JP2009171933A - Chip for biological specimen reaction, and method for biological specimen reaction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chip for biological specimen reaction, enabling a reaction treatment with a minute amount of a reaction liquid, and also capable of efficiently conducting a treatment of many specimens at a time. <P>SOLUTION: The chip comprises a transparent base plate 101 provided with a plurality of reaction vessels 103 and a transparent base plate 102 provided with a flow path 104 for injecting a reaction liquid thereinto. In this chip, the transparent base plate 102 is movable between a first position and a second position relative to the transparent base plate 101; wherein, when the transparent base plate 102 is disposed at the first position, the flow path 104 is connected to the respective reaction vessels 103, while when the transparent base plate 102 is disposed at the second position, the flow path is separated from the respective reaction vessels 103. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a biological sample reaction chip and a biological sample reaction method for performing a biological sample reaction such as nucleic acid amplification.

  A method of performing chemical analysis, chemical synthesis, bio-related analysis, or the like using a microfluidic chip in which a fine flow path is provided on a glass substrate or the like has attracted attention. Microfluidic chips are also called Micro Total Analytical System (Micro TAS), Lab-on-a-chip, etc., and require less samples and reagents than conventional devices, have shorter reaction times, and waste It is expected to be used in a wide range of fields, such as medical diagnosis, on-site analysis of the environment and food, and production of pharmaceuticals and chemicals. Since the amount of the reagent may be small, it is possible to reduce the cost of the inspection, and because the amount of the sample and the reagent is small, the reaction time is greatly shortened and the inspection can be made more efficient. In particular, when used for medical diagnosis, it is possible to reduce the number of specimens such as blood as a sample.

  As a method for amplifying a gene such as DNA or RNA used as a sample, a polymerase chain reaction (PCR) method is well known. In the PCR method, a mixture of target DNA and reagents is put in a tube, and a temperature control device called a thermal cycler is used to repeatedly react, for example, three steps of temperature changes of 55 ° C, 72 ° C, and 94 ° C with a period of several minutes. Therefore, only the target DNA can be amplified about twice as much per temperature cycle by the action of an enzyme called polymerase.

  In recent years, a method called real-time PCR using a special fluorescent probe has been put into practical use, and DNA can be quantified while performing an amplification reaction. Real-time PCR is widely used for research and clinical tests because of its high measurement sensitivity and reliability.

  However, in the conventional apparatus, the standard amount of reaction solution required for PCR is several tens of μl, and there is a problem that basically one reaction system can measure only one gene. There is a method of simultaneously measuring about four types of genes by inserting a plurality of fluorescent probes and distinguishing them by their colors, but the only way to measure more genes simultaneously is to increase the number of reaction systems. Since the amount of DNA extracted from the specimen is generally small and the reagents are expensive, it is difficult to measure a large number of reaction systems at the same time.

Patent Documents 1 and 2 disclose inventions in which a liquid specimen sample such as a PCR reaction solution or blood is accurately poured into a plurality of chambers using a rotary drive device.
In Patent Document 3, a microwell integrated on a semiconductor substrate is prepared, and PCR is performed in the well to amplify and analyze a large number of DNA samples at once with a small amount of sample. A method of performing is disclosed.
JP 2006-126010 A JP 2006-126011 A JP 2000-236876 A

  An object of the present invention is to obtain a biological sample reaction chip and a biological sample reaction method capable of performing a reaction process with a very small amount of a reaction solution and capable of efficiently processing many specimens at once. is there.

  The biological sample reaction chip according to the present invention includes a first substrate on which a plurality of reaction containers are formed, and a second substrate on which a reaction liquid introduction channel is formed, and the second substrate is The reaction solution introduction flow is movable between the first position and the second position with respect to the first substrate, and the second substrate is disposed at the first position. A path is connected to each of the reaction containers, and when the second substrate is disposed at the second position, the reaction liquid introduction channel is separated from each of the reaction containers. It is what.

According to the present invention, by supplying the reaction solution into the reaction vessel through the reaction solution introduction channel, it is possible to perform a reaction process with a very small amount of reaction solution that is difficult to quantify with a pipette. When the amount of the reaction solution is small, it is possible to reduce the cost of reagents and the like, and the reaction time is greatly shortened so that the processing efficiency can be improved. In addition, since processing can be performed in a large number of reaction vessels at a time, various types of inspections and the like can be performed efficiently.
In addition, when the reaction vessel is filled with the reaction solution, the second substrate is disposed at the first position, and when the reaction treatment is performed, the second substrate is disposed at the second position. Therefore, contamination between reaction vessels can be prevented.
Moreover, you may make it further provide the reaction liquid accommodating part connected to the flow path for reaction liquid introduction.

Further, it is desirable that a guide mechanism is provided on the first substrate and the second substrate along a direction in which the second substrate moves.
Furthermore, it is desirable that a stopper is provided so that the second substrate can be fixed to the first position and the second position.
Thereby, the second substrate can be reliably and easily moved between the first position and the second position.

  Further, it is desirable that a contact surface between the first substrate and the second substrate has liquid repellency. Thereby, liquid leakage from the reaction containers can be prevented, and contamination between the reaction containers can be prevented.

  Moreover, it is desirable that the contact surfaces of the first substrate and the second substrate are in close contact with each other. Thereby, liquid leakage from the reaction containers can be prevented, and contamination between the reaction containers can be prevented.

  In addition, by adjusting the contact force of the contact surface between when the second substrate is moved and when it is fixed, the liquid leakage prevention function can be enhanced and the second substrate can be easily moved. .

  In addition, it is preferable that a distance between the first position and the second position is smaller than a distance between adjacent reaction vessels.

  Moreover, it is desirable that each reaction container is coated with a reagent necessary for the reaction. Thereby, the user can perform a test | inspection etc. simply by filling a reaction liquid.

The biological sample reaction method according to the present invention is a biological sample reaction method using the above-described biological sample reaction chip, wherein the second substrate is disposed at the first position, and the reaction liquid introduction channel is provided. And filling the reaction vessel with the reaction solution, and placing the second substrate at the second position and executing a biological sample reaction process.
According to the present invention, by supplying the reaction solution into the reaction vessel through the reaction solution introduction channel, it is possible to perform a reaction process with a very small amount of reaction solution that is difficult to quantify with a pipette. When the amount of the reaction solution is small, it is possible to reduce the cost of reagents and the like, and the reaction time is greatly shortened so that the processing efficiency can be improved. In addition, since processing can be performed in a large number of reaction vessels at a time, various types of inspections and the like can be performed efficiently.
Further, when the reaction vessel is filled with the reaction solution, the second substrate is disposed at the first position, and when the reaction treatment is performed, the second substrate is disposed at the second position. Therefore, contamination between reaction vessels can be prevented.

Further, in the step of filling the reaction solution, the reaction solution can be filled into the reaction vessel using centrifugal force.
Further, in the step of filling the reaction solution, the reaction solution can be filled into the reaction vessel using capillary force.

The biological sample reaction process is a process including nucleic acid amplification, and the reaction solution contains a target nucleic acid, an enzyme for amplifying the nucleic acid, and nucleotides at a predetermined concentration. A primer can be applied in advance.
When performing real-time PCR processing, a fluorescent probe may be applied in advance in the reaction apparatus.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
Embodiment 1 FIG.
FIG. 1 is a top view showing a schematic configuration of a microreactor array (biological sample reaction chip) 10 according to Embodiment 1 of the present invention, and FIG. 2 is a cross-sectional view taken along a line CC in FIG. As shown in the figure, the microreactor array 10 includes a transparent substrate (first substrate) 101, a transparent substrate (second substrate) 102, a reaction vessel 103, a reaction solution introduction channel 104, a reaction solution storage unit 106, A reaction liquid supply port 107, a stopper 108, and a guide rail 109 are provided.

  As shown in FIG. 1, the microreactor array 10 is configured by bonding a transparent substrate 101 and a transparent substrate 102 together. A plurality of reaction vessels 103 are formed on the transparent substrate 101. On the transparent substrate 102, a reaction liquid introduction flow path 104 and a reaction liquid storage section 106 are formed. The transparent substrates 101 and 102 can be resin substrates, for example.

  The transparent substrate 102 can move in the direction of the arrow shown in FIG. 1 along the guide rail 109 and is fixed to the first position and the second position by the stopper 108. FIGS. 1A and 2A are diagrams illustrating a state in which the transparent substrate 102 is disposed at the first position, and FIGS. 1B and 2B are diagrams in which the transparent substrate 102 is the second. It is a figure which shows the state arrange | positioned in this position.

  As shown in FIG. 1A, when the transparent substrate 102 is disposed at the first position, the reaction vessel 103 is connected to the reaction liquid introduction flow path 104. In this state, the reaction vessels 103 are connected to each other via the reaction liquid introduction flow path 104, and thus the individual reaction vessels 103 are not separated. On the other hand, as shown in FIG. 1B, when the transparent substrate 102 is disposed at the second position, the reaction vessel 103 and the reaction solution introduction flow path 104 are separated, and thus the reaction vessels 103 are also connected to each other. To be separated. Since the moving distance from the first position to the second position is smaller than the distance between the adjacent reaction vessels 103, the reaction liquid is introduced when the transparent substrate 102 is moved from the first position to the second position. The front end of the flow path 104 does not come into contact with the adjacent reaction vessel 103.

  FIG. 3A is a cross-sectional view showing an example of the guide rail 109. The DD cross section of FIG. 1 is shown. By providing the guide rail 109, the transparent substrate 102 can be moved along the guide rail, and since the stopper 108 is provided, the transparent substrate 102 can be stopped at the first position and the second position. it can. Thus, the transparent substrate 102 can be moved between the first position and the second position reliably and easily.

  Instead of providing the guide rail 109, the transparent substrates 101 and 102 may be configured as shown in FIG. With this configuration, the transparent substrate 102 can be reliably and easily moved along a predetermined direction.

  The reaction vessel 103 is, for example, a circular shape having a diameter of 500 μm and a depth of 100 μm. The reaction liquid introduction flow path 104 has a cross section perpendicular to the direction in which the reaction liquid flows having a width of 100 μm and a depth of 100 μm. The distance between the adjacent reaction vessels 103 is sufficiently ensured so that mixing of the reaction liquid between the reaction vessels 103 can be prevented. The reaction vessel 103 and the reaction solution introduction channel 104 are preferably subjected to surface treatment so that the inner wall surface becomes lyophilic in order to prevent adsorption of bubbles. Further, it is desirable that the inner wall surfaces of the reaction vessel 103 and the reaction solution introduction channel 104 are subjected to surface treatment for suppressing nonspecific adsorption of biomolecules such as proteins.

  In addition, the surface of the transparent substrate 101 and the transparent substrate 102 that are in contact with each other is subjected to surface treatment, or the contact surface between the transparent substrate 101 and the transparent substrate 102 is provided with a sealing property so as to react. It is possible to prevent the reaction liquid from leaking from the container 103 and entering the other reaction container 103 through the substrate surface. Specifically, a method of coating the contact surface with silicone rubber or fluororesin can be considered.

  When the transparent substrate 102 is fixed at the first position or the second position, by using a pressing means (not shown), it is possible to increase the adhesion between the two substrates and prevent liquid leakage. When the transparent substrate 102 is moved, the transparent substrate 102 can be moved along the guide rail by reducing the applied pressure.

  Next, a method for filling the microreactor array 10 with the reaction solution will be described. In the step of filling the reaction solution, the transparent substrate 102 of the microreactor array 10 is fixed at the first position, and the reaction solution is supplied from the reaction solution supply port 107 to the reaction solution storage unit 108 using a pipette or the like.

The reaction solution contains target nucleic acid, polymerase, and nucleotide (dNTP) at a predetermined concentration suitable for the reaction.
As the target nucleic acid, for example, DNA extracted from a biological sample such as blood, urine, saliva, spinal fluid, or cDNA reversely transcribed from the extracted RNA can be used.
The primer may be contained in the reaction solution, but in the microreactor array of this example, each primer is applied in advance and stored in a dry state. Each reaction vessel 104 is coated with a different primer so that multiple PCRs can be performed simultaneously.

Next, the microreactor array 10 is rotated using a centrifuge 20 as shown in FIG.
As shown in FIG. 4, by fixing the microreactor array 10 on the rotary table 21 of the centrifuge 20 and rotating the centrifuge 20, the microreactor array 10 has a reaction container 103 to a reaction vessel 103. Centrifugal force is applied in the direction of heading.

  When centrifugal force is applied to the microreactor array 10, the reaction solution proceeds while filling the reaction solution introduction channel 104, and further fills the reaction vessel 103. Air having a lighter specific gravity than the reaction liquid is pushed into the reaction liquid introduction flow path 104 and replaced with the reaction liquid, thereby filling the reaction vessel 103 with the reaction liquid.

  Once the reaction solution is supplied to the microreactor array 10 according to the above procedure, next, PCR processing (biological sample reaction processing) is performed. In the step of performing the PCR process, the transparent substrate 102 is fixed at the second position, and the microreactor array 10 is installed in the thermal cycler to perform the PCR process. In general, first, the step of dissociating the double-stranded DNA at 94 ° C. is performed, then the step of annealing the primer at about 55 ° C. is performed, and then the temperature is increased using a thermostable DNA polymerase. The cycle including the step of replicating the complementary strand at 72 ° C. is repeated.

  In addition, when performing real-time PCR using the microreactor array 10, a primer and a fluorescent probe used for the PCR reaction are applied to the inner wall of the reaction vessel 103 in advance, and fluorescence is obtained using a CCD sensor or the like for each cycle. Measure strength. The amount of the initial target nucleic acid is calculated and measured from the number of cycles that reached a specific fluorescence intensity. The method for performing real-time PCR is not limited to the above. For example, when a double-stranded binding fluorescent dye such as SYBR (registered trademark) Green is used, a fluorescent probe is unnecessary.

As described above, according to the first embodiment, it is difficult to quantify with a pipette by supplying the reaction liquid into the reaction vessel 103 through the reaction liquid introduction flow path 104 using centrifugal force. Reaction processing with a small amount of reaction solution becomes possible. In addition, since processing can be performed in a large number of reaction vessels 103 at a time, various types of inspections and the like can be performed efficiently.
In addition, the transparent substrate 102 is disposed at the first position when the reaction vessel 103 is filled with the reaction solution, and the transparent substrate 102 is disposed at the second position during the PCR reaction process. Since it can isolate | separate, the contamination between reaction containers can be prevented.

  In the first embodiment, the microreactor array 10 is used as a reaction device for real-time PCR reaction, but can be used for various reactions using genes and biological samples. For example, an antigen, an antibody, a receptor, a protein such as an enzyme, a peptide (oligopeptide), or the like that specifically captures (for example, adsorbs, binds, etc.) a specific protein is coated in the reaction vessel 105 and It can also be used for processing for detecting a target protein.

(Modification)
FIG. 5 is a cross-sectional view showing a configuration of a microreactor array 30 according to a modification of the first embodiment. 3 shows the same cross section as that shown in FIG. As shown in the figure, the microreactor array 30 further includes a transparent substrate 110 between the transparent substrate 101 and the transparent substrate 102. The transparent substrate 110 is formed with a through-hole 111 that connects the reaction liquid introduction flow path 104 and the reaction vessel 103.

  The transparent substrate 110 and the transparent substrate 102 can move together in the direction of the arrow shown in the figure, and are fixed to the first position and the second position by the stopper 108. 5A shows a state where the transparent substrate 110 and the transparent substrate 102 are arranged at the first position, and FIG. 5B shows a state where the transparent substrate 110 and the transparent substrate 102 are arranged at the second position. It is a figure which shows the state currently performed.

  As shown in FIG. 5A, when the transparent substrate 110 and the transparent substrate 102 are disposed at the first position, the reaction vessel 103 is connected via the reaction liquid introduction flow path 104 and the through hole 111. . In this state, the reaction vessels 103 are connected to each other via the reaction liquid introduction flow path 104, and thus the individual reaction vessels 103 are not separated. On the other hand, as shown in FIG. 5B, when the transparent substrate 110 and the transparent substrate 102 are arranged at the second position, the through-hole 111 is separated from the reaction vessel 103, so that the reaction vessel 103 and the reaction liquid introduction The flow path 104 is separated, and therefore each reaction vessel 103 is also separated. Since the moving distance from the first position to the second position is smaller than the distance between the adjacent reaction vessels 103, the transparent substrate 110 and the transparent substrate 102 are moved from the first position to the second position. The tip of the reaction liquid introduction channel 104 does not come into contact with the adjacent reaction vessel 103.

  According to the microreactor array 30 according to this modified example, when the transparent substrate 110 and the transparent substrate 102 are moved to the second position, the opening of the transparent substrate 110 in contact with the transparent substrate 101 is only the opening of the through hole 111. Therefore, compared with the case where the opening of the reaction liquid introduction flow path 104 is in direct contact with the transparent substrate 101, the effect of preventing liquid leakage is high.

Embodiment 2. FIG.
FIG. 6 is a top view showing a schematic configuration of a microreactor array (biological sample reaction chip) 40 according to Embodiment 2 of the present invention, and FIG. 7 is a cross-sectional view taken along line EE of FIG.

  As shown in the figure, the microreactor array 40 includes transparent substrates 401, 402, 403a, 403b, and 403c, a reaction vessel 404, a reaction solution introduction channel 405, an exhaust channel 406, fine channels 407 and 408, a reaction. A liquid supply port 409 is provided.

  As shown in the figure, the microreactor array 40 is configured by bonding transparent substrates 401, 402, and 403a to c. In the transparent substrate 401, a reaction solution introduction channel 405 is formed. Further, an exhaust passage 406 is formed in the transparent substrate 402. The reaction liquid introduction channel 405 and the exhaust channel 406 are formed to have a width of 500 μm and a depth of 500 μm, for example.

  A plurality of reaction containers 404 are formed on the transparent substrate 403b. The reaction vessel 404 is formed to have a size of 500 μm × 750 μm and a depth of 100 μm, and the distance between the adjacent reaction vessels 404 is sufficiently secured so that mixing of the reaction liquid between the reaction vessels 404 can be prevented. Further, fine channels 407 and 408 are formed in the transparent substrates 403a and 403c, respectively. The transparent substrates 401, 402, and 403a to c can be resin substrates, for example.

  The transparent substrate 401 is movable in the direction of the arrow shown in FIG. 6, and is fixed at the first position and the second position. Further, the transparent substrate 402 is also movable in the direction of the arrow shown in FIG. 6, and is fixed at the third position and the fourth position. FIGS. 6A and 7A are diagrams showing a state in which the transparent substrate 401 is disposed at the first position and the transparent substrate 402 is disposed at the third position, and FIGS. 6B and 7B. ) Is a diagram illustrating a state in which the transparent substrate 401 is disposed at the second position and the transparent substrate 402 is disposed at the fourth position.

  As shown in FIGS. 6A and 7A, when the transparent substrate 401 is disposed at the first position and the transparent substrate 402 is disposed at the third position, the reaction vessel 404 has a reaction solution introduction channel. 405 and the exhaust channel 406 are connected to each other through fine channels 407 and 408, respectively. In this state, the reaction containers 404 are connected to each other via the reaction liquid introduction flow path 405 and the exhaust flow path 406, so that the individual reaction containers 404 are not separated. On the other hand, as shown in FIGS. 6B and 7B, when the transparent substrate 401 is disposed at the second position and the transparent substrate 402 is disposed at the fourth position, the reaction solution introduction flow path 405 and Since the exhaust flow path 406 is separated from the fine flow paths 407 and 408, respectively, the reaction container 404, the reaction liquid introduction flow path 405, and the exhaust flow path 406 are separated, and thus the reaction containers 404 are also separated from each other. As in the first embodiment, a guide rail and a stopper may be provided along the moving direction of the transparent substrate 401 and the transparent substrate 402.

  The reaction vessel 404, the reaction solution introduction channel 405, and the fine channels 407 and 408 are desirably subjected to surface treatment so that the inner wall surface becomes lyophilic to prevent the adsorption of bubbles. Further, it is desirable that the inner wall surfaces of the reaction vessel 404, the reaction solution introduction channel 405, and the fine channels 407 and 408 are subjected to a surface treatment that suppresses nonspecific adsorption of biomolecules such as proteins.

  In addition, the surface of the transparent substrate 401 and the transparent substrate 403a and the surface of the transparent substrate 402 and the transparent substrate 403c that are in contact with each other are subjected to surface treatment, or these contact surfaces are brought into close contact with each other. It is possible to prevent the reaction solution from leaking from the substrate and entering another reaction vessel 404 along the substrate surface. Specifically, a method of coating the contact surface with silicone rubber or fluororesin can be considered.

  When the transparent substrates 401 and 402 are fixed at predetermined positions, by using a pressurizing means (not shown), it is possible to increase the adhesion between the two substrates and prevent liquid leakage. When the transparent substrates 401 and 402 are moved, the transparent substrates 401 and 402 can be moved with a lower applied pressure.

  Next, a method for filling the microreactor array 40 with the reaction solution will be described with reference to FIG. In the step of filling the reaction liquid, the transparent substrate 401 of the microreactor array 40 is fixed at the first position, the transparent substrate 402 is fixed at the third position, and a pipette or the like is used from the reaction liquid supply port 409. The reaction solution is supplied to the reaction solution introduction channel 405.

The reaction solution contains target nucleic acid, polymerase, and nucleotide (dNTP) at a predetermined concentration suitable for the reaction.
As the target nucleic acid, for example, DNA extracted from a biological sample such as blood, urine, saliva, spinal fluid, or cDNA reversely transcribed from the extracted RNA can be used.
The primer may be contained in the reaction solution, but in the microreactor array of the present example, each reaction container 404 is coated in advance and stored in a dry state. Each reaction container 404 is coated with a different primer so that multiple PCRs can be performed simultaneously.

  As shown in FIG. 8A, when the reaction solution is supplied to the reaction solution introduction channel 405, the reaction solution proceeds while filling the reaction solution introduction channel 103 by capillary force. Further, as shown in FIG. 8B, when the reaction solution reaches the connection portion with the reaction vessel 404, the reaction solution enters the reaction vessel 404 by capillary force. Further, the reaction liquid that has entered the reaction vessel 404 stops advancing at the connection portion between the reaction vessel 404 and the exhaust passage 406 as shown in FIG.

Generally, when a liquid enters into a fine flow path, a capillary force P expressed by the following formula acts.
P = (lγcosθ) / S
Here, l is the circumferential length of the cross section perpendicular to the flow of the flow path, S is the area, γ is the surface tension, and θ is the contact angle. Assuming that γ and θ in each channel are constant, the magnitude relationship between the capillary forces in each channel is determined by the value of 1 / S. Here, the capillary force of the reaction solution introduction channel 405 is A, the capillary force of the connection portion between the reaction solution introduction channel 405 and the reaction vessel 404 is B, the capillary force of the reaction vessel 404 is C, and the reaction vessel 404 and the exhaust are exhausted. When the capillary force of the connecting portion of the flow channel 406 is D and the capillary force of the exhaust flow channel 406 is E, the following relationship is established.
A <B <C <D
E <D
Thus, since the capillary force increases in the order in the reaction solution introduction channel 405, the connection portion between the reaction solution introduction channel 405 and the reaction vessel 404, and the reaction vessel 404, the reaction solution is caused by the capillary force in the reaction vessel. Proceed into 404 and fill the reaction vessel 404. Further, since the capillary force in the exhaust channel 406 is smaller than the connection portion between the reaction vessel 404 and the exhaust channel 406, the reaction solution cannot travel from the reaction vessel 404 to the exhaust channel 406. .

  As described above, all reaction vessels 404 can be filled with the reaction solution by capillary force. The volume of the reaction vessel 404 is about 0.04 μl, and the amount of the filled reaction solution is also about 0.04 μl. This is very small compared to 20 μl, which is an example of a general amount of reaction solution in conventional PCR. Conventionally, a pipette is used for quantifying the reaction solution, but it is difficult to quantify a small amount of the reaction solution of about 0.04 μl with the pipette. However, according to this embodiment, a very small amount of reaction solution can be accurately introduced into the reaction vessel 404.

  When the reaction solution is supplied to the microreactor array 40 in the above procedure, PCR processing (biological sample reaction processing) is performed. In the PCR process, as shown in FIG. 8D, the transparent substrate 401 is fixed at the second position, the transparent substrate 402 is fixed at the fourth position, and the microreactor array 40 is used as a thermal cycler. Install and perform PCR treatment.

As described above, according to the second embodiment, it is difficult to quantify with a pipette by supplying a reaction solution into the reaction vessel 404 through the reaction solution introduction channel 405 using capillary force. Reaction processing with a small amount of reaction solution becomes possible. In addition, since processing can be performed in a large number of reaction vessels 404 at a time, various types of inspections and the like can be performed efficiently.
In addition, the transparent substrate 401 is disposed at the first position and the transparent substrate 402 is disposed at the third position when the reaction container 404 is filled with the reaction solution, and the transparent substrate 402 is disposed at the second position during the PCR reaction process. Since the individual reaction containers 404 can be separated at the time of the reaction process by arranging the at the fourth position, contamination between the reaction containers can be prevented.

  As in the first embodiment, the microreactor array 40 can be used for various reactions using genes and biological samples other than the real-time PCR reaction.

(Modification)
FIG. 9 is a top view illustrating a schematic configuration of a microreactor array 50 according to a modification of the second embodiment, and FIG. 10 is a cross-sectional view taken along line FF in FIG. As shown in the figure, the microreactor array 50 includes transparent substrates 501, 502, and 503. A reaction vessel 404 is formed on the transparent substrate 501, and fine channels 407 and 408 are formed on the transparent substrate 502. The transparent substrate 503 is formed with a reaction liquid introduction channel 405 and an exhaust channel 406.

  The transparent substrate 503 is movable in the direction of the arrow shown in the figure, and is fixed at the first position and the second position. 9A and 10A are diagrams illustrating a state in which the transparent substrate 503 is disposed at the first position, and FIGS. 9B and 10B illustrate that the transparent substrate 503 is the second. It is a figure which shows the state arrange | positioned in this position.

  As shown in FIGS. 9A and 10A, when the transparent substrate 503 is disposed at the first position, the reaction vessel 404 has a reaction liquid introduction channel 405 and an exhaust channel 406, respectively. The micro channels 407 and 408 are connected. In this state, the reaction containers 404 are connected to each other via the reaction liquid introduction flow path 405 and the exhaust flow path 406, so that the individual reaction containers 404 are not separated. On the other hand, as shown in FIGS. 9B and 10B, when the transparent substrate 503 is disposed at the second position, the reaction liquid introduction flow path 405 and the exhaust flow path 406 each have a fine flow. Since they are separated from the paths 407 and 408, the reaction vessel 404 is separated from the reaction solution introduction channel 405 and the exhaust channel 406, and thus the reaction vessels 404 are also separated from each other.

  According to the microreactor array 50 according to this modified example, the reaction liquid introduction channel 405 and the exhaust channel 406 are formed on the same transparent substrate 503, so the number of substrates is small and the apparatus configuration is simplified. Further, if the transparent substrate 503 is moved, there is an advantage that the positions of the reaction solution introduction channel 405 and the exhaust channel 406 can be moved.

It is a top view which shows schematic structure of the microreactor array by Embodiment 1 of this invention. 2 (A) and 2 (B) are CC cross-sectional views of FIGS. 1 (A) and 1 (B). It is sectional drawing which shows the example of a guide rail. It is a figure which shows schematic structure of the centrifuge which applies centrifugal force to a microreactor array. 6 is a cross-sectional view illustrating a schematic configuration of a microreactor array according to a modification of the first embodiment. FIG. It is a top view which shows schematic structure of the microreactor array by Embodiment 2 of this invention. 7A and 7B are EE cross-sectional views of FIGS. 6A and 6B. 6 is a diagram illustrating a method of filling a microreactor array according to Embodiment 2 with a reaction solution. FIG. 10 is a top view showing a schematic configuration of a microreactor array according to a modification of the second embodiment. FIG. FIGS. 10A and 10B are cross-sectional views taken along the line FF in FIGS. 9A and 9B.

Explanation of symbols

  10, 30, 40, 50 Microreactor array, 101, 102, 110 Transparent substrate, 103 Reaction vessel, 104 Reaction solution introduction channel, 106 Reaction solution storage portion, 107 Reaction solution supply port, 108 Stopper, 109 Guide rail, 111 through-hole, 20 centrifuge, 401, 402, 403a, 403b, 403c transparent substrate, 404 reaction vessel, 405 reaction liquid introduction flow path, 406 exhaust flow path, 407, 408 fine flow path, 409 reaction liquid supply port , 501, 502, 503 Transparent substrate

Claims (13)

A first substrate on which a plurality of reaction vessels are formed;
A second substrate on which a reaction liquid introduction channel is formed,
The second substrate is movable between a first position and a second position relative to the first substrate;
When the second substrate is disposed at the first position, the reaction liquid introduction flow path is connected to each of the reaction vessels,
The biological sample reaction chip, wherein when the second substrate is disposed at the second position, the reaction liquid introduction flow path is separated from each of the reaction containers.   The biological sample reaction chip according to claim 1, further comprising a reaction solution storage unit connected to the reaction solution introduction channel.   The biological sample reaction according to claim 1 or 2, wherein a guide mechanism is provided on the first substrate and the second substrate along a direction in which the second substrate moves. For chips.   The biological sample reaction according to any one of claims 1 to 3, further comprising a stopper provided so that the second substrate can be fixed to the first position and the second position. For chips.   The biological sample reaction chip according to claim 1, wherein a contact surface between the first substrate and the second substrate has liquid repellency.   The biological sample reaction chip according to any one of claims 1 to 4, wherein a contact surface between the first substrate and the second substrate is in close contact.   The biological sample reaction chip according to claim 6, wherein the contact force of the contact surface can be adjusted when the second substrate is moved and fixed.   The biological sample reaction according to any one of claims 1 to 7, wherein a distance between the first position and the second position is smaller than a distance between adjacent reaction containers. Chip.   The biological sample reaction chip according to any one of claims 1 to 8, wherein each of the reaction containers is coated with a reagent necessary for the reaction. A biological sample reaction method using the biological sample reaction chip according to any one of claims 1 to 9,
Placing the second substrate at the first position and filling the reaction vessel with the reaction solution through the reaction solution introduction channel;
Placing the second substrate at the second position and executing a biological sample reaction process.   The biological sample reaction method according to claim 10, wherein in the step of filling the reaction liquid, the reaction container is filled with the reaction liquid using centrifugal force.   The biological sample reaction method according to claim 10, wherein in the step of filling the reaction solution, the reaction solution is filled into the reaction vessel using capillary force. The biological sample reaction process is a process including nucleic acid amplification, and the reaction solution contains a target nucleic acid, an enzyme for amplifying the nucleic acid, and nucleotides at a predetermined concentration,
The biological sample reaction method according to claim 10, wherein a primer is previously applied to the reaction container.

Images