JP2014010109A - Microchip for nucleic acid amplification reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microchip for nucleic acid amplification reaction capable of performing simple and highly accurate analysis.SOLUTION: In a microchip for nucleic acid amplification reaction, a locking section for suppressing the movement of solid phase-like reagent containing a substance necessary for a nucleic acid amplification reaction is disposed in a reaction area. In the microchip for nucleic acid amplification reaction in which a locking section 3 is disposed in a reaction area 2, the movement of solid phase-like reagent can be suppressed or controlled by the locking section 3. Then, the solid phase-like reagent can be uniformly dissolved, and the solution and sample solution can be both uniformly and easily mixed. Thus, it is possible to provide the microchip for nucleic acid amplification reaction capable of performing simple and highly accurate analysis.

Description

  The present disclosure relates to a microchip for nucleic acid amplification reaction. More specifically, the present invention relates to a microchip for nucleic acid amplification reaction having a plurality of photodetection fields in one well serving as a reaction field for nucleic acid amplification reaction.

  In recent years, microchips having wells and flow paths for performing chemical and biological analysis on a silicon or glass substrate have been developed by applying microfabrication technology in the semiconductor industry. These microchips are beginning to be used in, for example, electrochemical detectors for liquid chromatography and small electrochemical sensors in medical settings.

  Such an analysis system using a microchip is called μ-TAS (micro-Total-Analysis System), a lab-on-chip, a biochip, and the like. As a technology that enables downsizing, integration, or downsizing of analyzers, it is attracting attention. Since μ-TAS can be analyzed with a small amount of sample and can be used as a disposable microchip, it is expected to be applied to biological analysis, especially for handling precious trace samples and many specimens. Has been.

  As an application example of μ-TAS, there is an optical detection device that introduces a substance into a plurality of regions arranged on a microchip and chemically detects the substance. As such an optical detection apparatus, for example, a reaction apparatus (for example, a real-time PCR apparatus) that optically detects a substance to be generated by causing a reaction between a plurality of substances such as a nucleic acid amplification reaction in a well on a microchip. and so on.

  Conventionally, in a microchip type nucleic acid amplification apparatus, a reagent and template DNA necessary for a nucleic acid amplification reaction are all mixed in advance, and this mixed solution is introduced into a plurality of wells arranged in the microchip to perform a reaction. Has been taken. However, this method requires a certain amount of time for the mixture to be introduced into the well, and during that time, the reaction proceeds in the mixture, facilitating non-specific nucleic acid amplification and improving the quantitativeness. There has been a problem of lowering.

  For example, Patent Document 1 discloses a microchip in which a plurality of reagents necessary for a nucleic acid amplification reaction are stacked and fixed in a predetermined order in a well. However, there is a need for a nucleic acid amplification reaction microchip capable of further simple and accurate analysis.

JP 2011-160728 A

  The main object of the present disclosure is to provide a nucleic acid amplification reaction microchip capable of simple and highly accurate analysis.

  In order to solve the above problems, the present disclosure provides a microchip for nucleic acid amplification reaction in which a reaction region is provided with a locking portion that suppresses the movement of a solid-phase reagent containing a substance necessary for nucleic acid amplification reaction. To do.

The locking portion may suppress movement in the longitudinal direction and / or thickness direction of the reaction region.
Further, the engaging portion may be a convex portion and / or a concave portion provided on the inner surface of the reaction region.
There may be a plurality of surfaces for performing light detection in the reaction region.
A sample solution may flow into the reaction region from a plurality of flow paths.
The solid phase reagent may be disposed on a surface where the light detection is performed.
The sample solution may have a channel through which the sample solution flows toward the solid phase reagent.

The convex portion may be one through which liquid can pass.
The convex portion may be provided in the reaction region, and the convex portion may be located in the middle in the short direction.
The convex part may be provided in the reaction region, and the convex part may be provided so as to face each other.
The convex portion may be fitted to the inner peripheral portion of the reagent.
The recess may be fitted to the outer periphery of the reagent.
Moreover, you may have the flow path into which the said sample solution flows toward the said solid-phase-type reagent.
Further, the reaction region may be at a position lower than a flow path into which the sample solution flows.
There may be a protrusion at a position where the reaction region flows from the flow path.
The solid-phase reagent may be arranged so as to occupy most of the area of the surface on which light detection is performed.

  The solid phase reagent may be a freeze-dried reagent. The solid phase reagent may be a tableting product. Two or more solid phase reagents may be used.

  According to the present disclosure, it is possible to provide a nucleic acid amplification reaction microchip capable of simple and highly accurate analysis.

It is a schematic diagram for demonstrating the structure of the microchip 1a which concerns on 1st embodiment of this indication. It is a schematic diagram for demonstrating the illustration of the cross section of the latching | locking part 3 of this indication. It is a schematic diagram for demonstrating the illustration when it has multiple combinations of the convex parts 31 and 31 which oppose in one well 2 of this indication. It is a mimetic diagram for explaining the illustration when it has two or more combinations of passage part 7 and convex part 31 in one well 2 of this indication. It is a schematic diagram for demonstrating the structure of the microchip 1b which concerns on 2nd embodiment of this indication. It is a schematic diagram for demonstrating the illustration when there are two or more things which the convex part 31 and the inner peripheral part R1 of the solid-phase reagent R fitted in one well 2 of this indication. It is a schematic diagram for demonstrating the illustration when there are two or more things which the convex part 31 and the inner peripheral part R1 of the solid-phase reagent R fitted in one well 2 of this indication. It is a schematic diagram for demonstrating the illustration when there exist two or more things which the recessed part 32 and the outer peripheral part R2 of the solid-phase reagent R fitted in one well 2 of this indication. It is a schematic diagram for demonstrating the illustration when there exist two or more things which the recessed part 32 and the inner peripheral part R2 of the solid-phase reagent R fitted in one well 2 of this indication. It is a schematic diagram when one solid-phase reagent R is accommodated in one well 2 of the present disclosure. In the microchip of this indication, it is a mimetic diagram for explaining the shape of a well from which solid phase reagent R is not from inside well 2. In the microchip of this indication, it is a mimetic diagram for explaining the illustration of channel 5.

  Hereinafter, preferred embodiments for carrying out the present disclosure will be described. In addition, embodiment described below shows typical embodiment of this indication, and, thereby, the range of this indication is not interpreted narrowly. The description will be made in the following order.

1. 1. Microchip for nucleic acid amplification reaction of the present disclosure 2. Configuration of a microchip for nucleic acid amplification reaction according to the first embodiment of the present disclosure 3. Configuration of a microchip for nucleic acid amplification reaction according to the second embodiment of the present disclosure 4. Configuration of a microchip for nucleic acid amplification reaction according to the second embodiment of the present disclosure 5. Configuration of nucleic acid amplification reaction microchip according to modified embodiment 1 of the present disclosure Configuration of microchip for nucleic acid amplification reaction according to modified embodiment 2 of the present disclosure

1. The microchip for nucleic acid amplification reaction of the present disclosure The microchip for nucleic acid amplification reaction (hereinafter also referred to as “microchip”) 1 of the present disclosure has a reaction region 2 that is a reaction field of the nucleic acid amplification reaction and is necessary for the nucleic acid amplification reaction. A locking portion 3 that suppresses the movement of the solid-phase reagent R containing various substances is provided. As the reaction region 2, for example, FIG. 1 shows wells 21a to 25a, FIG. 2 shows an example of a QQ cross section of the well 2, and FIGS. 3 and 4 show wells 201a to 205a. 5 shows wells 21b to 25b, and FIGS. 6 to 9 show wells 201b to 204b. FIG. 10 shows the well 2c. The shape of the well is not particularly limited, but is preferably rectangular and rounded at both ends. The length between both ends of the well is “well length (X)”, the width between both ends of the well is “well width (Y)” (length (X) ≧ width (Y)), and the depth of the well Is “well depth (Z)”.
The microchip 1 of the present disclosure includes, as a region into which a sample solution is introduced, an introduction unit 4 into which a liquid such as a sample is introduced from the outside, a reaction region 2 that serves as a reaction field for a nucleic acid amplification reaction, and an introduction unit 4 It is desirable that a flow path 5 (flow paths 51 to 55) for connecting the reaction area 2 and the reaction area 2 is provided.

Further, in one reaction region 2, there is a single surface 6 (hereinafter also referred to as “light detection side surface”) 6 on which light is incident and / or emitted to perform light detection of a nucleic acid amplification reaction in the reaction region. It is preferable that a plurality exist. The light detection side surface 6 is a surface on which a solid-phase reagent can be arranged. The solid-phase reagent R is preferably disposed on the surface 6 on the side where the light detection is performed. It is preferable that the area of one light detection side surface 6 is 12.5 to 400 mm ² .
The number of the light detection side surfaces 6 is not particularly limited, but is preferably plural. This number can be freely designed according to the arrangement of the locking portions 3, the size and number of solid phase reagents, irradiation light and detection light at the time of measurement, and the like. Further, light detection may be performed on the light detection side surface 6.
It is preferable that the solid phase reagent R is disposed so as to occupy most of the area of the surface 6 on the side where the light detection is performed, and is accommodated in the reaction region. It is preferable that a solid phase reagent R having an occupied area of preferably 70 to 100%, more preferably 80 to 95% of the area of the optical detection side surface 6 is disposed. Further, the thickness 61 refers to the length between the outer surface of the substrate layer 11 and the inner surface on which a solid phase reagent R (hereinafter also referred to as “reagent R”) can be disposed. The thickness 61 is preferably 0.5 to 2.0 mm.

The channel 5 is formed so that the sample solution flows into the reaction region 2. Each of the channels 51 to 55 preferably has a channel through which the sample solution flows toward the solid-phase reagent R. The inflow channel is preferably a channel in which the channel 5 is branched into a plurality. By connecting a plurality of flow paths to the reaction region 2, it is easy to uniformly dissolve a plurality of solid-phase reagents arranged in one reaction region.
Examples of the flow path 5 are shown in FIGS. 3, 4, and 12, but are not limited thereto.
For example, the flow path 5 is preferably provided with a branch flow path that allows the liquid to flow into the reaction region from the short positive direction and / or the short negative direction. The branched flow path of the flow path 5 is, for example, a direction substantially perpendicular to the longitudinal (X) direction or the short (Y) direction; an oblique direction with respect to the long (X) and short (Y) directions, etc. It is provided so that liquid may flow in from.
As shown in FIG. 12, by providing a branch channel for allowing the sample solution to flow from a direction substantially perpendicular to the longitudinal direction of the reaction region, it becomes easier for the liquid to flow equally into the reaction region. This facilitates uniform dissolution of the solid phase reagent in the reaction region.
At this time, as shown in FIG. 12A, a flow path for allowing the liquid to flow into the reaction region from the short positive direction or from the short negative direction may be provided. Further, as shown in FIG. 12C, a flow path for allowing the sample solution to flow into the reaction region alternately from the short positive direction and the negative direction or from both the short positive and negative directions may be provided. Further, as shown in FIG. 12B, a branch flow path may be provided so as to flow from the flow path 5 in an oblique direction before flowing into the reaction region in a substantially right angle direction.
In addition, as shown in FIG. 12D, it is preferable that the lengths of the respective channels are made equal so that the liquid in each channel 5 flows into the reaction region at the same time. As this flow path length, for example, the flow path length from the place where the introduction part or the flow path branches to the reaction region can be cited. By contacting the liquid with the solid-phase reagent at the same time, the plurality of solid-phase reagents R in the reaction region are dissolved at the same time, and the solution is easily homogenized.

  The locking portion 3 is preferably provided on the inner surface of the reaction region 2. Furthermore, it is preferable that the said latching | locking part 3 is formed by providing the convex 31 part and / or the recessed part 32 in the inner surface of the reaction area | region 2 (for example, refer FIG.2 and FIG.5). The convex portion 31 and the concave portion 32 may be formed, for example, in any of the longitudinal direction (X positive / negative direction), the short side direction (Y positive / negative direction), and the thickness direction (Z positive / negative direction).

The locking portion 3 can suppress the movement of the reagent R in the reaction region 2. Thereby, when a liquid (for example, sample solution) flows into the reaction region, it is prevented from moving to a place different from the surface on the light detection side where the solid-phase reagent is arranged. Further, by providing the locking portion 3, it is possible to adjust the arrangement of the solid phase reagent R in the reaction region 2. In addition, the locking portion 3 makes it possible to evenly arrange the reagent R in the reaction region 2. Thereby, when the sample solution flows into the reaction region 2, the reagent R can be evenly dissolved in the reaction region 2.
Preferably, it is preferable to provide a locking portion so as to suppress movement in the longitudinal direction and / or thickness direction of the solid phase reagent R. Thereby, even if a plurality of solid-phase reagents are arranged in one reaction region, it is easy to uniformly dissolve in the flowing sample solution.

Here, when a nucleic acid amplification reaction is performed by mixing a sample solution and a reagent solution, it is important to uniformly mix these solutions in order to make the reaction accurate and perform a highly accurate analysis. However, when the reaction well is designed to be large and a plurality of solid phase reagents are arranged, the time required for these solutions to be uniform varies depending on the position of the reagent arranged in the reaction well. For example, when the solid phase reagent is placed at the center of the reaction well and when it is placed at the end, it is shorter when the solid phase reagent is dissolved and the solution becomes uniform at the center. It takes time, and it takes a long time to place it at the end. Therefore, when the arrangement of the solid phase reagent is not controlled, there is a problem that the nucleic acid amplification reaction takes a long time or results in low reproducibility. Furthermore, when the concentration gradient of the reagent is large in the nucleic acid amplification method, there arises a problem in accuracy such as a decrease in sensitivity and specificity.
In order to uniformly dissolve the solution, there is a method of performing a mixing process such as stirring, vibration, ultrasonic treatment or the like on the solution. However, this requires a new process or apparatus and is not a simple method.

However, the microchip for nucleic acid amplification reaction in which the locking portion 3 of the present disclosure is installed in the reaction region 2 can suppress or control the movement of the solid phase reagent by the locking portion 3. . Furthermore, it is desirable to arrange the locking portion 3 so as to suppress movement in the longitudinal direction and / or thickness direction of the reaction region.
By doing so, it is possible to uniformly dissolve a plurality of solid phase reagents in the same reaction region without using physical treatment. Further, the solution can be made more efficient by adding a physical mixing process. Since the solid-phase reagent can be uniformly dissolved, this solution and the sample solution can be easily mixed uniformly. Thereby, it is possible to prevent the nucleic acid amplification reaction from taking a long time and to further improve reproducibility. In addition, it is possible to prevent a decrease in sensitivity and specificity in the nucleic acid amplification reaction.
Therefore, if the microchip for nucleic acid amplification reaction according to the present disclosure is used, simple and highly accurate analysis can be performed.

The microchip substrate (substrate layers 11 to 13) can be formed of glass or various plastics (PP, PC, COP, PMDS, etc.). The material of the microchip is desirably a material that is transparent to the measurement light emitted from the optical detection unit, has less autofluorescence, and has less wavelength error because of less wavelength dispersion.
Molding of the reaction region (well) 2, the locking portion 3, each flow path 5, and the passage portion 7 to the substrate of the microchip can be performed by wet etching or dry etching, nanoimprinting, injection molding, or machining. Is possible. The channel 5 may be formed to connect the main channel and the reaction region, and to arrange a branch channel for allowing the sample solution to flow from the main channel to the solid phase reagent R in the reaction region. .
The microchip can be formed by sealing the substrate on which the reaction region (well) 2, the locking portion 3, each flow path 5, the passage portion 7 and the like are formed with the same material or different materials. is there.

  The “nucleic acid amplification reaction” performed using the microchip according to the present disclosure includes a conventional PCR (Polymerase Chain Reaction) method in which a temperature cycle is performed and various isothermal amplification methods that do not involve a temperature cycle. Examples of isothermal amplification methods include LAMP (Loop-Mediated Isothermal Amplification) method, SMAP (SMartAmplification Process) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, ICAN (Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids) method. (Registered trademark), TRC (Transcription-Reverse transcription Concerted) method, SDA (Strand Displacement Amplification) method, TMA (Transcription-Mediated Amplification) method, RCA (Rolling Circle Amplification) method and the like. In addition, the “nucleic acid amplification reaction” broadly encompasses nucleic acid amplification reactions by temperature variation or isothermal for the purpose of nucleic acid amplification. These nucleic acid amplification reactions also include reactions involving quantification of amplified nucleic acids such as real-time PCR.

2. Configuration of Microchip for Nucleic Acid Amplification Reaction According to First Embodiment of Present Disclosure FIG. 1 is a schematic diagram illustrating the configuration of a microchip 1a according to the first embodiment of the present disclosure. 1A is a schematic top view, and FIG. 1B is a schematic cross-sectional view corresponding to the P1-P2 cross section of FIG. 1A. FIG. 2 is an illustration of a schematic cross-sectional view corresponding to the QQ cross section of FIG. 1A. The description of the same configuration as the nucleic acid amplification reaction microchip of the present disclosure described above will be omitted as appropriate.

In the microchip for nucleic acid amplification reaction indicated by reference numeral 1a in the figure, a reaction region 2 serving as a reaction field for the nucleic acid amplification reaction is restrained from moving the solid phase reagent R containing a substance necessary for the nucleic acid amplification reaction. A stop 3 is provided. A plurality of reaction regions 2 may be provided as necessary as shown in the wells 21a to 25a. When a plurality of reaction regions 2 are provided, the same or different reaction regions 2 may be provided.
Each of the channels 51 to 55 is preferably a channel through which the sample solution flows toward the solid-phase reagent R. This facilitates dissolution of the solid phase reagent.

Further, it is preferable that a plurality of surfaces (light detection side surfaces) 6 on the light detection side exist in the reaction region 2 as shown in FIG. 1B. The surface 6 on the side where the light detection is performed is preferably provided in the thickness direction. Further, it is preferable that the solid-phase reagent R is disposed on the light detection side surface 6. Two or more solid-phase reagents R may be used. The area in the XY direction of one reaction region 2 is preferably 12.5 to 400 mm ² , and the thickness 61 is preferably 0.5 to 2.0 mm.

The locking portion 3 is preferably a convex portion 31 formed on the inner surface of the reaction region 2. The convex portion 31 only needs to have a shape or a state through which a liquid (for example, a sample solution or a mixed solution of the reagent R) can pass. The passage portion 7 may be provided as a portion through which the liquid can pass. By providing the locking portion, when the reagent solution flows into the reaction region, the movement of the solid phase reagent to the adjacent light detection portion is restricted. And, by providing a locking part and partitioning the solid phase reagent so that they do not contact each other, the fluid can be moved, so that the dissolution of the reagent is easily uniform in one reaction region, and a plurality of light detections are performed. In this case, the error of each detection result is reduced.
The proportion of the cross-sectional area of the convex portion 31 in the total area of the QQ cross section of the reaction region 2 where the convex portion 31 is present is preferably 10 to 60%, more preferably 20 to 50%. The total area of the QQ cross section can be obtained by (well width (Y) × well depth (Z)) in the reaction region.

Examples illustrated in FIGS. 2A to I as the QQ cross section of the locking portion 3 of the present disclosure are not limited thereto.
As shown in FIG. 2A, it is preferable that the convex portions 31 are provided at both ends of the inner surface in the short direction, and the passage portion 7 is provided near the center. Furthermore, this convex part 31 may be provided so that a part protrudes in the thickness direction. Further, as shown in FIGS. 2B and 2C, it is preferable to provide a passage portion 7 in the vicinity of the center in the lateral direction and further in the thickness direction.
Moreover, as shown in FIG. 2D, it is preferable to provide the passing portions 7 and 7 at both ends of the inner surface in the short direction, and the convex portion 31 near the center. Furthermore, this convex part 31 may be provided so that a part protrudes in the thickness direction. Moreover, as shown to FIG. 2E and F, it is preferable to provide the passage part 7 in the both ends of a transversal direction, and also the thickness direction.
Moreover, as shown to FIG. 2G, the convex part 31 is provided in the inner surface of the reaction area | region 2 at the both ends of a transversal direction, and its center vicinity, and several passage is carried out between the convex part 31 of a center, and the convex part 31 of both ends. It is preferable to provide the part 7. Furthermore, this convex part 31 may be provided so that a part protrudes in the thickness direction.
Moreover, as shown in FIG. 2H, it is preferable that the convex portions 31 are provided in a lattice shape in the short side direction and the thickness direction, and the passing portion 7 is provided between the lattices. The lattice-like convex portions 31 may be provided so that a part thereof protrudes in the thickness direction.

  Further, as shown in FIG. 2I, it is preferable to provide a fibrous or membrane-like one that has water permeability and allows the solubilized reagent R to pass therethrough. For example, a nonwoven fabric, a woven fabric, etc. are mentioned. Examples of the raw material include aramid, glass, cellulose, nylon, vinylon, polyester, polyolefin, rayon, low density polyethylene resin, ethylene vinyl acetate resin, synthetic rubber, copolymerized polyamide resin, and copolymerized polyester resin. Among these, you may use 1 type or in combination of 2 or more types.

  Moreover, it is also possible to use combining suitably the structure of FIG. Moreover, you may provide the convex part 31 and the passage part 7 on the basis of the thickness direction.

Moreover, as shown to FIG. 3A-C, it is preferable to provide in the reaction area | region 2 so that the convex part 31 may oppose. It is preferable to provide the convex part 31 which opposes a transversal direction and / or a thickness direction on the inner surface of the reaction region 2. The passage portion 7 is preferably provided between the convex portions 31 and 31. In addition, the shape of the QQ cross section of FIG. 3A-C has the example structure shown to FIG.
Examples of the shape of the convex portions 31 that face each other include polygonal shapes such as triangles and squares; semi-elliptical shapes; semi-circular shapes and the like.

Moreover, as shown to FIG. 4A-B, it is preferable that the convex part 31 is provided in the reaction area | region 2 so that it may be located in the middle of a transversal direction and / or a thickness direction. And it is preferable that the passing part 7 is provided in the both ends of this convex part 31. FIG. In addition, the shape of the QQ cross section of FIG. 4A-B has the structure of an example shown to FIG.
Examples of the shape of the convex portion at the intermediate position include a polygonal shape such as a triangle and a square; a semi-elliptical shape; a semicircular shape; In the case of a polygonal shape, it may be rounded.
3A to 3C and 4A to 4B, the flow path 5 may be provided so that the sample solution flows into the reaction region 2 from the short positive direction and / or the short negative direction. . It is preferable that the flow path 5 is formed so that the sample solution flows from either the short positive direction or the short negative direction because the microchip can be downsized.

  In the microchip 1a of the present disclosure, the substrate layer 11 is bonded to the substrate layer 12 on which the reaction regions (wells) 21 to 25, the locking portions 3, the flow paths 5, the passage portions 7, and the like are formed. The substrate layer 13 is bonded to the substrate (see FIG. 1B). In the microchip 1a, when the bonding of the substrate layer 11 and the substrate layer 12 is performed under a negative pressure with respect to the atmospheric pressure, the reaction regions (wells) 21 to 25, the locking portion 3, each flow path 5, and the passage portion 7 can be hermetically sealed so as to have a negative pressure (1/100 atm) with respect to the atmospheric pressure. In the microchip 1a, the region into which the sample solution is introduced is set to a negative pressure with respect to the atmospheric pressure, whereby the sample solution is sucked by the negative pressure inside the microchip when the sample solution is introduced, and a fine channel structure is formed. In addition, the sample solution can be introduced into the microchip 1a in a shorter time.

  The material of the substrate layers 11, 12 and 13 can be glass or various plastics. Preferably, the substrate layers 12 and 13 are made of a material having gas impermeability. By using the substrate layers 12 and 13 constituting the outer surface of the microchip 1a as materials having gas impermeability such as PC, the sample solution introduced into the wells 21 to 25 is vaporized by heating in the nucleic acid amplification reaction. Further, it is possible to prevent disappearance (liquid loss) through the substrate layer 11. Further, when the region of the microchip 1a into which the sample solution is introduced is hermetically sealed as a negative pressure with respect to the atmospheric pressure, the negative pressure inside the microchip 1a is prevented by preventing air from penetrating from the outside. Therefore, it is preferable that the substrate layers 12 and 13 are made of a material having gas impermeability.

As the material of the substrate layer having gas impermeability, glass, plastics, metals, ceramics, and the like can be adopted. As plastics, PMMA (polymethyl methacrylate: acrylic resin), PC (polycarbonate), PS (polystyrene), PP (polypropylene), PE (polyethylene), PET (polyethylene terephthalate), diethylene glycol bisallyl carbonate, SAN resin ( Styrene-acrylonitrile copolymer), MS resin (MMA-styrene copolymer), TPX (poly (4-methylpentene-1)), polyolefin, SiMA (siloxanyl methacrylate monomer) -MMA copolymer, SiMA- Fluorine-containing monomer copolymers, silicone macromers (A) -HFBuMA (heptafluorobutyl methacrylate) -MMA terpolymers, disubstituted polyacetylene-based polymers, and the like can be given. Examples of the metals include aluminum, copper, stainless steel (SUS), silicon, titanium, tungsten, and the like. Examples of ceramics include alumina (Al ₂ O ₃ ), aluminum nitride (AlN), silicon carbide (SiC), titanium oxide (TiO ₂ ), zirconia oxide (ZrO ₂ ), and quartz.

  The substrate layer 11 is preferably made of an elastic material. In the microchip 1a, the substrate layer 11 that seals the introduction part 4 is made of an elastic material, so that a part of a puncture member such as a needle can be penetrated into the introduction part 4 from the outside of the microchip 1a. Become. When a sample solution is filled in a syringe or the like to which a needle is connected in advance and the substrate layer 11 is pierced with the needle, the sealed introduction portion 4 is connected only to the inside of the syringe, and the sample solution is not generated. Can be introduced into the microchip 1a.

  Further, when the region where the sample solution is introduced is hermetically sealed as a negative pressure with respect to the atmospheric pressure, the point between the outside of the microchip 1 a and the introduction unit 4 is reached when the tip of the needle reaches the introduction unit 4. The sample solution in the syringe is automatically sucked into the introduction unit 4 due to the pressure difference.

  By forming the substrate layer 11 with an elastic material, the puncture site is naturally sealed by the self-sealing property of the substrate layer 11 when the needle is pulled out from the introduction portion 4 after the sample solution is introduced. Can be. In the present technology, natural sealing of the puncture site of the needle due to elastic deformation of the substrate layer is defined as “self-sealing property” of the substrate layer.

  Examples of the material for the substrate layer having elasticity include acrylic elastomers, urethane elastomers, fluorine elastomers, styrene elastomers, epoxy elastomers, natural rubbers, etc., in addition to silicone elastomers such as polydimethylsiloxane (PDMS). .

  In the case of optically analyzing the substance held in each well of the microchip 1a according to the present disclosure, the material of each substrate layer is light transmissive, has less autofluorescence, and has a small wavelength dispersion. Therefore, it is preferable to select a material with a small optical error.

  Next, the reagent R accommodated in the well of the microchip 1a will be described. As shown in FIG. 1, it is preferable to arrange at least one solid-phase reagent R on each light detection side surface 6 in the well 2. It is preferable to arrange two or more types of reagents R on the light detection side surface 6 in a planar or three-dimensional manner. The two or more types of reagents Ra and Rb at this time contain at least a part of a substance necessary for obtaining an amplified nucleic acid chain in the nucleic acid amplification reaction. Specifically, oligonucleotide primers (hereinafter also referred to as “primers”), nucleic acid monomers (dNTPs), enzymes, and reaction buffers that are complementary to at least a part of the base sequence of DNA, RNA or the like to be amplified. Ingredients included. In addition, although not directly required for nucleic acid amplification reaction, a probe equipped with a label such as a fluorescent label for detecting the amplified nucleic acid chain, a detection reagent that intercalates into a double-stranded nucleic acid, etc. As a substance necessary for chain detection, it can be a component contained in the reagents Ra and Rb.

  Components necessary for the nucleic acid amplification reaction contained in the reagent Ra and the reagent Rb may have different compositions. For example, the reagent Ra may be a reagent solution that contains a primer and does not contain an enzyme (first reagent solution), and the reagent Rb may be a reagent solution that contains an enzyme and does not contain a primer (second reagent solution). it can. In this way, the reagent Ra containing the primer does not contain the enzyme, and the reagent Rb containing the enzyme does not contain the primer, so that the primer and the enzyme are not mixed until the sample solution is introduced into the well. Occurrence is suppressed. The reagent Ra may include an enzyme-containing reagent solution (second reagent solution), and the reagent Rb may include a primer-containing reagent solution (first reagent solution). , Rb can be of any composition. The shapes of the reagents Ra and Rb are not limited, and any shape may be used as long as the volume can be accommodated in the well 2. It is preferable that the shape corresponds to the above-described locking portion 3 (specifically, the convex portion 31 or the concave portion 32). Moreover, the reagents Ra and Rb having the same composition may be accommodated in a plurality of wells provided in the microchip 1a, and the reagents Ra and Rb having different compositions may be accommodated in each well.

The solid-phase reagent R only needs to contain the necessary reagent and be molded into a desired shape and accommodated in the well 2. In order to produce the reagent R, it is possible to dry and solidify the reagent solution and mold it, or to mold it from a powdery reagent raw material.
As a drying method, for example, freeze-drying is suitable. In addition, it is preferable that freeze-drying includes steps of preliminary freezing, primary drying (sublimation freezing), and secondary drying (removing bound water).
In preliminary freezing, the freezing temperature may be equal to or lower than the eutectic point (temperature at which the reagent solution freezes), but it is frozen at about -40 ° C for the purpose of preventing enzyme deactivation and freezing the reagent solution completely. It is desirable to make it. In the primary drying, the reagent solution frozen in the preliminary freezing step is dried. At this time, by drying the reagent solution below the eutectic point, dissolution during drying is prevented, and water contained in the reagent solution can be sublimated. The degree of vacuum in the primary drying is desirably 100 Pa or less, for example. Since the boiling point of water at 100 Pa is about −20 ° C., it is close to the eutectic point of the reagent solution described above, and dissolution of the reagent solution during drying is prevented. The vacuum degree of primary drying should just select an appropriate value according to the eutectic point of the prepared reagent liquid. In the secondary drying, water in a molecular state attached to components contained in the reagent solution after the primary drying is removed. The dryness of the reagent solution may be increased by heating to a temperature at which the components contained in the reagent solution do not deactivate or denature.

In addition, after freeze-drying, it may be compressed by applying pressure with a tableting machine or the like so that the dissolution rate does not decrease.
In order to mold while maintaining solubility, an excipient, a disintegrant, a binder, a lubricant, and the like may be added to the reagent solution. Examples of excipients include D-mannitol, glycerin, lactose, starch, dextrin, sucrose, and crystalline cellulose. Examples of the binder include starch paste and hydroxypropyl cellulose. Examples of disintegrants include crospovidone, carmellose, carmellose calcium, hydroxypropyl cellulose, crystalline cellulose, powdered cellulose, corn starch, xylitol, and lubricants such as magnesium stearate. Those additives that do not affect the nucleic acid amplification reaction are suitable.

The solid phase reagent R may be formed from a powdered reagent with a tableting machine or the like.
Mix the excipient with the reagent to make the reagent powder. Examples of the method for forming a powder include fluidized bed granulation, high-speed stirring granulation, dry granulation, spray drying (spray drying), and freeze drying. A tableting method and a molding method are conceivable for molding the powder.
In the direct tableting method, a certain amount is put into the above-mentioned molds having different planar shapes, and solidified by applying an optimum pressure. In order to maintain solubility even after solidification, the above-mentioned excipients, disintegrants, saccharides and the like may be added.
In the molding method, a mixed powder containing a drug is granulated with water or a water / alcohol binder, and compressed under low pressure while being wet. Thereafter, the solvent is evaporated to increase the pores.

The use of the microchip 1a of the present disclosure will be described. First, a sample solution obtained by collecting a sample is injected from the introduction unit 4. The injected sample solution flows into each of the channels 51 to 55. The well 25a connected to the flow channel 55 will be described. The sample solution flows from the flow channel 55 to each branch flow channel, and further flows toward each reagent R in the well 25a. Then, a nucleic acid amplification reaction is performed in the reaction region, and the reaction is measured by light detection in real time on a light-detectable surface (for example, surface 6 on the light detection side).
The surface capable of detecting light is not particularly limited as long as it is a surface capable of detecting light within the reaction region 2. For example, the part between the latching | locking part 3 and the latching | locking part 3, the location between the edge of the reaction region 2 and the latching | locking part 3, the surface 6 by the side of a light detection surface, etc. are mentioned.
In addition, when there are a plurality of locations where light is detected in one well, it is possible to obtain and display the average value of these by a statistical method, or display the measured values separately. Is possible.
As described above, the reagent R is evenly arranged and locked by the convex portion 31 in the reaction well 2, while the passage portion 7 makes it easy for the liquid to pass through and mix in the well. Thereby, each reagent R can be melt | dissolved equally. Furthermore, the reaction can be prevented from being prolonged and reproducibility can be improved. It is possible to prevent a decrease in sensitivity and specificity, and it is possible to improve quantitativeness. Therefore, according to the present disclosure, simple and highly accurate analysis is possible.
In addition, in the present disclosure, the location of light detection and the arrangement position of the reagent do not have to be one-to-one, and the reagent is uniformly dissolved, so that the entire chip can be detected by something like an image sensor or a part of the well It is also possible to detect only by PD.

3. Configuration of Microchip for Nucleic Acid Amplification Reaction According to Second Embodiment of Present Disclosure FIG. 5 is a schematic diagram illustrating the configuration of a microchip 1b according to the second embodiment of the present disclosure. 5A is a schematic top view, and FIG. 5B is a schematic cross-sectional view corresponding to the P1-P2 cross section of FIG. 5A. 6-9B are illustrations of cross-sectional schematic diagrams corresponding to the MM cross section of FIG. 5A. The description of the same configuration as the above-described nucleic acid amplification reaction microchip will be omitted as appropriate.

  In the figure, the microchip for nucleic acid amplification reaction indicated by reference numeral 1b has a solid phase reagent R containing a substance necessary for the nucleic acid amplification reaction in the reaction region 2 (wells 21b to 25b) serving as a reaction field of the nucleic acid amplification reaction. The latching | locking part 3 which suppresses a movement of is provided. A plurality of reaction regions 2 may be provided as necessary as shown in the wells 21a to 25b. When a plurality of reaction regions 2 are provided, the same or different reaction regions 2 may be provided.

  Furthermore, as shown in FIG. 5B, it is preferable that the reaction region 2 has a plurality of surfaces (light detection side surfaces) 6 on the light detection side. Light for performing light detection passes through the light detection side surface 6. Examples of the light detection side surface 6 include the area between the broken lines in FIGS. The broken line is the same as the width of the reaction region in the short direction. The solid phase reagent R is preferably disposed on the surface 6 on which the light detection is performed. The solid phase reagent R may be two or more.

The locking part 3 is preferably formed in the reaction region 2 and fitted with a solid-phase reagent R. Further, the locking portion 3 is preferably a convex portion 31 or a concave portion 32. Thereby, it becomes possible to suppress the movement of the reagent.
The convex part 31 should just be formed so that it may fit in the inner peripheral part R1 of the solid-phase reagent R, and the state which has not penetrated the inner peripheral part of the solid-phase reagent R (refer FIG. 6B) Or any of the states of penetration (refer to Drawing 7B) may be sufficient. The outer periphery of the reagent R may be in contact with the inner surface of the well in the short direction. However, the passage portion 7 is provided between the inner surface of the well and the reagent R, so that the reagent solution flows into the well. It is preferable because it is easy.
Note that the solid reagent R may have any shape as long as it fits the convex portion 31. And the ratio for which the cross-sectional area of the convex part 31 accounts among the total area of the reagent R of a MM cross section becomes like this. Preferably it is 10 to 60%, More preferably, it is 20 to 50%. In addition, the total area (MM cross section) of the reagent R can be calculated by the height of the reagent (Z) × the width of the reagent (Y).

The concave portion 32 only needs to be formed so as to be fitted to the outer peripheral portion R2 of the solid phase reagent R, and surrounds a part of the outer peripheral portion R2 of the solid phase reagent R (see FIG. 8B). Or any of the state (refer FIG. 9B) which surrounded all may be sufficient. The concave portion 32 may be ring-shaped, which makes it possible to surround the solid phase reagent R. When enclosing the entire outer peripheral portion R2 of the solid phase reagent R, it is preferable to provide the concave portion 32 with a passage portion 7 through which a liquid can pass. The passage unit 7 may adopt the same configuration as described in the first embodiment of the present disclosure.
In addition, the shape of the solid reagent R should just be a thing fitted to the recessed part 32. FIG. And the ratio for which the cross-sectional area of the reagent R accounts among the total area of the recessed part 32 of a MM cross section becomes like this. Preferably it is 10 to 60%, More preferably, it is 20 to 50%. In addition, the cross-sectional area (MM cross section) of the recessed part 32 can be calculated | required by the width (Y) x thickness 61 (Z) of a well. The distance between the broken lines as shown in FIGS. 8 and 9 is the width (Y) of the well.

The use of the microchip 1b of the present disclosure will be described. A sample solution is injected from the introduction part 4. The injected sample solution flows into each of the channels 51 to 55. The well 25b connected to the flow path 55 will be described. The sample solution flows from the flow channel 55 to each branch flow channel, and further flows toward each reagent R in the well 25b. Then, a nucleic acid amplification reaction is performed, and the reaction at each light detection side surface 6 is measured by light detection in real time.
Reagents R are evenly arranged in the reaction well 2 by the recesses 32, and the diffusion distance for achieving uniformity can be shortened. Thereby, each reagent R can be dissolved evenly promptly. Furthermore, the reaction can be prevented from being prolonged and reproducibility can be improved. It is possible to prevent a decrease in sensitivity and specificity, and it is possible to improve quantitativeness.

4). Configuration of the microchip for nucleic acid amplification reaction according to the third embodiment of the present disclosure The nucleic acid amplification reaction microchip according to the third embodiment of the present disclosure is a combination of the first embodiment and the second embodiment of the present disclosure. It is. The description of the same configuration as the nucleic acid amplification reaction microchip of the present disclosure described above will be omitted as appropriate.
The locking portion 3 of the present disclosure is provided as a convex portion 31 and / or a concave portion 32 on the inner surface of the reaction region 2. As described above, the convex portions 31 may be provided so as to face each other in the lateral direction and / or the thickness direction. Further, as described above, the convex portion 31 may be provided so as to be positioned in the middle in the short direction. Moreover, you may provide the above-mentioned convex part 31 and the recessed part 32 so that it may fit to the inner peripheral part R1 and outer peripheral part R2 of the solid-phase reagent R. FIG.
A plurality of reaction regions 2 having different locking portions 3 may be provided in the nucleic acid amplification reaction microchip according to the third embodiment of the present disclosure.

5. Configuration of Microchip for Nucleic Acid Amplification Reaction According to Modified Embodiment 1 of the Present Disclosure FIG. 10 is a part of a schematic diagram illustrating the configuration of the microchip 1d according to the modified embodiment 1 of the present disclosure, and shows the upper surface of the well 2C. It is a schematic diagram. The well 2 </ b> C is for placing the solid phase reagent R in the reaction region 2, which is smaller than the number of the light detection side surfaces 6. FIG. 10 is a diagram showing a state in which most of the reaction region 2 is filled with one solid phase reagent R. FIG.
The description of the same configuration as the above-described nucleic acid amplification reaction microchip will be omitted as appropriate. The above-described configuration may be incorporated as appropriate.
The area in the XY direction of one reaction region 2 is preferably 12.5 to 400 mm ² , and the thickness 61 is preferably 0.5 to 2.0 mm. Further, it is preferable that the solid phase reagent R having an occupied area of 70 to 100%, more preferably 80 to 95% of the area in the XY direction of the surface 6 for optical detection is filled. is there. As an example of the capacity of the reaction region 2, the capacity of the reaction region 2 is preferably 6.25 to 800 μL, and more preferably 25 to 200 μL.

6). Configuration of Nucleic Acid Amplification Reaction Microchip According to Modified Embodiment 2 of the Present Disclosure FIG. 11 is a part of a schematic diagram illustrating the configuration of the microchip 1e according to the modified embodiment 2 of the present disclosure. It is an illustration of the cross-sectional schematic diagram corresponding to a -M cross section. By adjusting the positional relationship between the flow path and the well, the shape of the entrance of the well, and adjusting the spatial space in the reaction area, the reagent is prevented from coming out of the reaction well when the solid-phase reagent breaks. It becomes possible. Thereby, it becomes easy to uniformly dissolve a plurality of reagents existing in one well. The description of the same configuration as the above-described nucleic acid amplification reaction microchip will be omitted as appropriate. The above-described configuration may be incorporated as appropriate.
11A and 11B show a part of a microchip having a well that is a dead-end type.
As shown in FIG. 11A, the reaction region 2 is preferably at a position lower than the flow path 5 into which the sample solution flows. Furthermore, it is preferable that the gap between the solid-phase reagent R and the well 2 is small in the thickness direction.
Further, as shown in FIG. 11B, it is preferable that there is a protrusion at a position where the reaction region 2 flows from the flow path 5. Accordingly, it is preferable to provide a shield only at the inlet portion where the sample solution flows from the flow path into the well.
FIGS. 11C and 11D show a part of a microchip having wells that are penetrating through which the sample solution flowing from the flow path 5 can flow from the short positive direction and the negative direction.
As shown in FIG. 11C, the reaction region 2 is preferably at a position lower than the flow path 5 into which the sample solution flows. Furthermore, it is preferable that the gap between the solid-phase reagent R and the well 2 is small in the thickness direction.
Further, as shown in FIG. 11D, it is preferable that there is a protrusion at a position where the reaction region 2 flows from the flow path 5. Accordingly, it is preferable to provide a shield only at the inlet portion where the sample solution flows from the flow path into the well.

The present technology can employ the following configurations.
[1] A nucleic acid amplification reaction microchip in which a reaction part is provided with a locking part that suppresses the movement of a solid-phase reagent containing a substance necessary for a nucleic acid amplification reaction.
[2] The microchip for nucleic acid amplification reaction according to [1], wherein the engaging portion is a convex portion and / or a concave portion provided on the inner surface of the reaction region.
[3] The microchip for nucleic acid amplification reaction according to the above [1] or [2], wherein the convex portion is capable of passing a liquid.
[4] The microchip for nucleic acid amplification reaction according to any one of [1] to [3], wherein a plurality of light detection side surfaces exist in the reaction region.
[5] The microchip for nucleic acid amplification reaction according to any one of [1] to [4], wherein a sample solution flows into the reaction region from a plurality of flow paths.
[6] The microchip for nucleic acid amplification reaction according to any one of [1] to [5], wherein the solid-phase reagent is disposed on a surface where the light detection is performed.
[7] The microchip for nucleic acid amplification reaction according to any one of [1] to [6], further including a channel through which the sample solution flows toward the solid-phase reagent.
[8] The microchip for nucleic acid amplification reaction according to any one of [1] to [7], wherein the locking portion suppresses movement in the longitudinal direction and / or thickness direction of the reaction region.
[9] The microchip for nucleic acid amplification reaction according to any one of [1] to [8], wherein the convex portion is provided in the reaction region, and the convex portion is located in the middle of the lateral direction. .
[10] The microchip for nucleic acid amplification reaction according to any one of [1] to [9], wherein the convex portion is provided in the reaction region and the convex portion is opposed to the convex portion.

[11] The microchip for nucleic acid amplification reaction according to any one of [1] to [10], wherein the convex portion is fitted to an inner peripheral portion of the reagent.
[12] The microchip for nucleic acid amplification reaction according to any one of [1] to [11], wherein the concave portion is fitted to an outer peripheral portion of the reagent.
[13] The microchip for nucleic acid amplification reaction according to any one of [1] to [12], further including a channel through which the sample solution flows toward the solid-phase reagent.
[14] The microchip for nucleic acid amplification reaction according to any one of [1] to [13], wherein the reaction region is at a position lower than a flow path into which the sample solution flows.
[15] The microchip for nucleic acid amplification reaction according to any one of [1] to [14], wherein a protrusion is provided at a position where the reaction region flows from the flow path.
[16] The nucleic acid amplification reaction microchip according to any one of [1] to [15], wherein the solid-phase reagent is arranged so as to occupy most of the area of the surface on which light detection is performed. .
[17] The microchip for nucleic acid amplification reaction according to any one of [1] to [16], wherein the solid phase reagent is a freeze-dried reagent.
[18] The microchip for nucleic acid amplification reaction according to any one of [1] to [17], wherein the solid-phase reagent is a tableting product.
[19] The microchip for nucleic acid amplification reaction according to any one of [1] to [18], wherein the solid phase reagent is two or more.

  1a, 1b microchip; 2, 2a, 2b well; 3, 31, 32 locking part; 4 introduction part; 5 flow path; 6 photodetection side; 7 passage part; R, Ra, Rb solid phase reagent; R1 inner periphery; R2 outer periphery

Claims (18)

  A microchip for nucleic acid amplification reaction, wherein a reaction part is provided with a locking part for suppressing movement of a solid-phase reagent containing a substance necessary for nucleic acid amplification reaction.   The microchip for nucleic acid amplification reaction according to claim 1, wherein the locking portion suppresses movement in the longitudinal direction and / or thickness direction of the reaction region.   The microchip for nucleic acid amplification reaction according to claim 1, wherein the locking portion is a convex portion and / or a concave portion provided on the inner surface of the reaction region.   The microchip for nucleic acid amplification reaction according to claim 2 or 3, wherein one or a plurality of surfaces on the side where light detection is performed exist in the reaction region.   The microchip for nucleic acid amplification reaction according to claim 4, wherein the sample solution flows into the reaction region from a plurality of flow paths.   The nucleic acid amplification reaction microchip according to claim 1, wherein the solid-phase reagent is disposed on a surface on the light detection side.   The nucleic acid amplification reaction microchip according to claim 1, further comprising a flow path through which the sample solution flows toward the solid-phase reagent.   The microchip for nucleic acid amplification reaction according to claim 3, wherein the convex portion is provided in the reaction region, and the convex portion is located in the middle in the short direction.   The nucleic acid amplification reaction microchip according to claim 3, wherein the convex portion allows liquid to pass through.   The microchip for nucleic acid amplification reaction according to claim 3, wherein the convex portions are provided in the reaction region, and the convex portions are provided to face each other.   The microchip for nucleic acid amplification reaction according to claim 3, wherein the convex portion is fitted to an inner peripheral portion of the reagent.   The microchip for nucleic acid amplification reaction according to claim 3, wherein the concave portion is fitted to an outer peripheral portion of the reagent.   The microchip for nucleic acid amplification reaction according to claim 3, wherein the reaction region is at a position lower than a flow path into which the sample solution flows.   The microchip for nucleic acid amplification reaction according to claim 5, wherein a protrusion is provided at a position where the reaction region flows from the flow path.   The microchip for nucleic acid amplification reaction according to claim 6, wherein the solid phase reagent is arranged so as to occupy most of the area of the surface on the light detection side.   The nucleic acid amplification reaction microchip according to claim 6, wherein the solid phase reagent is a freeze-dried reagent.   The microchip for nucleic acid amplification reaction according to claim 6, wherein the solid-phase reagent is a tableting product. The microchip for nucleic acid amplification reaction according to claim 6, wherein the solid phase reagent is two or more.

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