JP2017063779A - Temperature regulating apparatus for pcr and nucleic acid amplification apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a temperature regulating apparatus for PCR which makes it possible to reduce temperature variations among PCR reaction units in a microchip having a plurality of PCR reaction units.SOLUTION: The PCR temperature regulating apparatus 61 for a microchip 1 has a microchannel, and comprises: a Peltier element 62; and a thermally conductive material layer 64 disposed on the Peltier element 62 and in contact with the microchip 1.SELECTED DRAWING: Figure 6

Description

  The present invention relates to a PCR temperature control device and a nucleic acid amplification device.

  Patent Document 1 discloses a microfluidic chip suitable for performing PCR (Polymerase Chain Reaction) and the like. In the microfluidic chip described in Patent Document 1, a heat transfer member is arranged from the outer surface of the microchannel to the outer surface of the chip. By contacting the heat transfer member with a cooling heat source, the microfluidic can be heated or cooled.

JP 2007-278789 A

  The inventors of the present application have examined an apparatus that can perform a multi-item inspection at the same time by performing PCR using, for example, a microchip having a plurality of PCR reaction units. However, in this case, if temperature variation occurs between the PCR reaction units, amplification in each PCR reaction unit may not be complete, or nucleic acids other than the amplification target may be amplified, making it difficult to perform an appropriate test. I found a new problem. In addition, there is a need for downsizing the apparatus in the PCR inspection using such a microchip. In order to reduce the size, for example, it is conceivable to simplify the temperature adjustment device and adjust the reaction temperature of the PCR with only one heater. In such a case, temperature unevenness occurs at the center and the end of the microchip. Sometimes it became prominent.

  An object of the present invention is to provide a temperature control device for PCR and a nucleic acid amplification device that can reduce temperature variation between PCR reaction units.

  The PCR temperature control device according to the present invention is a PCR temperature control device for a microchip having a microchannel, and is arranged on the Peltier element and the Peltier element, and is in contact with the microchip. A heat conductive material layer.

  In another specific aspect of the temperature control device for PCR according to the present invention, the thermally conductive material layer is a metal.

  In still another specific aspect of the temperature control apparatus for PCR according to the present invention, the heat conductive material layer includes a first heat conductive member and a second heat conductive member, and the second heat conductive member. Are stacked on the first heat conducting member, and the heat conductivity of the second heat conducting member is greater than the heat conductivity of the first heat conducting member.

  In still another specific aspect of the temperature control device for PCR according to the present invention, the thermal conductive material layer includes an anisotropic thermal conductive member having a thermal conductivity in a plane direction higher than that in a thickness direction, and the different thermal conductivity member. A heat conductive member made of metal laminated on the isotropic heat conductive member. Preferably, the anisotropic heat conducting member is graphite.

  On the other specific situation of the temperature control apparatus for PCR which concerns on this invention, the thickness of the said heat conductive material layer exists in the range of 0.01 mm or more and 1 mm or less.

  In still another specific aspect of the PCR temperature control device according to the present invention, the PCR device further includes an introduction device for introducing a predetermined amount of the sample into the microchip.

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

  In a specific aspect of the nucleic acid amplification device according to the present invention, the microchip has a plurality of PCR reaction units.

  In another specific aspect of the nucleic acid amplification device according to the present invention, the volumes of the plurality of PCR reaction units are in the range of 0.5 μL or more and 20 μL or less, respectively.

  In another specific aspect of the nucleic acid amplification device according to the present invention, among the plurality of PCR reaction units, a primer is accommodated in at least two or more PCR reaction units, and accommodated in the at least two or more PCR reaction units. The difference between the maximum value and the minimum value of the Tm value of the primer is 10 ° C. or less.

  In still another specific aspect of the nucleic acid amplification device according to the present invention, the microchip includes a weighing unit that introduces a certain amount of sample into the PCR reaction unit.

  According to the PCR temperature control device and the nucleic acid amplification device according to the present invention, in a microchip having a plurality of PCR reaction units, temperature variations between the PCR reaction units can be reduced. Therefore, according to the PCR temperature control device and the nucleic acid amplification device of the present invention, the accuracy in the multi-item PCR test can be increased.

It is a perspective view which shows the external appearance of the microchip which comprises the nucleic acid amplifier which concerns on one Embodiment of this invention. In the microchip which comprises the nucleic acid amplifier which concerns on one Embodiment of this invention, it is a typical top view which shows the flow-path structure provided in the chip | tip main body. 1 is a partially cutaway schematic cross-sectional view for explaining a PCR reaction part and a valve in an embodiment of the present invention. FIG. 4 is a partially cutaway schematic cross-sectional view for explaining a state in which the valve is closed in the structure shown in FIG. 3. It is a partial notch schematic sectional drawing for demonstrating the other example of the sealing means in the microchip which comprises the nucleic acid amplifier which concerns on one Embodiment of this invention. 1 is a schematic configuration diagram showing a nucleic acid amplification device according to an embodiment of the present invention. It is a schematic block diagram which expands and shows the part in which the heat conductive material layer which comprises the temperature control apparatus for PCR in FIG. 6 is provided. It is a schematic block diagram which expands and shows the modification of the heat conductive material layer which comprises the temperature control apparatus for PCR in FIG.

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

  FIG. 6 is a schematic configuration diagram showing a nucleic acid amplification device according to an embodiment of the present invention. FIG. 7 is an enlarged schematic configuration diagram showing a portion where the heat conductive material layer constituting the PCR temperature adjusting device 61 in FIG. 6 is provided.

  The nucleic acid amplification device 71 includes a PCR temperature control device 61 and the microchip 1. The PCR temperature control device 61 is a PCR temperature control device for the microchip 1. More specifically, the PCR temperature control device 61 is a temperature control device used when performing PCR using the microchip 1 having a microchannel. Therefore, the temperature control target of the PCR temperature control device 61 is the microchip 1 (the reaction solution thereof). The PCR temperature control device 61 includes a Peltier element 62, a heat radiating member 63, and a heat conductive material layer 64.

  The Peltier element 62 is provided for heating or cooling the microchip 1. The Peltier element 62 has a first surface 62a and a second surface 62b facing the first surface 62a.

  A heat conductive material layer 64 is laminated on the first surface 62 a of the Peltier element 62. The microchip 1 is laminated on the surface of the heat conductive material layer 64 opposite to the Peltier element 62 side. Therefore, by providing the heat conductive material layer 64, it is possible to reduce the temperature unevenness of the surface (the lower surface of the microchip) in contact with the heat conductive material layer 64 in the microchip 1. Since the temperature unevenness of the surface in contact with the heat conductive material layer 64 in the microchip 1 can be reduced, the temperature variation between the PCR reaction parts of the microchip 1 can be reduced. Therefore, in the PCR temperature control device 61 and the nucleic acid amplification device 71, it is possible to increase the accuracy in a multi-item PCR test.

  On the other hand, a heat radiating member 63 is connected to the second surface 62 b side of the Peltier element 62. The heat dissipation member 63 is provided to dissipate heat generated in the Peltier element 62. As the heat radiating member 63, for example, a heat sink made of metal can be used.

  In addition, the heat radiating member 63 should just be arrange | positioned at the 2nd surface 62b side of the Peltier element 62. FIG. The heat radiating member 63 may be disposed so as to be in direct contact with the second surface 62b, or may be connected to the second surface 62b via another member.

  In order to detect the temperature of the upper surface of the heat conductive material layer 64, a temperature sensor 65 is provided. The temperature sensor 65 can be configured by an appropriate temperature-sensitive element that can detect the temperature of the upper surface of the heat conductive material layer 64. The temperature sensor 65 is connected to the control device 66 and gives a signal based on the detected temperature to the control device 66.

  A Peltier element driving circuit 67 is connected to the control device 66. The Peltier element driving circuit 67 is driven by the control device 66 to drive the Peltier element 62. Specifically, the Peltier element driving circuit 67 applies a voltage for heating or cooling the Peltier element 62 to the Peltier element 62. Thereby, the Peltier element 62 is heated or cooled.

  Although not illustrated in the present embodiment, the nucleic acid amplification device 71 includes a detection mechanism that amplifies the nucleic acid by a PCR reaction and then detects the amplified nucleic acid. The PCR temperature adjusting device 61 may further include an introduction device such as a dispenser that introduces a certain amount of sample into the microchip 1. In that case, since the same amount of sample can be introduced into each PCR reaction part, temperature variation among a plurality of PCR reaction parts can be further effectively reduced. However, the microchip 1 may have a weighing unit that introduces a certain amount of sample into the PCR reaction unit. Even in such a case, the same amount of sample can be introduced into each PCR reaction section, so that temperature variations among a plurality of PCR reaction sections can be further effectively reduced.

  Hereinafter, the heat conductive material layer 64 constituting the PCR temperature adjusting device 61 and the microchip 1 used in the PCR temperature adjusting device 61 will be described in more detail.

(Heat conduction material layer)
A metal can be used as a material constituting the heat conductive material layer 64. The metal is preferably made of Al or an alloy of Al. In that case, the temperature variation between the PCR reaction units can be more effectively reduced.

  The thickness of the heat conductive material layer 64 is preferably in the range of 0.01 mm or more and 1 mm or less. In that case, the temperature variation between the PCR reaction units can be more effectively reduced.

The thermal conductivity of the heat conductive material layer 64 is desirably in the range of 0.5 W · m ⁻¹ · K ⁻¹ or more and 2000 W · m ⁻¹ · K ⁻¹ or less. In that case, temperature variations between the PCR reaction units can be further reduced.

  As long as the heat conductive material layer 64 is excellent in heat conductivity as described above, an appropriate heat conductive material can be used. The heat conductive material layer 64 is not limited to the above metal, and a carbon material such as grease or graphite is used. Also good.

  Further, as shown in a modified example in FIG. 8, the heat conductive material layer 64 may include a first heat conductive member 64A and a second heat conductive member 64B. In the modified example, a second heat conductive member 64B having a different thermal conductivity from that of the first heat conductive member 64A is laminated on the first heat conductive member 64A. Accordingly, the first heat conducting member 64A is disposed on the Peltier element 62 side, and the second heat conducting member 64B is disposed on the microchip 1 side. In this modification, the heat conductive material layer 64 has a structure in which two heat conductive members are stacked, but may have a structure in which three or more heat conductive members are stacked.

The first heat conducting member 64A and the second heat conducting member 64B may be made of the same material, but may be made of different materials. When composed of different materials, the thermal conductivity λ ₁ of the second thermal conductive member 64 B on the microchip 1 side and the thermal conductivity λ ₂ of the first thermal conductive member 64 A on the Peltier element 62 side are λ The relationship is preferably ₁ ≧ λ ₂ , and more preferably the relationship of λ ₁ > λ ₂ . In this case, temperature unevenness in the surface direction can be more effectively suppressed. Examples of the combination of the first and second heat conducting members 64A and 64B include a combination in which Al is used for the first heat conducting member 64A and Cu is used for the second heat conducting member 64B. Can do. In this case, for example, the first and second heat conducting members 64A and 64B can be formed by depositing or sputtering Cu on an Al plate.

  The first heat conducting member 64A disposed on the Peltier element 62 side can be made of an anisotropic heat conducting material. The anisotropic heat conductive material is a heat conductive material whose thermal conductivity in the plane direction, which is a direction parallel to the first surface 62a, is higher than the thermal conductivity in the thickness direction. When such an anisotropic heat conductive material is used, the temperature unevenness in the surface direction can be more effectively suppressed. Therefore, temperature variations among a plurality of PCR reaction units can be further effectively reduced. In the present specification, the thickness direction refers to the stacking direction of the Peltier element 62 and the heat conductive material layer 64, and the plane direction is orthogonal to the stack direction of the Peltier element 62 and the heat conductive material layer 64. It means the surface direction to be performed.

  As the anisotropic heat conductive material, graphite can be suitably used. However, as the anisotropic heat conductive material, a heat conductive material including a resin and metal nanowires or nanofillers oriented in a specific direction in the resin may be used.

  As a preferable combination of the first and second heat conducting members 64A and 64B, the first heat conducting member 64A is a graphite sheet, and the second heat conducting member 64B is a combination of metal plates. In that case, temperature variations among a plurality of PCR reaction units can be further effectively reduced.

(Microchip)
Inside the microchip 1, there is provided a microchannel through which a microfluid is conveyed. A plurality of PCR reaction units are provided in a part of the microchannel. Therefore, the microchip 1 is a PCR testing microchip having a plurality of PCR reaction units. The plurality of PCR reaction units are provided for amplifying a nucleic acid and detecting the amplified nucleic acid. The volumes of the plurality of PCR reaction units are in the range of 0.5 μL or more and 20 μL or less, respectively. As in this embodiment, by setting the volume of the plurality of PCR reaction units within the above range, the accuracy in a multi-item PCR test can be further enhanced.

  Of the plurality of PCR reaction units, at least two or more PCR reaction units contain a primer that binds to the nucleic acid to be amplified. Further, the difference between the maximum value and the minimum value of the Tm value (melting temperature) of the primer in the at least two PCR reaction parts is 10 ° C. or less. That is, the difference in Tm value between the primers accommodated in the at least two PCR reaction parts is 10 ° C. or less. As in this embodiment, by setting the difference between the maximum value and the minimum value of the Tm value (melting temperature) of the primer to 10 ° C. or less, it is possible to reduce the temperature variation between the PCR reaction parts, and to perform a multi-item PCR test. The accuracy in the can be further increased.

  The difference between the maximum value and the minimum value of the Tm values of the primers accommodated in the at least two or more PCR reaction units is preferably 5 ° C. or less. Specifically, the Tm value of the primer is preferably 60 ° C. or higher and 70 ° C. or lower. In that case, the precision in the multi-item PCR test can be further increased.

  The microchip 1 may have a chip body made of a laminate of a plurality of substrates. Hereinafter, a specific example of a microchip having a chip body made of a laminate of a plurality of substrates will be described.

  FIG. 1 is a perspective view showing the appearance of a microchip constituting a nucleic acid amplification device 71 according to an embodiment of the present invention.

  The microchip 1 has a chip body 2. The chip body 2 is configured by stacking a plurality of substrates 3 to 7. As a material constituting the substrates 3 to 7, materials such as synthetic resin, rubber, or metal can be used. The substrates 4 and 6 are made of rubber having flexibility. As such rubber, silicone rubber is used in this embodiment. The thickness of the substrate 6 is preferably in the range of 0.05 to 0.5 mm, and the thickness of the substrate 7 is preferably in the range of 0.1 to 0.3 mm.

  On the substrate 5 located in the center in the thickness direction, a microchannel described later is formed. Moreover, the board | substrates 3 and 7 located in the thickness direction outermost side consist of a comparatively thin synthetic resin board which can be bent by the chemical operation mentioned later in this embodiment. One substrate 3 has an introduction portion 8 opened. Further, a discharge portion 9 is also opened on the outer surface of the substrate 3. The discharge unit 9 may be opened on the lower surface of the substrate 7.

  The substrate 5 can be formed of hard synthetic resin or glass. The substrate 5 is provided with a flow path structure schematically shown in FIG. As shown in FIG. 2, the first microchannel 12 is connected to the introduction portion 8. The first microchannel 12 is branched into second microchannels 12a to 12c.

  Three weighing units 13a to 13c are connected to the second microchannel 12a. One ends of third microchannels 14a to 14c having a smaller cross-sectional area than the weighing units 13a to 13c are connected to the tips of the weighing units 13a to 13c. The first to third PCR reaction units 15a to 15c are connected to the other ends of the third microchannels 14a to 14c. Discharge flow paths 16a to 16c as fourth micro flow paths are connected to the side of the first to third PCR reaction units 15a to 15c opposite to the side to which the third micro flow paths 14a to 14c are connected. Has been. The discharge channels 16 a to 16 c are joined to the fifth micro channel 17.

  The fifth microchannel 17 is connected to the seventh microchannel 19 via the sixth microchannel 18 having a smaller cross-sectional area than the fifth microchannel 17. A seventh microchannel 19 is connected to the discharge unit 9.

  Similarly, the weighing units 23a, 23b, and 23c are connected to the second microchannel 12b. The weighing units 23a, 23b, and 23c are connected to the third microchannels 24a, 24b, and 24c, respectively. The fourth to sixth PCR reaction units 25a, 25b, and 25c are connected to the third microchannels 24a, 24b, and 24c, respectively. And the discharge flow paths 26a-26c as a 4th micro flow path are connected to the 4th-6th PCR reaction part 25a-25c. The discharge channels 26 a to 26 c are joined to the fifth micro channel 27. A discharge flow channel 27 as a fifth micro flow channel is connected to the seventh micro flow channel 19 via a sixth micro flow channel 28.

  Similarly, the weighing units 33a, 33b, and 33c are connected to the second microchannel 12c. Seventh to ninth PCR reaction units 35a to 35c are connected to the weighing units 33a to 33c via third microchannels 34a to 34c, respectively. And the discharge flow paths 36a-36c as a 4th micro flow path are connected to the 7th-9th PCR reaction part 35a-35c. The discharge flow paths 36 a to 36 c are joined to the fifth micro flow path 37. The fifth microchannel 37 is connected to the seventh microchannel 19 via the sixth microchannel 38.

  On the other hand, downstream ends of the second microchannels 12 a to 12 c are connected to the discharge unit 9.

  The volumes of the weighing units 13a to 13c, 23a to 23c, and 33a to 33c are made equal.

  The first to ninth PCR reaction units 15a to 15c, 25a to 25c, and 35a to 35c each contain a primer whose Tm value has been adjusted in advance.

  For example, ΔTm (= TmH−TmL) which is the difference between TmH and TmL when the highest Tm value is TmH and the lowest Tm value is TmL among the Tm values of the primers between a plurality of PCR reaction parts. Is adjusted so that ΔTm ≦ 10 ° C. The Tm values may be adjusted to be the same.

  Moreover, what is necessary is just to adjust GC content in order to make Tm value of the primer couple | bonded with a nucleic acid into +/- 10 degrees C or less between several primers. For example, when the Tm value is 60 ° C., the GC content ratio between the primers is 45 to 50%, and the ratio when the Tm values of the primer 1 and the primer 2 are Tm1 and Tm2, respectively, is Tm1 / Tm2 = 0. .8 to 1.2 is sufficient.

  For example, the primers shown in Table 1 below can be used as long as they are primers targeting chlamydia 16S RNA. Tm value approximates by making GC content comparable.

  Preferably, the first to ninth PCR reaction units 15a to 15c, 25a to 25c, and 35a to 35c contain a fluorescent substance in order to facilitate detection of the amplified nucleic acid in the PCR reaction unit. May be.

  As shown in FIG. 2, in the channel structure 11, the weighing units 13a to 13c, 23a to 23c, 33a to 33c, and the first to ninth PCR reaction units 15a to 15c, 25a to 25c, and 35a to 35c. Are connected by the above-described microchannel.

  When performing a PCR test, a liquid sample is introduced from the introduction unit 8. This introduction can be performed using a micropipette or a pump that projects a microfluid. As this liquid specimen, a buffer solution containing DNA or the like is used.

  The liquid sample passes through the first microchannel 12 and reaches the second microchannels 12a to 12c. And it will fill in the balance parts 13a-13c, 23a-23c, and 33a-33c. After the liquid sample fills the weighing units 13 a to 13 c, 23 a to 23 c, and 33 a to 33 c, the surplus liquid sample moves through the second microchannels 12 a to 12 c and reaches the discharge unit 9. Therefore, it is possible to weigh a certain amount of specimen according to the volumes of the weighing units 13a to 13c, 23a to 23c, and 33a to 33c.

  Furthermore, gas is fed into the second microchannels 12a to 12c using a micropump or the like and pressurized. Then, a certain amount of sample weighed in the weighing units 13a to 13c, 23a to 23c, and 33a to 33c is supplied to the first to ninth PCR reaction units 15a to 15c, 25a to 25c, and 35a to 35c. To do.

  Next, the sample is stopped in the first to ninth PCR reaction units 15a to 15c, 25a to 25c, and 35a to 35c by closing a valve as a sealing means.

  3 and 4 are partially cutaway schematic cross-sectional views for explaining a valve as the sealing means.

  FIG. 3 shows a portion where the first PCR reaction unit 15a in FIG. 2 is provided. The substrate 5 is provided with a microchannel and a first PCR reaction unit 15a. A valve 41 is provided on the upstream side of the first PCR reaction unit 15a. A valve 42 is provided on the downstream side of the first PCR reaction unit 15a. The valve 41 includes through holes 41a and 41c provided in the substrate 4, and a connection flow path 41b provided in the substrate 4 and connecting the through holes 41a and 41c.

  The connection flow path 41 b has a height direction dimension from the upper surface of the substrate 4 to an intermediate position of the substrate 4. A channel sealing support 41d is provided below the connection channel 41b. The channel sealing support portion 41 d is constituted by a part of the substrate 4. The substrate 4 is made of silicone rubber as described above. Therefore, when a force is applied in the thickness direction, the film can be deformed so as to become thinner.

  The valve 42 includes through holes 42a and 42c, a connection channel 42b, and a channel sealing support 42d. This valve 42 is the same as the valve 41 except that it is provided on the substrate 6 side.

  As described above, the valves 41 and 42 are closed when a certain amount of specimen is accommodated in the first PCR reaction unit 15a. Specifically, as shown by an arrow A in FIG. 3, the substrate 3 is pressurized toward the substrate 7 side. This pressurizing operation may be performed with a finger or may be performed using an appropriate jig.

  As a result, the substrates 4 and 6 made of silicone rubber are subjected to compressive stress and are deformed so as to be thin. Therefore, the connection flow paths 41b and 42b are closed, and the valves 41 and 42 are closed as shown in FIG. Therefore, a liquid sample is reliably accommodated in the first PCR reaction unit 15a.

  As shown in FIG. 6, the microchip 1 is disposed on the heat conductive material layer 64 of the PCR temperature adjusting device 61. The lower surface of the substrate 7 constituting the microchip 1 is in contact with the heat conductive material layer 64. As described above, the PCR temperature adjusting device 61 is configured by laminating the heat radiating member 63, the Peltier element 62, and the heat conducting material layer 64 in this order.

  Further, the temperature of the first PCR reaction unit 15a is controlled by the Peltier element 62 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. The Peltier element 62 can be heated or cooled by being given a voltage by the Peltier element driving circuit 67 as described above.

  In the remaining second to ninth PCR reaction units 15b to 15c, 25a to 25c, and 35a to 35c, PCR can be simultaneously performed by the Peltier element 62 as described above.

  In the first to ninth PCR reaction units 15a to 15c, 25a to 25c, and 35a to 35c, primers are accommodated in advance. The PCR reaction units 15a to 15c, 25a to 25c, and the PCR reaction units 15a to 15c, 25a to 25c, 35a to 35c, so that the Tm value of the primer that binds to the nucleic acid is ± 10 ° C or less between the plurality of primers. 25a to 25c and 35a to 35c. A single chip body 2 can be used to simultaneously identify a plurality of bacteria and the cause of the disease can be identified effectively and easily.

  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, the first to ninth PCR reaction units 15a to 15c, 25a to 25c, and 35a to 35c are desirably previously accommodated with fluorescent substances for detecting the amplified nucleic acid. Thereby, the nucleic acid amplified by the fluorescence method can be easily detected.

  In the microchip 1 of the present embodiment, as described above, from introduction of a liquid sample to weighing of a certain amount of sample, PCR reaction, and inspection can be performed simultaneously and efficiently.

  In the microchip 1, the valves 41 and 42 are shown as sealing means. However, instead of the valves 41 and 42, a micropump is provided in the chip body 2 and the micropump and the PCR reaction unit are connected to each other. You may use the structure which accommodated curable resin in the flow-path part. That is, a micropump 51 may be provided in the chip body 2 as shown in a partially cutaway schematic cross-sectional view in FIG. As the micropump 51, a micropump containing a gas generating agent that generates a gas by irradiation with light can be used. However, the structure of the micropump is not particularly limited.

  A micro flow path 52 is connected to the micro pump 51. A curable resin 53 is accommodated in the microchannel 52. The micro flow channel 52 is connected to the micro flow channel portions on the upstream side and the downstream side of the first PCR reaction unit 15a. When closing the channel, the micropump 51 is driven to push the curable resin 53 to the upstream and downstream microchannel portions of the first PCR reaction unit 15a, and the upstream and downstream microchannel portions. Is closed by curing the curable resin 53. As described above, a sealing unit using the micropump 51 and the curable resin 53 may be used.

  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.

  As Peltier element 62, Taisei make, model number: UT-3030CE-M was used. A model number: SK434 / 50SA manufactured by Fixcher was fixed to the Peltier element 62 as a heat sink.

  As the Peltier element driving circuit 67, OMRON Corporation model number: E5CC-QX2DSM-002 was used. As the temperature sensor 65, model number: NER-CF2-0305 manufactured by Nethshin Co., Ltd. was used.

  Further, as shown in Table 2 below, in Examples 1 and 4 to 6, an aluminum plate was used as the first heat conductive member 64A. However, the thickness of the first heat conductive material layer 64A is set to the respective sizes shown in Table 2. In Examples 2 and 3, a copper plate or a silver plate having a thickness of 0.3 mm was used as the first heat conducting member 64A. In Examples 1 to 6, the heat conductive material layer 64 was configured only by the first heat conductive member 64A.

  In Examples 7 and 8, the heat conductive material layer 64 including the first heat conductive member 64A and the second heat conductive member 64B laminated on the first heat conductive member 64A was used. In Example 7, a graphite sheet was used for the first heat conducting member 64A, and an aluminum plate was used for the second heat conducting member 64B. In Example 7, an aluminum plate was used as the second heat conductive member 64B, and the first heat conductive member 64A was formed by applying grease. The thicknesses of the first and second heat conducting members 64A and 64B are the thicknesses and sizes shown in Table 2. In Comparative Example 1, the heat conductive material layer 64 was not used.

(Evaluation of Examples and Comparative Examples)
Evaluation of temperature unevenness Temperature control was performed so that the temperature of the heating surface in the PCR temperature adjusting device 61 was 60 ° C. When the heat conductive material layer 64 is laminated, the temperature of the surface of the heat conductive material layer 64 is the temperature of the first surface 62a of the Peltier element 62 when the heat conductive material layer 64 is not laminated. Was measured. The planar shape of the measurement surface is a rectangle, and its dimensions are 30 mm × 30 mm. In a square area of 20 mm × 20 mm located at the center of the measurement surface, the temperature of each vertex of the square and the five points at the center of the square was measured with a thermo camera. Among the five points, the highest temperature was T1 (° C.) and the lowest temperature was T2 (° C.). The temperature unevenness was defined as follows.

  Temperature unevenness (° C) = T1 (° C)-T2 (° C)

  The evaluation results of Examples 1 to 8 and Comparative Example 1 are shown in Table 2 below.

  As is clear from Table 2, it can be seen that the temperature unevenness is small according to Examples 1 to 8 as compared with Comparative Example 1 in which the heat conductive material layer 64 is not provided. From Examples 1 and 4 to 6, it can be seen that temperature unevenness can be further effectively reduced by setting the thickness of the aluminum plate to 0.5 mm or more.

  In Example 2 and Example 3, since a 0.3 mm copper plate or silver plate was used as the heat conductive material layer 64, temperature unevenness can be effectively reduced. It can also be seen that it is desirable to use a silver plate as in Example 3 to reduce temperature unevenness.

  In Examples 7 and 8, a graphite sheet or grease is used for the first heat conducting member 64A and an aluminum plate is used for the second heat conducting member 64B. However, by using a graphite sheet or grease together with the aluminum plate, It can be seen that the temperature unevenness can be reduced more effectively. It can also be seen that it is desirable to use a graphite sheet as in Example 7 to reduce temperature unevenness.

DESCRIPTION OF SYMBOLS 1 ... Microchip 2 ... Chip main body 3-7 ... Board | substrate 8 ... Introducing part 9 ... Discharge part 11 ... Channel structure 12 ... 1st microchannel 12a-12c ... 2nd microchannel 13a-13c, 23a- 23c, 33a to 33c ... weighing units 14a to 14c, 24a to 24c, 34a to 34c ... third micro flow paths 15a to 15c, 25a to 25c, 35a to 35c ... first to ninth PCR reaction parts 16a to 16c, 26a to 26c, 36a to 36c ... discharge channels 17, 27, 37 ... fifth micro channels 18, 28, 38 ... sixth micro channels 19 ... seventh micro channels 41, 42 ... valves 41a, 41c, 42a, 42c ... through holes 41b, 42b ... connection channels 41d, 42d ... channel sealing support 51 ... micro pump 52 ... micro channel 53 ... curable resin 61 ... PCR temperature control Device 62 ... Peltier element 62a ... first surface 62b ... second surface 63 ... heat radiation member 64 ... heat conduction material layer 64A ... first heat conduction member 64B ... second heat conduction member 65 ... temperature sensor 66 ... control Device 67 ... Peltier device driving circuit 71 ... Nucleic acid amplification device

Claims (12)

A temperature control device for PCR for a microchip having a microchannel,
Peltier element,
A thermally conductive material layer disposed on the Peltier element and in contact with the microchip;
A temperature control device for PCR, comprising:   The temperature control device for PCR according to claim 1, wherein the heat conductive material layer is a metal. The heat conductive material layer has a first heat conductive member and a second heat conductive member,
The second heat conducting member is laminated on the first heat conducting member;
The temperature control device for PCR according to claim 1, wherein the thermal conductivity of the second thermal conductive member is larger than the thermal conductivity of the first thermal conductive member.   The heat conductive material layer includes an anisotropic heat conductive member whose thermal conductivity in the plane direction is higher than the heat conductivity in the thickness direction, and a heat conductive member made of metal laminated on the anisotropic heat conductive member. The temperature control apparatus for PCR of Claim 1 which has.   The temperature control apparatus for PCR according to claim 4, wherein the anisotropic heat conducting member is graphite.   The temperature control apparatus for PCR according to any one of claims 1 to 5, wherein a thickness of the heat conductive material layer is in a range of 0.01 mm or more and 1 mm or less.   The PCR temperature control device according to any one of claims 1 to 6, further comprising an introduction device for introducing a predetermined amount of the sample into the microchip. A microchip having a microchannel;
The temperature control device for PCR according to any one of claims 1 to 7,
A nucleic acid amplification apparatus comprising:   The nucleic acid amplification device according to claim 8, wherein the microchip has a plurality of PCR reaction units.   The nucleic acid amplification device according to claim 9, wherein volumes of the plurality of PCR reaction units are in a range of 0.5 μL or more and 20 μL or less, respectively. Primers are accommodated in at least two or more PCR reaction parts among the plurality of PCR reaction parts,
The nucleic acid amplification device according to claim 9 or 10, wherein a difference between a maximum value and a minimum value of Tm values of the primers accommodated in the at least two PCR reaction units is 10 ° C or less.   The nucleic acid amplification device according to any one of claims 9 to 11, wherein the microchip includes a weighing unit that introduces a predetermined amount of sample into the PCR reaction unit.

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