JP2017138137A - Pcr microchip and nucleic acid amplifier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a PCR microchip that allows a rapid PCR.SOLUTION: The present invention relates to a PCR microchip 1 including: a micro passage; a heating surface heated by a temperature regulator 11; a reaction chamber 4 for performing a PCR; a base plate 2 having a first surface 2a and a second surface 2b, the first surface 2a having at least one concave part 2c; and a cover member 3 on the first surface 2a of the base plate 2, the reaction chamber 4 having the cover member 3 and the concave part 2c, being distant from the heating surface by less than 0.2 mm, and having a capacity in the range from 1 μL to 10 μL, both inclusive.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a PCR microchip and a nucleic acid amplification apparatus.

  Conventionally, microchips are used for various inspections and diagnoses by PCR (Polymerase Chain Reaction).

  Patent Document 1 below discloses a microchip that can be used for PCR. The microchip of Patent Document 1 includes a substrate on which a channel groove and a reaction chamber recess are formed, and a cover member. In Patent Document 1, a reaction chamber is formed by thermally bonding a cover member to the surface of a substrate provided with a recess.

International Publication No. 2012/060186

  By the way, when performing various tests and diagnoses by PCR, there is a problem that it takes time to feed back the determination result. In order to shorten the time for feedback of determination results, it is necessary to increase the speed of PCR. However, the microchip as in Patent Document 1 has not been able to sufficiently increase the PCR speed. Therefore, there is a need for a microchip that can increase the speed of PCR.

  An object of the present invention is to provide a microchip for PCR and a nucleic acid amplification apparatus that enable PCR to be performed at high speed.

  The microchip for PCR according to the present invention is a microchip for PCR having a microchannel, a heating surface heated by a temperature control device, and a reaction chamber in which PCR is performed, and the first microchips are opposed to each other. A substrate having at least one recess on the first surface, and a cover member provided on the first surface of the substrate, the reaction A chamber is constituted by the cover member and the recess, a distance between the heating surface and the reaction chamber is less than 0.2 mm, and a volume of the reaction chamber is 1 μL or more and 10 μL or less.

  By configuring in this way, the reaction chamber can be heated or cooled more efficiently, and the speeding up of PCR can be realized. In addition, the PCR microchip can be made smaller than the conventional one.

  In the PCR microchip according to the present invention, the number of reaction chambers is preferably 5 or more and 20 or less.

  By comprising in this way, the test | inspection of many items can be performed at high speed.

  The PCR microchip according to the present invention may further include an adhesive layer disposed between the first surface of the substrate and the cover member.

  In this case, since it is not necessary to apply heat as in the case of fusion or the like, the PCR microchip can be more easily produced.

  In a specific aspect of the microchip for PCR according to the present invention, a distance between the first surface of the substrate and the heating surface is 0.18 mm or more.

  In this case, since the joining portion of the substrate and the cover member is separated from the heating surface, deterioration of the joining portion and leakage of the reaction liquid due to heating during PCR can be further prevented, and the reliability of the inspection is further improved. Can be increased.

  In another specific aspect of the microchip for PCR according to the present invention, an adhesive strength between the first surface of the substrate and the cover member at 105 ° C. is 1 N / inch or more.

  By comprising in this way, deterioration of the junction part by the heating at the time of performing PCR and the leakage of a reaction liquid can be prevented further, and the reliability of a test | inspection can be improved further.

  In the PCR microchip according to the present invention, the substrate may have a through-hole connecting the reaction chamber and the outside.

  By comprising in this way, a reaction liquid can be poured even after joining a board | substrate and a cover member.

  The PCR microchip according to the present invention may contain reagents in the reaction chamber at a ratio of 80% or more of the capacity of the reaction chamber.

  With such a configuration, a sufficient amount of reaction solution is ensured even if the reaction chamber has a small capacity, so that PCR can be performed more rapidly and reliably.

  The nucleic acid amplification device of the present invention includes a PCR microchip configured according to the present invention and a temperature control device.

  With such a nucleic acid amplification apparatus, PCR can be performed at high speed.

  ADVANTAGE OF THE INVENTION According to this invention, the microchip for PCR and the nucleic acid amplifier which enable it to perform PCR at high speed can be provided.

1 is a schematic configuration diagram showing a nucleic acid amplification device according to a first embodiment of the present invention. 1 is a schematic plan view showing a microchannel structure provided in a PCR microchip constituting a nucleic acid amplification device according to a first embodiment of the present invention. It is a schematic block diagram which shows the modification of the nucleic acid amplification apparatus which concerns on the 1st Embodiment of this invention. It is a figure for demonstrating the method of filling the reagent to the microchip for PCR. It is a schematic block diagram for demonstrating the temperature control apparatus which comprises the nucleic acid amplifier which concerns on the 1st Embodiment of this invention. It is a schematic block diagram which shows the nucleic acid amplifier which concerns on the 2nd Embodiment of this invention. It is a schematic block diagram which shows the modification of the nucleic acid amplifier which concerns on the 2nd Embodiment of this invention. It is a schematic block diagram which shows the nucleic acid amplifier which concerns on the 3rd Embodiment of this invention. It is a schematic block diagram which shows the modification of the nucleic acid amplifier which concerns on the 3rd Embodiment of this invention.

  Hereinafter, the present invention will be clarified by describing specific embodiments of the present invention with reference to the drawings.

[First Embodiment]
FIG. 1 is a schematic configuration diagram showing a nucleic acid amplification apparatus according to the first embodiment of the present invention.

  The nucleic acid amplification device 10 includes a PCR microchip 1 and a temperature control device 11. The PCR microchip 1 is a microchip used for PCR, having a microchannel, a heating surface heated by the temperature control device 11, and a reaction chamber 4 in which PCR is performed. The PCR microchip 1 is provided on a temperature control device 11. In the nucleic acid amplification device 10, PCR is performed by heating or cooling the PCR microchip 1 by the temperature control device 11. The heating surface is a surface in contact with the temperature adjusting device 11 when provided on the temperature adjusting device 11 in the microchip 1.

  The PCR microchip 1 has a substrate 2 and a cover member 3. The substrate 2 has a first surface 2a and a second surface 2b facing each other. The first surface 2a of the substrate 2 is provided with at least one recess 2c. More specifically, at least one recess 2 c is provided on the first surface 2 a side of the substrate 2. A cover member 3 is provided on the first surface 2 a of the substrate 2. A reaction chamber 4 for performing PCR is constituted by a recess 2 c and a cover member 3. When a plurality of recesses 2 c are provided, the plurality of reaction chambers 4 are formed by the plurality of recesses 2 c facing the cover member 3. The plurality of reaction chambers 4 are each connected to a microchannel. In the present embodiment, the PCR microchip 1 is placed on the temperature control device 11 from the substrate 2 side. In the present embodiment, the second surface 2b of the substrate 2 is a heating surface.

  In the PCR microchip 1, the distance between the heating surface and the reaction chamber 4 is less than 0.2 mm. Thus, in the PCR microchip 1, the reaction chamber 4 is brought close to the temperature control device 11. Therefore, the heat of the temperature control device 11 is easily transferred to the reaction chamber 4. Therefore, the PCR microchip 1 and the nucleic acid amplification device 10 can increase the speed of PCR. Note that the distance between the heating surface and the reaction chamber 4 refers to the shortest distance between the heating surface and the reaction chamber 4. For example, when the heating surface is the second surface 2b of the substrate 2, it indicates the length of a perpendicular line dropped from the bottom 2d of the recess 2c to the heating surface. In addition, when the heating surface is the outer surface 3 a of the cover member 3, it indicates the length of a perpendicular drawn from the first surface 2 a of the substrate 2 to the outer surface 3 a of the cover member 3. Further, from the viewpoint of further increasing the speed of PCR, the distance between the heating surface and the reaction chamber 4 is preferably 0.02 mm or more, and preferably less than 0.2 mm.

  The volume of the reaction chamber 4 is 1 μL or more and 10 μL or less. In the PCR microchip 1, the capacity of the reaction chamber 4 is within the above range, and is smaller than the capacity of the reaction chamber in the prior art, so that heat from the temperature control device 11 is easily transferred to the reaction chamber 4. Therefore, also from this point, the PCR microchip 1 and the nucleic acid amplification device 10 can speed up PCR. When the PCR microchip 1 has a plurality of reaction chambers 4, the capacity of the reaction chamber 4 refers to the capacity of each reaction chamber 4. Therefore, when the PCR microchip 1 has a plurality of reaction chambers 4, the capacity of each reaction chamber 4 is 1 μL or more and 10 μL or less, respectively. Moreover, when the PCR microchip 1 has a plurality of reaction chambers 4, it can be used for simultaneous inspection of multiple items.

  In the PCR microchip 1, the distance between the first surface 2a of the substrate 2 and the heating surface is 0.18 mm or more. The inventors of the present application have found that when the PCR microchip 1 is formed by bonding the substrate 2 and the cover member 3, there are the following problems. That is, when the PCR reaction is performed, the heat of the temperature control device 11 is transmitted not only to the reaction chamber 4 but also to the bonding portion between the substrate 2 and the cover member 3, so that the bonding strength is weakened. It has been found that the reaction solution may peel from the reaction chamber 4 and leak out from the reaction chamber 4. Even in such a case, by setting the volume of the reaction chamber 4 to 1 μL or more and 10 μL or less, the bonding surface on the outer periphery of the reaction chamber 4 becomes larger than the volume of the reaction chamber 4. It becomes difficult to peel from the substrate 2. Furthermore, by setting the distance between the first surface 2a of the substrate 2 and the heating surface within the above range, it is possible to further prevent a decrease in bonding strength and a leakage of the reaction liquid due to a temperature change, thereby improving the reliability of the inspection. It can be further increased. Note that the distance between the first surface 2a of the substrate 2 and the heating surface refers to the shortest distance between the first surface 2a and the heating surface. When the second surface 2b of the substrate 2 is a heating surface as in the present embodiment, the distance between the first surface 2a of the substrate 2 and the heating surface is equal to the thickness of the substrate 2.

  The adhesive strength between the first surface 2a of the substrate 2 and the cover member 3 at 105 ° C. is preferably 1 N / inch or more. In this case, since the cover member 3 is not easily peeled off by heat, liquid leakage from the reaction chamber 4 is less likely to occur, and the reliability of fluorescence detection can be further enhanced.

  Hereinafter, the PCR microchip 1 and the temperature control device 11 will be described in more detail.

(Microchip for PCR)
The PCR microchip 1 has a microchannel through which a microfluid is conveyed. A plurality of reaction chambers 4 are provided in the middle of the microchannel. Specifically, a channel structure shown in FIG. 2 is provided in the PCR microchip 1.

  As shown in FIG. 2, a micro flow path 6 is connected to the introduction part 5. The micro flow path 6 is branched into the micro flow paths 6a to 6c. The reaction chambers 4a to 4i are connected to the micro flow paths 6a to 6c via the weighing units 7a to 7i. The micro flow paths 6 a to 6 c are connected to the discharge unit 8.

  The volumes of the reaction chambers 4a to 4i are 1 μL or more and 10 μL or less, respectively. Since the capacity of the reaction chambers 4a to 4i is within the above range and is small, heat from the temperature control device 11 is easily transferred to each of the reaction chambers 4a to 4i, so that PCR can be speeded up. The number of reaction chambers is preferably 5 or more, and preferably 20 or less. More preferably, it is 8 or more, more preferably 15 or less. When the number of reaction chambers is in the above range, simultaneous inspection of multiple items can be performed with higher accuracy.

  Further, the height of each of the reaction chambers 4a to 4i is preferably 0.1 mm or more, and preferably 0.9 mm or less. More preferably, it is 0.3 mm or more, and more preferably 0.6 mm or less. By setting the height of the reaction chambers 4a to 4i within the above range, the entire reaction chamber can be heated more efficiently, and the PCR can be further accelerated. The height of the reaction chambers 4a to 4i refers to the distance between the bottom 2d of the recess 2c of the substrate 2 shown in FIG. 1 and the first surface 2a.

  Returning to FIG. 1, the plurality of reaction chambers 4 are configured by closing the plurality of concave portions 2 c of the substrate 2 by the cover member 3. Although not shown, the microchannel is configured by closing a microchannel groove provided on the first surface 2 a side of the substrate 2 by the cover member 3.

  More specifically, the cover member 3 is bonded to the first surface 2 a of the substrate 2. The first surface 2a of the substrate 2 and the cover member 3 are bonded by thermal fusion. Thereby, the plurality of recesses 2c and the microchannel groove of the substrate 2 are closed. In addition, the 1st surface 2a of the board | substrate 2 and the cover member 3 may be adhere | attached with adhesives, such as a photocurable adhesive agent. Therefore, an adhesive layer 9 may be provided between the first surface 2a of the substrate 2 and the cover member 3 as shown in FIG.

  A film can be used for the cover member 3. It does not specifically limit as a material which comprises a film, For example, polyolefin etc. can be used. The cover member 3 may be formed by laminating and fusing the same type or different types of films. Further, the cover member 3 itself may be a pressure sensitive adhesive tape. In that case, the first surface 2a of the substrate 2 can be bonded by pressure-sensitive bonding without using an adhesive. In the PCR microchip of the present invention, in addition to the cover member 3 as a sealing material, a gas generating tape for liquid feeding and the like may be further provided.

  The material constituting the substrate 2 is not particularly limited, and examples thereof include synthetic resin, rubber, and metal. Preferably, a resin having high moldability such as polyolefin is used.

  The substrate 2 can be formed by, for example, injection molding. Further, the substrate 2 may be formed by in-mold forming in a state where the film as the cover member 3 is put in a mold and integrated by heat fusion.

  The total light transmittance of the cover member 3 is 90% or more. Moreover, the cover member 3 does not have autofluorescence. Thus, the reliability of fluorescence detection can be further enhanced by increasing the total light transmittance of the cover member 3 on the side opposite to the temperature control device 11 side or by using a material that does not have autofluorescence. it can.

Further, the water vapor transmission rate of the substrate 2 and the cover member 3 at a temperature of 37.7 ° C. and a humidity of 90% RH is preferably 150 g / m ² / day or less. In this case, bubbles are hardly generated in the reaction chamber 4, and the reliability of fluorescence detection can be further enhanced.

  In the PCR microchip 1, the reagent can be accommodated in the reaction chamber 4 at a rate of 80% or more of the capacity of the reaction chamber 4. When the reagent is accommodated at a rate of 80% or more of the capacity of the reaction chamber 4, the reliability of fluorescence detection can be further enhanced. From the viewpoint of further enhancing the reliability of fluorescence detection, it is preferable that the reagent can be accommodated in the reaction chamber 4 at a ratio of 90% or more of the capacity of the reaction chamber 4. Moreover, it is preferable that the reagent is accommodated at a ratio of 90% or more of the capacity of the reaction chamber 4. More preferably, it is 95% or more of the capacity of the reaction chamber 4, and more preferably 99% or more. The reason why the reagent can be filled at such a ratio will be explained as follows.

  For example, in the method of pasting the cover member 3 after filling the recess 2c of the substrate 2 with the reagent, the liquid may spill during the pasting, or heating or light irradiation during the pasting may adversely affect the reagent. Reagents can only be filled to a volume of 50% or less. On the other hand, in the PCR microchip 1, as shown in FIG. 4, after bonding the cover member 3, the through hole 2 e is formed from the second surface 2 b side of the substrate 2 opposite to the cover member 3. Since the reagent is injected through, it is possible to inject the reagent at a higher filling rate. That is, even if the capacity of the reaction chamber 4 is reduced, highly reliable PCR can be performed. In the present invention, the capacity of the through hole 2 e used for reagent injection is not included in the capacity of the reaction chamber 4. Further, the through-hole 2e may be closed after the reagent is injected. For example, a film may be disposed on the surface of the substrate 2 or the inside of the through hole 2e may be filled with resin and sealed.

(Temperature control device)
FIG. 5 is a schematic configuration diagram for explaining a temperature control device constituting the nucleic acid amplification device according to the first embodiment of the present invention.

  The temperature control device 11 has a Peltier element 12. The Peltier element 12 has a third surface 12a and a fourth surface 12b facing the third surface 12a.

  A heat conductive material layer 13 is laminated on the third surface 12a. Since the heat conductive material layer 13 is laminated, in this embodiment, the temperature unevenness of the heating surface can be further effectively reduced. As the heat conductive material layer 13, a metal plate such as Al or Al alloy having high heat conductivity can be used. On the upper surface of the heat conductive material layer 13, the PCR microchip 1 is laminated as a temperature control object. The PCR microchip 1 is laminated on the heat conductive material layer 13 from the substrate 2 side shown in FIG.

  On the other hand, a heat radiating member 14 is connected to the fourth surface 12 b side of the Peltier element 12. The heat radiating member 14 is provided to dissipate heat generated in the Peltier element 12. As the heat radiating member 14, for example, a heat sink made of metal can be used.

  In addition, the heat radiating member 14 should just be arrange | positioned at the 4th surface 12b side of the Peltier device 12. FIG. The heat dissipation member 14 may be disposed so as to be in direct contact with the fourth surface 12b, or may be connected to the fourth surface 12b via another member.

  A temperature sensor 15 is provided to detect the temperature of the upper surface of the heat conductive material layer 13. The temperature sensor 15 can be configured by an appropriate temperature-sensitive element that can detect the temperature of the upper surface of the heat conductive material layer 13. The temperature sensor 15 is connected to the control device 16 and gives a signal based on the detected temperature to the control device 16.

  A Peltier element driving circuit 17 is connected to the control device 16. The Peltier device driving circuit 17 is driven by the control device 16 to drive the Peltier device 12. Specifically, the Peltier element driving circuit 17 applies a voltage for heating or cooling the Peltier element 12 to the Peltier element 12. Thereby, the Peltier element 12 is heated or cooled.

  In this way, the temperature of the reaction chamber 4 shown in FIG. That is, heating and cooling are performed between a plurality of temperature ranges for performing PCR. Thereby, the nucleic acid is amplified by PCR reaction.

  In the nucleic acid amplification device 10, after the nucleic acid is amplified by the PCR reaction, the amplified nucleic acid is detected. The detection method is not particularly limited, and a method using an intercalator, a method using a Q probe, and the like can be used. Preferably, it is desirable that a fluorescent material for detecting the amplified nucleic acid is accommodated in the plurality of reaction chambers 4 in advance. Thereby, the nucleic acid amplified by the fluorescence method can be easily detected.

[Second Embodiment]
FIG. 6 is a schematic configuration diagram showing a nucleic acid amplification device according to the second embodiment of the present invention. As shown in FIG. 6, in the nucleic acid amplification device 20, the PCR microchip 1 is placed on the temperature control device 11 from the cover member 3 side. In the present embodiment, the outer surface 3a of the cover member 3 is a heating surface.

  In the second embodiment, the distance between the heating surface and the reaction chamber 4 is less than 0.2 mm. The total light transmittance of the cover member 3 is 90% or more. Moreover, the cover member 3 does not have autofluorescence. Other points are the same as in the first embodiment.

  Also in the second embodiment, the distance between the heating surface and the reaction chamber 4 is less than 0.2 mm, and the reaction chamber 4 is brought close to the temperature adjustment device 11. Easy to get to. Therefore, it is possible to speed up PCR. From the viewpoint of further increasing the speed of PCR, the thickness of the cover member 3 is preferably 0.02 mm or more, and preferably 0.2 mm or less.

  Further, as in the first embodiment, the reaction chamber 4 has a capacity of 1 μL or more and 10 μL or less, and the reaction chamber 4 is small in size, so that heat from the temperature control device 11 is easily transmitted. Therefore, also from this point, it is possible to increase the speed of PCR.

  As in the first embodiment, in the PCR microchip 1, the distance between the first surface 2a of the substrate 2 and the heating surface is 0.18 mm or more. By comprising in this way, the fall of the joint strength of the board | substrate 2 and the cover member 3 and the leakage of a reaction liquid can be prevented further, and the reliability of a test | inspection can be improved further. Moreover, in this embodiment, since the distance from the joining surface of the substrate 2 and the cover member 3 to the heating surface can be adjusted regardless of the thickness of the substrate 2, the height of the reaction chamber 4 (recess 2c) The distance between the heating surface and the reaction chamber 4 can be set freely. In the present embodiment, the distance between the first surface 2 a of the substrate 2 and the heating surface is equal to the thickness of the cover member 3.

  The adhesive strength between the first surface 2a of the substrate 2 and the cover member 3 at 105 ° C. is preferably 1 N / inch or more. In this case, since the cover member 3 is not easily peeled off by heat, liquid leakage from the reaction chamber 4 is less likely to occur, and the reliability of fluorescence detection can be further enhanced.

  Further, as in the second embodiment, by increasing the total light transmittance of the substrate 2 on the side opposite to the temperature control device 11 side, or by using a material that does not have autofluorescence for the substrate 2, The reliability of detection can be further increased. Therefore, also in the nucleic acid amplification device 20 and the PCR microchip 1 of the second embodiment, it is possible to increase the speed of PCR and to improve the reliability of fluorescence detection. Also in the second embodiment, an adhesive layer 9 may be provided between the first surface 2a of the substrate 2 and the cover member 3 as shown in a modified example in FIG. In this case, the distance between the first surface 2 a of the substrate 2 and the heating surface is equal to the sum of the thickness of the cover member 3 and the thickness of the adhesive layer 9.

[Third Embodiment]
FIG. 8 is a schematic configuration diagram showing a nucleic acid amplification device according to the third embodiment of the present invention. As shown in FIG. 8, in the nucleic acid amplification device 30, the substrate 2 having a plurality of recesses 2c is configured by sealing one surface of a substrate member 2A having a through hole with a cover member 3A. The other points are the same as in the second embodiment.

  Also in the third embodiment, the distance between the heating surface and the reaction chamber 4 is less than 0.2 mm, and the reaction chamber 4 is brought close to the temperature adjustment device 11. Easy to get to. Therefore, it is possible to speed up PCR. Similarly to the first embodiment, the capacity of the reaction chamber 4 is 1 μL or more and 10 μL or less, and the size of the reaction chamber 4 is small, so that heat from the temperature control device 11 is easily transmitted. Therefore, also from this point, it is possible to increase the speed of PCR.

  Moreover, as shown in a modified example in FIG. 9, the substrate member 2A having a through hole and the cover member 3A may be bonded together with an adhesive. That is, the board | substrate 2 which has the several recessed part 2c may be comprised by 2A of board | substrate members which have a through-hole, adhesive layer 9A, and cover member 3A. Even in that case, the same effects as those of the PCR microchip and the nucleic acid amplification device of the first to third embodiments can be obtained.

[Examples and Comparative Examples]
Next, the present invention will be clarified by giving specific examples and comparative examples of the present invention. In addition, this invention is not limited to a following example.

Example 1
In Example 1, the PCR microchip 1 shown in FIG. 6 was produced as follows.

  First, as a material constituting the substrate 2, a cycloolefin polymer (manufactured by Nippon Zeon Co., Ltd., trade name “Zeonor 1420R”) is used, and this is injection-molded to have 10 recesses 2c and a thickness of 0.5 mm. A substrate 2 was prepared. Subsequently, a 0.16 mm thick cycloolefin polymer film (manufactured by Nippon Zeon Co., Ltd., trade name “Zeonor 14F”) was used as the cover member 3 and bonded to the substrate 2 by heat sealing at 140 ° C. A PCR microchip 1 having 10 reaction chambers 4 and each reaction chamber 4 having a capacity of 5 μL was obtained, and the obtained PCR microchip 1 was placed on the temperature control device 11 side. The member to be covered is the cover member 3. That is, the outer surface 3a of the cover member 3 is a heating surface.

(Example 2)
In the same manner as in Example 1, except that the substrate 2 and the cover member 3 were bonded together by a photo-curing adhesive (trade name “3017E” manufactured by Three Bond Co., Ltd.) instead of heat fusion. Chip 1 was obtained. The thickness of the photocurable adhesive was 0.02 mm. In the obtained PCR microchip 1, the member disposed on the temperature control device 11 side is the cover member 3.

(Example 3)
The cover member 3 is not a film, but a pressure-sensitive adhesive tape having a thickness of 0.1 mm (product name “9795R” manufactured by 3M), and the substrate 2 and the pressure-sensitive adhesive tape are bonded together by pressure-sensitive adhesion. Except for the above, a microchip for PCR 1 was obtained in the same manner as in Example 1. In the obtained PCR microchip 1, the member disposed on the temperature control device 11 side is the cover member 3.

Example 4
The cover member 3 is not a film but a pressure-sensitive adhesive tape having a thickness of 0.1 mm (product name “9795R” manufactured by 3M), and the substrate 2 and the cover member 3 are bonded together by pressure-sensitive adhesion. Obtained a microchip 1 for PCR in the same manner as in Example 1. In the obtained PCR microchip 1, the member disposed on the temperature control device 11 side is the substrate 2. That is, the second surface 2b of the substrate 2 is a heating surface. In the substrate 2, the distance between the heating surface and the reaction chamber 4 is 0.1 mm.

(Example 5)
As a material constituting the substrate 2, polypropylene having a thickness of 0.5 mm (PP, manufactured by Nippon Polypro Co., Ltd., trade name “MA3H”) is used, and as the cover member 3, polypropylene having a thickness of 0.16 mm (PP, manufactured by Acrysanday Co., Ltd., product) The PCR microchip 1 was obtained in the same manner as in Example 1 except that the name “PF-11”) was used. In the obtained PCR microchip 1, the member disposed on the temperature control device 11 side is the cover member 3.

(Comparative Example 1)
A PCR microchip was obtained in the same manner as in Example 1 except that the number of reaction chambers 4 obtained was one and the material constituting the substrate 2 was injection-molded so that the volume was 50 μL. In the obtained PCR microchip, the member disposed on the temperature control device 11 side is the cover member 3.

(Comparative Example 2)
The number of reaction chambers 4 obtained is two, the material constituting the substrate is injection-molded so that the volume of each reaction chamber 4 is 20 μL, and the cover member 3 is a cycloolefin polymer film having a thickness of 0.2 mm ( A PCR microchip was obtained in the same manner as in Example 1 except that the product name “ZEONOR 14F” manufactured by Nippon Zeon Co., Ltd. was used. The member to be arranged is the cover member 3.

(Evaluation of Examples and Comparative Examples)
Evaluations shown in the following (1) to (3) were performed on the PCR microchips obtained in the examples and comparative examples. The results are shown in Table 1 below.

(1) Ct value It evaluated by using FAM as a fluorescent pigment | dye, applying excitation light with a wavelength of 494 nm, and detecting the light with a wavelength of 534.5 nm. Moreover, the Crossing Point method was used as an analysis method. In the PCR using the microchips of Examples 1 to 5, the Ct value is small compared to the case of using the microchips of Comparative Examples 1 and 2, and high-speed PCR can be realized.

(2) Peel strength (adhesive strength)
The peel strength at the interface between the substrate 2 and the cover member 3 was measured using a product name “RTC-1310A” manufactured by Orientec. The measurement was performed by chucking the end portions of the substrate 2 and the cover member 3 in an atmosphere of 105 ° C., and peeling them off at a speed of 300 mm / min so that the surfaces of both sides become 90 °.

(3) Gas leak test The pressure in the reaction chamber during heating at 105 ° C. was measured using a product name “FL-610” manufactured by Fukuda. If the pressure at 5 minutes after the temperature of the surface of the substrate 2 and the cover member 3 in contact with the heater reached 105 ° C., the pressure was 1.15 atm or more, and if it was less than 1.15 atm, the test was rejected.

  In the PCR using the microchips of Examples 1 to 5, the Ct value is smaller than in the case of using the microchips of Comparative Examples 1 and 2, and it can be seen that high-speed PCR can be realized.

  Further, from the results of the gas leak test in Examples 1 to 5, the distance between the first surface 2a of the substrate 2 and the heating surface is 0.18 mm or more, or the first surface 2a of the substrate 2 and the cover member It can be seen that if at least one of the peel strength (adhesive strength) at the interface with 3 is 1 N / inch or more is satisfied, the sealing property is excellent. In Comparative Examples 1 and 2, since the capacity of each reaction chamber 4 is larger than 10 μL, it is considered that good sealing performance could not be obtained.

DESCRIPTION OF SYMBOLS 1 ... PCR microchip 2 ... Board | substrate 2a, 2b ... 1st, 2nd surface 2c ... Recess 2d ... Bottom 2e ... Through-hole 2A ... Substrate member 3, 3A ... Cover member 3a ... Outer surface 4, 4a-4i ... Reaction chamber 5 ... Introducing section 6, 6a-6c ... Micro flow path 7a-7i ... Weighing section 8 ... Discharging section 9 ... Adhesive layers 10, 20, 30 ... Nucleic acid amplification apparatus 11 ... Temperature control apparatus 12 ... Peltier element 12a , 12b ... third and fourth surfaces 13 ... heat conducting material layer 14 ... heat dissipation member 15 ... temperature sensor 16 ... control device 17 ... Peltier element drive circuit

Claims (8)

A microchip for PCR having a microchannel, a heating surface heated by a temperature control device, and a reaction chamber in which PCR is performed,
A substrate having a first surface and a second surface facing each other, wherein the first surface is provided with at least one recess;
A cover member provided on the first surface of the substrate,
The reaction chamber is constituted by the cover member and the recess;
The distance between the heating surface and the reaction chamber is less than 0.2 mm;
A PCR microchip, wherein the reaction chamber has a volume of 1 μL or more and 10 μL or less.   The PCR microchip according to claim 1, wherein the number of reaction chambers is 5 or more and 20 or less.   The PCR microchip according to claim 1, further comprising an adhesive layer disposed between the first surface of the substrate and the cover member.   The PCR microchip according to any one of claims 1 to 3, wherein a distance between the first surface of the substrate and the heating surface is 0.18 mm or more.   The PCR microchip according to any one of claims 1 to 4, wherein an adhesive strength between the first surface of the substrate and the cover member at 105 ° C is 1 N / inch or more.   The PCR microchip according to any one of claims 1 to 5, wherein the substrate has a through hole that connects the reaction chamber and the outside.   The PCR microchip according to any one of claims 1 to 6, wherein a reagent is accommodated in the reaction chamber at a rate of 80% or more of the capacity of the reaction chamber. The microchip for PCR according to any one of claims 1 to 7,
A temperature control device;
A nucleic acid amplification apparatus comprising:

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