical reaction in the reaction part, a heat source is formed so as to be in contact with the second film, and the temperature of the reaction part is adjusted via the second film .

Furthermore, in the reaction chip of the present invention according to claim 2, the heat conductive film is a film having a layer made of metal .

  Furthermore, the reaction chip according to the third aspect of the present invention includes a detection unit (for example, the detection unit 13 and the detection recess 13a in the embodiment) capable of optical analysis on the surface of the base material. It is said.

  Furthermore, in the reaction chip according to the fourth aspect of the present invention, the detection section is concave.

  Furthermore, the reaction chip of the present invention according to claim 5 is a reagent storage unit that can store a reaction reagent on the surface of the base material (for example, reagent storage unit 11 and reagent storage recess 11a in the embodiment). It is characterized by having.

  Furthermore, in the reaction chip according to the sixth aspect of the present invention, the reagent container is concave.

  Furthermore, in the reaction chip of the present invention as set forth in claim 7, the reaction part is for enzyme reaction.

  Furthermore, in the reaction chip of the present invention according to claim 8, the enzyme reaction is a polymerase chain reaction.

According to the reaction chip of the present invention as set forth in claim 1, since the reaction part is in the form of a channel, the supply of the solution to the reaction part and the recovery of the solution from the reaction part are facilitated, and the groove part is formed. Since the reaction part is formed by a film whose thermal conductivity is increased by reducing the thickness relative to the base material to be processed, the temperature state of the entire solution stored in the reaction part can be easily and uniformly set. Can be controlled.
Furthermore, according to the reaction chip of the present invention as set forth in claim 2, since the reaction part is formed of a heat conductive film, the temperature state of the entire solution stored in the reaction part can be more easily determined. It can be controlled uniformly.

Furthermore, according to the reaction chip of the present invention as set forth in claim 3, at least a process for causing a desired reaction and a detection process can be continuously and efficiently performed on a single substrate. it can.
Furthermore, according to the reaction chip of the present invention as set forth in claim 4, the detection part can be easily formed on the surface of the substrate.
Furthermore, according to the reaction chip of the present invention as set forth in claim 5, at least a process for containing a reaction reagent and a process for causing a desired reaction are continuously and efficiently performed on a single substrate. Can be executed.
Furthermore, according to the reaction chip of the present invention as set forth in claim 6, it is possible to easily form the reagent container on the surface of the base material.

Furthermore, according to the reaction chip of the present invention as set forth in claim 7, the enzyme reaction can be easily and uniformly generated with respect to the entire solution in the reaction part.
Furthermore, according to the reaction chip of the present invention described in claim 8, the polymerase chain reaction can be easily and uniformly generated with respect to the entire solution in the reaction part.

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

  As shown in FIG. 1, for example, the biochemical reaction device 1 according to the present embodiment includes a reagent storage device 2 that executes a reagent storage step of storing a reaction reagent in a reaction chip 10, and a polymerase chain that is an enzyme reaction, for example. Consists of a reaction device 3 that executes a reaction step that causes a predetermined reaction such as a reaction (PCR: Polymerase Chain Reaction), and a detection device 4 that executes a detection step that detects a sample such as DNA by optical analysis, for example. Has been.

  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 solution in a 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 convex shape). 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. In addition, 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.

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.
In addition, the recessed part by cutting or metal mold | die formation etc. may be formed toward the groove part 21 from the surface 10A side of the base material 10a, and the wall thickness by the side of the surface 10A of the flow path 22 may be made thin. In this way, when the heat source for reaction is arranged at a position facing the surface 10A (for example, a position above the surface 10A), heat is quickly and uniformly transferred into the flow path 22.

  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.
The metal film 23 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 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 S11 shown in FIG. 5, as a reaction liquid supply process, the reaction solution 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 includes, for example, DNA extracted from blood or the like, or pre-purified template DNA, 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 a sealing process, the mineral oil is supplied from the opening 24 toward the inside of the reaction unit 12 storing the reaction solution, for example, as shown in FIGS. 6 (a) and 6 (b). Next, the mineral oil M is overlaid on the liquid surface of the reaction solution R exposed to the atmosphere inside the reaction unit 12 to seal the inside of the reaction unit 12. Note that the opening 24 may be covered with an appropriate film (for example, a film equivalent to the film 23 or the like) without being overlaid with mineral oil.

  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 DNA is heat denatured.

  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.
The film 23 may be formed by laminating an adhesive layer or applying a sealant to a resin film, a metal film, or a film obtained by laminating these films.

  Below, the test result which actually used the reaction chip 10 mentioned above for the test is demonstrated.

The groove part 21 and the through-holes 25 and 25 were formed in the base material 10a of the resin plate (PP made from Novatec, 3 mm thickness) which consists of polypropylene by cutting. ABI PRISM Optical Cover (manufactured by ABI, thickness 100 um) was pasted as the film 23 so as to seal the opening of the groove portion 21, thereby producing a reaction chip 10 having a hollow flow path 22.
Then, 5 ul of mineral oil, 5 ul of reaction solution, and 5 ul of mineral oil were put into the reaction part 12 through the opening 24 in this order, and the film 23 was stuck on both the openings 24 and 24 and sealed.
Then, a series of reaction generation steps were performed on the reaction chip 10.
From this test, it was found that an amplification product was obtained with the same efficiency as when a series of reaction generation steps were performed using a polypropylene microtube instead of the reaction chip 10.

As described above, according to the reaction chip 10 according to the present embodiment, since the reaction part 12 of the reaction chip 10 has a flow path shape, supply of the solution to the reaction part 12 and recovery of the solution from the reaction part 12 are performed. Becomes easy. In addition to this, since the reaction part 12 is formed by the film 23 whose thermal conductivity is increased by reducing the thickness relative to the base material 10a forming the groove part 21, the reaction part 12 has The temperature state of the entire stored solution can be easily and uniformly controlled.
Furthermore, since the film 23 is formed of a heat conductive film, the temperature state of the entire solution stored in the reaction unit 12 can be more easily and uniformly controlled.
Moreover, since the reaction chip 10 is configured to include 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 process, reaction process, and detection process 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 modification shown in FIGS. 8A to 8D, the reaction unit 12 is provided on the back surface 10B of the base material 10a. The groove portion 21, the film 23 for sealing the opening portion of the groove portion 21 by covering the opening end 21a of the groove portion 21, and the base material 10a are penetrated in the thickness direction and provided on the surface 10A of the base material 10a. Two through holes 25, 25 connected to the two openings 24, 24 and opened inside the groove 21 and at positions where the two openings 24, 24 do not interfere with each other on the surface 10A of the substrate 10a. Provided and connected to the groove 21 The two groove portions 31, the second film 32 and may be configured with affixed on the bottom surface 31A of the second groove 31 that.
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.
In this way, when a heat source for reaction is disposed at a position facing the surface 10A of the base material 10a (for example, a position above the surface 10A), heat is quickly and uniformly transferred into the flow path 22. Is done.

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 solution in the reaction unit 12.

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 of starting the extension reaction step after the reaction solution becomes relatively high temperature in order to suppress the occurrence of primer misannealing and oligomerization) is used. 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 the reaction unit according to the embodiment of the present invention, and shows a state in which mineral oil is overlaid on the liquid surface of the reaction solution inside the reaction unit. (B) is sectional drawing of the reaction part which concerns on embodiment of this invention, Comprising: It is a figure which shows the state which overlaid the mineral oil on the liquid level of the reaction solution 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.

Explanation of symbols

10 reaction chip 10a base material 11a reagent storage recess (reagent storage)
12 reaction part 13 detection part 13a detection recessed part (detection part)
21 Groove 21a Open end 22 Channel 23 Film 24 Open

Claims (8)

A reaction chip used for biochemical reaction,
A channel-shaped reaction section comprising a groove provided on the surface of the substrate and a heat conductive film covering at least a part of the opening end of the groove;
A second groove connected to the groove provided on the surface of the base material at a position that does not interfere with the opening of the groove on the surface opposite to the side on which the thermally conductive film is disposed ;
A second film comprising a thermally conductive film affixed to the bottom surface of the second groove,
Equipped with a,
The second groove and the second film are formed such that a heat source is in contact with the second film when the biochemical reaction is performed in the reaction part, and the reaction is performed via the second film. A reaction chip characterized in that the temperature of the part is adjusted . The reaction chip according to claim 1, wherein the heat conductive film is a film having a layer made of metal. The reaction chip according to claim 1, further comprising a detection unit capable of optical analysis on the surface of the base material. The reaction chip according to claim 3, wherein the detection unit is concave. The reaction chip according to any one of claims 1 to 4, further comprising a reagent storage unit capable of storing a reaction reagent on a surface of the base material. The reaction chip according to claim 5, wherein the reagent container is concave. The reaction chip according to any one of claims 1 to 6, wherein the reaction unit is for enzyme reaction. The reaction chip according to claim 7, wherein the enzyme reaction is a polymerase chain reaction.

Images