JP2009270922A - Biosample reaction method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a biosample reaction method processing a reaction in a minute amount of a reaction fluid and also efficiently processing many specimens at one time. <P>SOLUTION: The biosample reaction method, using a microreactor array 10 which includes a plurality of reaction vessels 103, a reaction fluid introducing channel 104 connected to each reaction vessel 103, a reaction fluid storage part 105 connected to the reaction fluid introducing channel 104 and a waste fluid storage part 105, includes: a first step of filling the reaction fluid introducing channel 104 and the reaction vessels 103 with a reaction fluid by a centrifugal force; and a second step of sending the reaction fluid in the reaction fluid introducing channel 104 to the waste fluid storage part 105 by a centrifugal force. Between the first step and the second step, the angles of the direction of the flow channels from the reaction fluid introducing channel 104 to the waste fluid storage part 105 against the direction of the centrifugal force are different. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a biological sample reaction method 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.
Patent Document 3 discloses that microwells integrated on a semiconductor substrate are prepared, and PCR is performed in the wells to amplify and analyze a large number of DNA samples at once with a small amount of samples. 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 method that can perform a reaction process with a very small amount of a reaction solution and that can efficiently process a large number of specimens at a time.

  The biological sample reaction method according to the present invention includes a plurality of reaction vessels, a reaction solution introduction channel connected to each of the reaction vessels, a reaction solution storage unit connected to the reaction solution introduction channel, A biological sample reaction method using a biological sample reaction chip provided with a waste liquid storage section connected to the reaction liquid introduction flow path, wherein the reaction liquid introduction flow path and the reaction container by centrifugal force A first step of filling the reaction solution into the second step, a second step of sending the reaction solution in the reaction solution introduction channel to the waste liquid storage unit by centrifugal force, and a biological sample reaction process. 3 processes.

According to the present invention, by supplying the reaction solution into the reaction vessel through the reaction solution introduction channel using centrifugal force, it is possible to perform a reaction process with a very small amount of the reaction solution that is difficult to quantify with a pipette. Become. 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, after filling the reaction liquid into the reaction liquid introduction channel and the reaction container, the reaction liquid in the reaction liquid introduction flow path is sent to the waste liquid container, so that the individual reaction containers can be separated. Therefore, contamination between reaction vessels can be prevented.

In the first step, the direction of the flow path from the reaction liquid introduction flow path to the waste liquid storage portion has an angle α with respect to the direction of centrifugal force. In the second process, The direction of the flow path from the reaction liquid introduction flow path to the waste liquid container has an angle β with respect to the direction of the centrifugal force, and 90 degrees ≦ α ≦ 180 degrees and 0 degrees ≦ β <90 degrees. Good.
According to the present invention, by changing the direction in which the centrifugal force is applied in the first step and the second step, the reaction vessel is efficiently filled with the reaction solution by a simple method, and the reaction in the reaction solution introduction flow path is performed. Only liquid can be delivered.

The direction of the flow path from the reaction liquid introduction flow path to the waste liquid storage section has an angle α with respect to the direction of centrifugal force, and 90 degrees ≦ α ≦ 180 degrees, and the first step Then, the waste liquid container may be isolated from the outside, and in the second step, the waste liquid container may be communicated with the outside.
According to the present invention, the reaction liquid is efficiently filled into the reaction vessel by a simple method by communicating the waste liquid container with the outside in the second step, and only the reaction liquid in the reaction liquid introduction channel is sent out. can do.

Further, in the second step, the capacity of the waste liquid storage unit may be made larger than that in the first step.
According to the present invention, by adjusting the capacity of the waste liquid storage unit, it is possible to efficiently fill the reaction vessel into the reaction vessel by a simple method and send out only the reaction solution in the reaction solution introduction channel.

  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 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. 1A 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. 1B is a cross-sectional view taken along the line CC in FIG. FIG. As shown in the figure, the microreactor array 10 includes transparent substrates 101 and 102, a reaction vessel 103, a reaction liquid introduction flow path 104, a flow path 104a from the reaction liquid introduction flow path 104 to the waste liquid storage section 105, and waste liquid storage. Unit 105, reaction solution storage unit 106, and reaction solution supply port 107.

  As shown in FIG. 1, the microreactor array 10 is configured by bonding a transparent substrate 101 and a transparent substrate 102 together. The transparent substrate 101 is formed with a plurality of reaction vessels 103, a reaction solution introduction channel 104, a channel 104 a, a waste solution storage unit 105, and a reaction solution storage unit 106. A reaction liquid supply port 107 is formed in the transparent substrate 102. The transparent substrates 101 and 102 can be resin substrates, for example.

  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 channel 104 has a cross section perpendicular to the direction in which the reaction liquid flows having a width of 200 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.

  Next, a method for filling the microreactor array 10 with the reaction solution will be described. In the step of filling the reaction solution, first, the reaction solution is supplied from the reaction solution supply port 107 to the reaction solution storage unit 106 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 device 50 as shown in FIG.
As shown in FIG. 2, by fixing the microreactor array 10 on the rotary table 51 of the centrifuge 50 and rotating the centrifuge 50, the microreactor array 10 is transferred from the reaction solution storage unit 106 to the reaction vessel 103. Centrifugal force is applied in the direction of heading.

  First, the microreactor array 10 is fixed to the centrifuge 50 and rotated so that a centrifugal force is applied in the direction shown in FIG. When the centrifuge 50 is rotated in the state shown in FIG. 3A, a centrifugal force is applied to the microreactor array 10, so that the reaction liquid proceeds while filling the reaction liquid introduction flow path 104, and the reaction vessel 103 is further passed through. Fill. 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.

  When centrifugal force is applied to the microreactor array 10 in the state shown in FIG. 3A, the reaction liquid is not sent to the waste liquid storage unit 105. This is because, as shown in the figure, the direction of the flow path from the reaction liquid introduction flow path 104 to the waste liquid storage section 105 forms an angle of 135 degrees with respect to the direction of the centrifugal force. This is because the component in the direction of the flow path toward the portion 105 is 0 or less. If the angle formed by the direction of the flow path 104a from the reaction liquid introduction flow path 104 to the waste liquid storage section 105 and the direction of the centrifugal force is 90 degrees or more and 180 degrees or less, the reaction liquid is not sent to the waste liquid storage section 105. . As described above, since the reaction liquid does not flow toward the waste liquid storage unit 105, it is possible to fill the reaction liquid in all the reaction containers 103.

  Next, the rotation of the centrifuge 50 is stopped once, and this time, the microreactor array 10 is fixed to the centrifuge 50 so that a centrifugal force is applied in the direction shown in FIG. By rotating the centrifugal device 50 again, the reaction solution in the reaction solution introduction channel 104 is sent out to the waste solution storage unit 105 this time. This is because, in the state of FIG. 3B, the direction of the flow path 104a from the reaction liquid introduction flow path 104 to the waste liquid storage section 105 forms an angle of 45 degrees with respect to the direction of the centrifugal force. This is because the component in the direction of the flow path toward the waste liquid storage unit 105 of the centrifugal force becomes 0 or more. If the angle between the direction of the flow path from the reaction liquid introduction flow path 104 to the waste liquid storage section 105 and the direction of the centrifugal force is not less than 0 degrees and less than 90 degrees, the reaction liquid is sent to the waste liquid storage section 105. . The reaction solution in the reaction solution introduction channel 104 is sent to the waste solution storage unit 105, but the reaction solution in the reaction vessel 103 remains in the reaction vessel 103.

  In this way, each reaction vessel 103 can be separated by sending the reaction solution in the reaction solution introduction channel 104 to the waste solution storage unit 105.

  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. Specifically, the microreactor array 10 is installed in a thermal cycler to perform PCR processing. 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.
Further, after filling the reaction liquid introduction flow path 104 and the reaction vessel 103 with the reaction liquid by centrifugal force, the direction in which the centrifugal force is applied is changed, and the reaction liquid in the reaction liquid introduction flow path 104 is again applied by the centrifugal force. Since the individual reaction containers 103 can be separated during the reaction process, contamination between the 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, antigens, antibodies, receptors, proteins such as enzymes, peptides (oligopeptides), etc. that specifically capture (for example, adsorb, bind, etc.) a specific protein are coated in the reaction vessel 103, and the reaction solution is used. It can also be used for processing for detecting a target protein.

Embodiment 2. FIG.
4A is a top view showing a schematic configuration of a microreactor array (biological sample reaction chip) 20 according to Embodiment 2 of the present invention, and FIG. 4B is a cross-sectional view taken along the line CC in FIG. 4A. FIG. As shown in the figure, the microreactor array 20 includes transparent substrates 101 and 102, a reaction vessel 103, a reaction liquid introduction flow path 104, a flow path 104a from the reaction liquid introduction flow path 104 to the waste liquid storage section 105, and waste liquid storage. Part 105, reaction liquid storage part 106, reaction liquid supply port 107, external communication hole 201, and external communication path 202.

  As shown in FIG. 4, the microreactor array 20 is configured by bonding a transparent substrate 101 and a transparent substrate 102 together. In the transparent substrate 101, a plurality of reaction vessels 103, a reaction liquid introduction channel 104, a channel 104a, a waste liquid storage unit 105, a reaction liquid storage unit 106, and an external communication path 202 are formed. A reaction liquid supply port 107 and an external communication hole 201 are formed in the transparent substrate 102. The upper surface of the external communication hole 201 is sealed with a thin film or the like. The transparent substrates 101 and 102 can be resin substrates, for example.

  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 channel 104 has a cross section perpendicular to the direction in which the reaction liquid flows having a width of 200 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.

  Next, a method for filling the microreactor array 10 with the reaction solution will be described. In the step of filling the reaction solution, first, the reaction solution is supplied from the reaction solution supply port 107 to the reaction solution storage unit 106 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, as in the first embodiment, the microreactor array 20 is rotated using a centrifuge device 50 as shown in FIG.
First, the microreactor array 20 is fixed to the centrifuge 50 and rotated so that a centrifugal force is applied in the direction shown in FIG. When the centrifuge 50 is rotated in the state shown in FIG. 5A, a centrifugal force is applied to the microreactor array 20, so that the reaction liquid proceeds while filling the reaction liquid introduction flow path 104, and the reaction vessel 103 is further passed through. Fill. 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.

  When a centrifugal force is applied to the microreactor array 20 in the state shown in FIG. 5A, the reaction solution is not sent to the waste liquid storage unit 105. This is because, as shown in the figure, the direction of the flow path from the reaction liquid introduction flow path 104 to the waste liquid storage section 105 forms an angle of 135 degrees with respect to the direction of the centrifugal force. This is because the component in the direction of the flow path toward the portion 105 is 0 or less. If the angle formed by the direction of the flow path 104a from the reaction liquid introduction flow path 104 to the waste liquid storage section 105 and the direction of the centrifugal force is 90 degrees or more and 180 degrees or less, the reaction liquid is not sent to the waste liquid storage section 105. . As described above, since the reaction liquid does not flow toward the waste liquid storage unit 105, it is possible to fill the reaction liquid in all the reaction containers 103.

  Next, the rotation of the centrifugal device 50 is stopped once, and then, as shown in FIG. 5B, a hole for venting air is opened using a needle or the like in the seal portion on the upper surface of the external communication hole 201. Furthermore, by rotating the centrifugal device 50 again, the reaction solution in the reaction solution introduction channel 104 is sent out to the waste solution storage unit 105 this time. At this time, the reaction solution in the reaction solution introduction channel 104 is sent to the waste solution storage unit 105, but the reaction solution in the reaction vessel 103 remains in the reaction vessel 103. Note that the external communication hole 201 is desirably disposed at a position away from the waste liquid storage unit 105 so that the reaction liquid sent into the waste liquid storage unit 105 does not leak.

  In this way, each reaction vessel 103 can be separated by sending the reaction solution in the reaction solution introduction channel 104 to the waste solution storage unit 105.

  When the reaction solution is supplied to the microreactor array 10 by the procedure as described above, the opening of the microreactor array 20 is sealed with a tape or the like, and the PCR process (biological sample reaction process) is performed as in the first embodiment.

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 103 through the reaction solution introduction channel 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.
Further, after filling the reaction liquid introduction flow path 104 and the reaction vessel 103 with the reaction liquid by centrifugal force, the external communication hole 201 connected to the waste liquid storage section 105 via the external communication path 202 is opened, and the centrifugal separation is performed again. Since the reaction liquid in the reaction liquid introduction flow path 104 is sent out to the waste liquid storage unit 103 by force, individual reaction containers 103 can be separated during the reaction process, and therefore, contamination between reaction containers. Can be prevented.

  In the second embodiment, the microreactor array 20 is used as a reaction device for real-time PCR reaction, but it can be used for various reactions using genes and biological samples. For example, antigens, antibodies, receptors, proteins such as enzymes, peptides (oligopeptides), etc. that specifically capture (for example, adsorb, bind, etc.) a specific protein are coated in the reaction vessel 103, and the reaction solution is used. It can also be used for processing for detecting a target protein.

Embodiment 3 FIG.
6A is a top view showing a schematic configuration of a microreactor array (biological sample reaction chip) 30 according to Embodiment 3 of the present invention, and FIG. 6B is a cross-sectional view taken along the line CC in FIG. 6A. FIG. As shown in the figure, the microreactor array 30 includes transparent substrates 101 and 102, a reaction vessel 103, a reaction liquid introduction channel 104, a waste liquid storage part 105, a reaction liquid storage part 106, a reaction liquid supply port 107, and a waste liquid storage part. A capacity adjustment unit 301 is provided.

  As shown in FIG. 6, the microreactor array 30 is configured by bonding a transparent substrate 101 and a transparent substrate 102 together. The transparent substrate 101 is formed with a plurality of reaction vessels 103, a reaction solution introduction channel 104, a waste solution storage unit 105, and a reaction solution storage unit 106. A waste liquid storage unit capacity adjustment unit 301 is provided at the opening of the waste liquid storage unit 105. A reaction liquid supply port 107 is formed in the transparent substrate 102. The transparent substrates 101 and 102 can be resin substrates, for example.

  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 channel 104 has a cross section perpendicular to the direction in which the reaction liquid flows having a width of 200 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.

  Next, a method for filling the microreactor array 10 with the reaction solution will be described. In the step of filling the reaction solution, first, the reaction solution is supplied from the reaction solution supply port 107 to the reaction solution storage unit 106 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, as in the first embodiment, the microreactor array 30 is rotated using a centrifuge device 50 as shown in FIG.
First, the microreactor array 30 is fixed to the centrifuge 50 and rotated so that a centrifugal force is applied in the direction shown in FIG. When the centrifuge 50 is rotated in the state shown in FIG. 7A, a centrifugal force is applied to the microreactor array 30, so that the reaction solution proceeds while filling the reaction solution introduction flow path 104, and the reaction vessel 103 is further passed through. Fill. 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.

  At this time, the waste liquid storage section capacity adjustment section 301 of the waste liquid storage section 105 is fixed at the position shown in FIG. In the state of FIG. 7B, the capacity of the waste liquid storage unit 105 is minimized. When a centrifugal force is applied to the microreactor array 30 in this state, the reaction solution is sent to the waste liquid storage unit 105 by the centrifugal force, but the reaction sent to the waste liquid storage unit 105 because the capacity of the waste liquid storage unit 105 is small. The liquid is a small amount, and the remaining reaction liquid is filled in the reaction vessel 103.

  Next, the rotation of the centrifugal device 50 is stopped once, and as shown in FIG. 7C, the position of the waste liquid storage unit capacity adjustment unit 301 is shifted to increase the capacity of the waste liquid storage unit 105. Furthermore, by rotating the centrifugal device 50 again, the reaction solution in the reaction solution introduction channel 104 is sent out to the waste solution storage unit 105 this time. At this time, the reaction solution in the reaction solution introduction channel 104 is sent to the waste solution storage unit 105, but the reaction solution in the reaction vessel 103 remains in the reaction vessel 103.

  In this way, each reaction vessel 103 can be separated by sending the reaction solution in the reaction solution introduction channel 104 to the waste solution storage unit 105.

As described above, according to the third embodiment, it is difficult to quantify with a pipette by supplying the reaction solution into the reaction vessel 103 through the reaction solution introduction channel 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.
Further, after filling the reaction liquid introduction flow path 104 and the reaction container 103 with the reaction liquid by centrifugal force, the position of the waste liquid storage section capacity adjustment section 301 is changed to increase the capacity of the waste liquid storage section 105, and the centrifugal force is again applied. As a result, the reaction liquid in the reaction liquid introduction flow path 104 is sent to the waste liquid storage unit 105, so that the individual reaction containers 103 can be separated during the reaction process, and contamination between the reaction containers is prevented. be able to.

  In the third embodiment, the microreactor array 20 is used as a reaction device for real-time PCR reaction, but it can be used for various reactions using genes and biological samples. For example, antigens, antibodies, receptors, proteins such as enzymes, peptides (oligopeptides), etc. that specifically capture (for example, adsorb, bind, etc.) a specific protein are coated in the reaction vessel 103, and the reaction solution is used. It can also be used for processing for detecting a target protein.

FIG. 1A is a top view showing a schematic configuration of a microreactor array according to Embodiment 1 of the present invention, and FIG. 1B is a cross-sectional view taken along the line CC in FIG. It is a figure which shows schematic structure of the centrifuge which applies centrifugal force to a microreactor array. 3 is a diagram for explaining a method of filling a microreactor array according to Embodiment 1 with a reaction solution. FIG. 4A is a top view showing a schematic configuration of the microreactor array according to Embodiment 2 of the present invention, and FIG. 4B is a cross-sectional view taken along the line CC in FIG. 4A. 6 is a diagram illustrating a method of filling a microreactor array according to Embodiment 2 with a reaction solution. FIG. 6A is a top view showing a schematic configuration of the microreactor array according to Embodiment 3 of the present invention, and FIG. 6B is a cross-sectional view taken along the line CC in FIG. 6A. FIG. 10 is a diagram for explaining a method for filling a microreactor array according to Embodiment 3 with a reaction solution.

Explanation of symbols

  10 Microreactor array, 101, 102 Transparent substrate, 103 Reaction vessel, 104 Reaction liquid introduction flow path, 104a flow path, 105 Waste liquid storage section, 106 Reaction liquid storage section, 107 Reaction liquid supply port, 50 Centrifugal device, 201 External Communication hole, 202 External communication path, 301 Waste liquid storage unit capacity adjustment unit

Claims (6)

A plurality of reaction vessels, a reaction solution introduction channel connected to each of the reaction vessels, a reaction solution storage unit connected to the reaction solution introduction channel, and a reaction solution introduction channel A biological sample reaction method using a biological sample reaction chip comprising:
A first step of filling the reaction solution into the reaction solution introduction channel and the reaction vessel by centrifugal force;
A second step of sending the reaction liquid in the reaction liquid introduction flow path to the waste liquid container by centrifugal force;
And a third step of performing a biological sample reaction process. In the first step, the direction of the flow path from the reaction liquid introduction flow path to the waste liquid storage portion has an angle α with respect to the direction of centrifugal force,
In the second step, the direction of the flow path from the reaction liquid introduction flow path to the waste liquid container has an angle β with respect to the direction of centrifugal force,
The biological sample reaction method according to claim 1, wherein 90 degrees ≦ α ≦ 180 degrees and 0 degrees ≦ β <90 degrees. The direction of the flow path from the reaction liquid introduction flow path to the waste liquid container has an angle α with respect to the direction of centrifugal force, and 90 degrees ≦ α ≦ 180 degrees,
In the first step, the waste liquid container is isolated from the outside,
The biological sample reaction method according to claim 1, wherein in the second step, the waste liquid container is communicated with the outside.   2. The biological sample reaction method according to claim 1, wherein in the second step, the volume of the waste liquid storage unit is made larger than that in the first step.   The biological sample reaction method according to any one of claims 1 to 4, wherein a reagent necessary for the reaction is applied to each of the reaction containers. 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 any one of claims 1 to 5, wherein a primer is previously applied to the reaction container.

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