JP3980045B2 - DNA amplification equipment - Google Patents

Description

  The present invention relates to an apparatus for performing a DNA amplification reaction to amplify a small amount of DNA.

  In recent years, the polymerase chain reaction (hereinafter abbreviated as PCR) has been actively used as a means for amplifying a small amount of DNA. In this method, for example, as disclosed in JP-A-6-292579, first, a sample containing a target nucleic acid is subjected to two types of nucleotide sequences containing both base sequences of each strand of the target DNA. Primer, thermostable DNA synthase and four deoxyribonucleoside triphosphates (dATP, dCTP, dGTP and dTTP) are added. Next, the step of dissociating the DNA (denaturation) only by controlling the temperature of the system, the step of binding the single-stranded DNA generated by the dissociation with the primer (annealing), and the single-stranded DNA to which the primer is bound Each step of the step of synthesizing a complementary strand with a DNA synthase (extension) is performed using as a template, and this is repeated one or more times.

  As described above, PCR can efficiently amplify a small amount of DNA, but in addition to this PCR, an attempt has been made to automate further including pretreatment and posttreatment. For example, in the apparatus disclosed in JP-A-6-327476, whole blood is first centrifuged and extracted with a phenol and chloroform solution to purify nucleic acid components in the whole blood (pretreatment stage). Next, the purified nucleic acid component is dissolved in a buffer, the above-mentioned two kinds of primers, heat-resistant DNA synthase and four kinds of deoxyribonucleoside triphosphates are added, and the target nucleic acid is amplified by carrying out thermal cycling. (PCR stage). Finally, the reaction solution containing the amplified nucleic acid is treated by gel electrophoresis, and impurities such as primers and by-products are removed to obtain the target nucleic acid (post-treatment stage).

  In addition, some attempts have been made on the reaction vessel itself for performing PCR. For example, an apparatus disclosed in Japanese Patent Laid-Open No. 7-75544 discloses a thermal cycle necessary for a DNA synthesis reaction by providing a capillary tube through two thermostats and simply flowing a mixed solution containing a specimen into the capillary tube. Is realized. In this method, since the amplification reaction is carried out in the capillary, the temperature gradient between the outside and inside of the liquid flow is reduced, and there is an advantage that the temperature in the system can be equalized more quickly.

  In the conventional DNA amplification apparatus, the reaction vessel is surrounded by a thermostatic bath or a metal block, and the reaction vessel itself is heated and cooled to perform a thermal cycle necessary for the DNA amplification reaction. Therefore, in these apparatuses, the temperature of the reaction solution is not directly controlled but indirectly controlled via the reaction vessel. In addition, in these apparatuses, the temperature of the reaction solution cannot be directly measured because the reaction solution is very small. For this reason, the conventional DNA amplifying apparatus cannot precisely control the temperature of the reaction solution and lacks reliability. In the apparatus disclosed in Japanese Patent Laid-Open No. 7-75544, a temperature gradient is prevented from being formed in the reaction solution by performing the reaction in the capillary. However, this apparatus cannot directly measure the temperature of the reaction solution, and the temperature control is still inaccurate.

  In addition, the apparatus disclosed in the above-mentioned Japanese Patent Application Laid-Open No. 6-327476 realizes full automation that automatically performs all processes including pre-processing and post-processing. All operations are performed mechanically. For this reason, an apparatus becomes large-sized and cost becomes very high. Similarly, the apparatus disclosed in Japanese Patent Application Laid-Open No. 7-75544 also uses a mechanical pump to circulate the reaction solution, which has the disadvantage of increasing the size and cost of the apparatus.

Accordingly, an object of the present invention is to provide a DNA amplification apparatus capable of precisely controlling the temperature of a reaction solution and efficiently performing a DNA amplification reaction. In particular, the present invention makes it easier to remove bubbles present in the sample solution, and prevents uneven temperature distribution in the sample solution due to the presence of bubbles, thereby making the DNA amplification reaction more accurate and efficient. An object of the present invention is to provide a DNA amplifying apparatus that can be used in the above.

  Another object of the present invention is to provide a DNA amplifying device capable of reducing mechanical working parts, thereby reducing the size of the entire device and reducing the cost.

  The DNA amplification apparatus according to the present invention comprises two first and second planar reaction cells formed on an inorganic substrate, a sample introduction unit for introducing a sample solution into the first reaction cell, the second A sample discharge unit for discharging a sample solution from the reaction cell, the sample introduction unit and the first reaction cell, the first reaction cell and the second reaction cell, and the second reaction cell and the sample discharge Three flow paths that communicate with each other, at least one temperature measuring unit provided on the bottom surface of each of the first and second reaction cells, and the back surfaces of the first and second reaction cells of the inorganic substrate And a sample solution transporting means.

  The inorganic substrate used for this apparatus is not specifically limited, For example, a silicon wafer, another semiconductor substrate, glass, or metal plates, such as aluminum and stainless steel, can be used. However, it is preferable to use a silicon wafer from the viewpoint that the temperature measurement unit can be integrally formed and the technique of fine processing is established.

  The production method of the reaction cell, the sample introduction part, the discharge part, and the flow path formed on the inorganic substrate differs depending on the type of the substrate, and may be produced by a method usually used for each material. For example, when a silicon wafer is used as the inorganic substrate, the etching may be performed by a method usually performed in the field of semiconductor manufacturing. Etching may be isotropic etching, anisotropic etching, or dry etching. However, anisotropic wet etching using potassium hydroxide, tetramethylammonium (TMA) or the like is preferable in terms of the smoothness of the bottom surface after etching.

  The temperature measuring unit provided on the bottom surface of the reaction cell may be formed integrally when forming the reaction cell, or the temperature measuring means may be separately fixed after forming the reaction cell. As the temperature measuring means, a diode type using a pn junction, various thermistors, a thermocouple, or the like can be used. In the case where the inorganic substrate is a silicon wafer, it is preferable because a diode-type temperature measuring unit can be directly formed on the substrate.

  Any heating means can be used as long as it can heat the sample solution to a temperature necessary for the DNA amplification reaction, and is not particularly limited. Since it is necessary, those capable of fine temperature control are preferable.

  By the way, since dissolved air exists in the sample solution, fine bubbles may be generated during the heat treatment. Since air has high heat insulating properties, the presence of bubbles tends to make the temperature of the solution non-uniform around the bubbles. In particular, in the apparatus according to the present invention, the reaction cell has a shallow planar shape and has a small capacity, so that the influence of bubbles on temperature control is increased. DNA amplification reaction by PCR requires strict temperature control, and efficiency and accuracy decrease unless the temperature error falls within 0.1 ° C.

  In addition, the DNA amplification reaction includes a step of heating at a high temperature. During heating at this high temperature, the pressure inside the reaction cell increases due to the expansion of the solution. At this time, if bubbles exist in the solution, the pressure increase due to the expansion of the bubbles exceeds that of the solution, and the reaction cell may be damaged or the sample solution may leak.

  Therefore, it is preferable to remove dissolved air in the solution as much as possible.

  As an example of a method for dealing with bubbles generated in a liquid flow path, there can be mentioned an electrolyte analyzer System E4A manufactured by Beckman. The instruction manual of this apparatus describes that the apparatus is provided with a filter called a DEBUBBLER FILTER. In general, it is known that a hydrophobic filter such as a Teflon (registered trademark) filter allows gas to pass through but water does not pass through it. The liquid is directly passed through a holder that holds the water to remove bubbles in the liquid.

  However, it is practically impossible to incorporate the above-described debris filter into a micro device such as the DNA amplification device according to the present invention. Furthermore, it is conceivable to cut out small pieces from the hydrophobic filter used in this debabilizer filter and attach them to a fine flow path on the substrate to provide a bubble removal function. Have difficulty.

  Therefore, the present inventor has also intensively studied a bubble removing mechanism that can be integrated into a minute apparatus, and in the DNA amplification apparatus of the present invention, the first reaction cell and the second reaction cell are combined. It has been found that this problem can be solved by forming a gas discharge hole in the communicating flow path.

  The cross-sectional shape of the gas discharge hole is not particularly limited, and may be a rectangle or a circle, for example. The diameter of the gas discharge hole is preferably 0.2 to 1.0 μm at the smallest. Furthermore, it is preferable that at least the inner peripheral surface of the gas discharge hole has hydrophobicity, and it is more preferable that the peripheral edge of the opening also has hydrophobicity. In order to impart hydrophobicity to the inner peripheral surface of the gas discharge hole and the periphery of the opening, for example, a predetermined region may be covered with a hydrophobic film such as a silane-treated film. In addition, when the substrate in which the gas discharge hole is formed is a single crystal silicon substrate, pure single crystal silicon itself has hydrophobicity, so that pure single crystal silicon, that is, bare single crystal in a predetermined region. The crystalline silicon may be exposed. Note that the “bare single crystal silicon” mentioned here does not exclude that a thin natural oxide film that does not lose hydrophobicity is formed on the surface of pure single crystal silicon. In addition, the inner peripheral surface of the gas discharge hole is preferably a tapered inner peripheral surface having an opening area that decreases from the flow path to the outside.

In another aspect of the present invention, a DNA amplification apparatus includes a reaction cell formed on a heat-conductive substrate, a sample introduction unit for introducing a sample solution into the reaction cell, and discharging the sample solution from the reaction cell. A DNA amplifying apparatus comprising a sample discharge section for heating and a heating means for heating the reaction cell, characterized by comprising a rotor for stirring the sample solution in the reaction cell .

  The method of forming the inorganic substrate, temperature measuring unit and heating means, reaction cell, sample introduction unit and discharge unit, flow path and the like that can be used in this apparatus is the same as described above.

  The rotor used in this apparatus is not particularly limited, but it is preferable to use magnetic particles. For example, although the same effect can be obtained even if the rotating blade is installed in the reaction cell, the structure becomes complicated. In this regard, in the case of magnetic particles, by using magnetism, a driving part such as a motor can be provided at a position away from the reaction cell, and the structure of the apparatus main body can be simplified. Although it is conceivable to use a commonly used magnetic rotor, it is necessary to develop a very small rotor to fit in a shallow reaction cell, and high costs are expected along with technical difficulties.

  As described above, according to the present invention, there is provided a DNA amplification apparatus capable of precisely controlling the temperature of a reaction solution and efficiently performing a DNA amplification reaction.

  In addition, according to the present invention, there is provided a DNA amplification device capable of reducing mechanical working parts, thereby reducing the size of the entire device and reducing the cost.

  Furthermore, according to the present invention, bubbles present in the sample solution can be easily removed, and the temperature distribution in the sample solution due to the presence of bubbles can be prevented, thereby making the DNA amplification reaction more accurate and efficient. Provided is a DNA amplification device that can be used.

  Embodiments of the present invention will be described below with reference to the drawings.

  1 is a plan view showing the upper surface of the first embodiment of the DNA amplification device according to the present invention, FIG. 2 is a plan view showing the lower surface of the device shown in FIG. 1, and FIG. 3 is a cross-sectional view of the device shown in FIG. Each figure is shown. As shown in the figure, in this first embodiment, two planar reaction cells 102 and 103, a sample introduction unit 104 and a sample discharge unit 105 are formed on the upper surface of the inorganic substrate 101, and the sample introduction unit 104 and The reaction cell 102 communicates with the flow channel 106, the reaction cells 102 and 103 communicate with the flow channel 107, and the reaction cell 103 and the sample discharge unit 105 communicate with each other through the flow channel 108. Temperature measuring sections 109 and 110 are provided on the bottom surfaces of the reaction cells 102 and 103, respectively, and heating means 111 and 112 are provided on the lower surface of the inorganic substrate 101 at positions corresponding to the reaction cells 102 and 103, respectively. A lid plate 113 is bonded to the upper surface of the inorganic substrate 101, and the reaction cells 102 and 103, the sample introduction unit 104, the sample discharge unit 105, and the flow paths 106, 107, and 108 formed on the upper surface of the substrate 101 A space is formed by the cover plate and the bottom and side surfaces of each cover plate.

  As a material of the inorganic substrate 101, a silicon wafer, another semiconductor substrate, a glass substrate, a metal plate such as aluminum, stainless steel, or the like can be used. When a silicon wafer is used as the inorganic substrate 101, the temperature measuring units 109 and 110 can be formed integrally, which is preferable. When another substrate is used, after the reaction cell is formed on the substrate, a separate temperature measuring means, for example, a thermistor may be fixed at a predetermined position.

  Hereinafter, the manufacturing method of this embodiment will be described using a silicon wafer as an example of the inorganic substrate 101. The reaction cells 102 and 103, the sample introduction unit 104, the sample discharge unit 105, and the channels 106, 107 and 108 provided on the upper surface of the inorganic substrate 101 are formed by etching a silicon wafer. As an etching method, any method that is usually used for etching a silicon wafer can be used. For example, isotropic etching, anisotropic etching, or dry etching can be used. However, in view of good smoothness of the bottom surface after etching, anisotropic wet etching using potassium hydroxide, tetramethylammonium (TMA) or the like is preferable. That is, by using anisotropic etching, the smoothness of the bottom surface can be improved, and turbulent flow generated when a sample solution is flowed, and physical adsorption of contents, bubbles, etc. to the bottom surface can be suppressed. The planar shape of the reaction cells 102 and 103 is not limited to a hexagon as shown in the figure, and may be any shape, for example, a square or a circle, as long as the sample solution can be easily moved. However, when the etching of the silicon wafer is performed by anisotropic etching, it is preferable that the silicon wafer has a quadrilateral or more polygonal shape. This is to prevent the solution from remaining in the reaction cell when the sample solution is moved because it is difficult to form a smooth curve by anisotropic etching.

  In addition, when moving the water-soluble liquid, if the inner wall is hydrophilic, the resistance decreases and the flow easily occurs. Therefore, the inner wall is formed by forming an oxide film on the silicon wafer by a method such as thermal oxidation. It is preferable to apply a treatment to make the surface hydrophilic.

  As described above, the temperature measuring units 109 and 110 provided on the bottom surfaces of the reaction cells 102 and 103 may be formed integrally with the reaction cell or may be separately fixed with a measuring means. As the temperature measuring means, a diode type using a pn junction, various thermistors, thermocouples, etc. can be used, but since it can be directly formed on a silicon wafer, a diode type measuring means should be used. preferable. FIG. 4 shows a cross section of an example of a diode-type temperature measuring unit. This temperature measurement unit forms a reaction cell on a silicon wafer, and then forms a pn junction type diode composed of three layers 114, 115 and 116 having different conductivity types on the bottom surface of the reaction cell by a conventional method. In order to extract the measurement signal, the insulating layers 117 and 118 and the metal wirings 119 and 120 can be provided. There must be at least one temperature measuring unit in one reaction cell, and more precise measurement is possible when a plurality of temperature measuring units are provided in one reaction cell.

  The cover plate 113 bonded to the upper surface of the inorganic substrate 101 may be any inorganic substrate, for example, a silicon wafer or a glass substrate can be used. In particular, since the glass substrate is transparent, it is preferable in that the state in the reaction cell can be visually recognized.

  Any heating means 111 and 112 may be used as long as they can perform the thermal cycle necessary for the DNA amplification reaction. For example, an electric heater, a Peltier element, or an infrared lamp can be used. In the figure, the heating means 111 and 112 are fixed at positions corresponding to the reaction cells 102 and 103 on the back surface of the inorganic substrate 101, respectively. For example, one heating means may be fixed to the entire back surface. From the viewpoint of uniformly heating the inside of the reaction cell, it is preferable that the heating means be capable of plane heating.

  The sample introduction unit 104 and the sample discharge unit 105 are connected to a means for introducing a sample solution to be amplified into the DNA amplification device or discharging the sample solution after the DNA amplification reaction. The means for introducing or discharging the sample solution and the connecting method thereof are not particularly limited. For example, as shown in FIG. 5, the tube is placed at a position corresponding to the sample introducing portion 104 and the sample discharging portion 105 of the cover plate 113. A joint (not shown) may be installed, a tube 122 may be connected to the tube joint, and one of the tubes 122 may be connected to the sample solution transport means 123. In the configuration shown in FIG. 5, the introduction of the sample solution into the DNA amplification device, the discharge of the sample solution from the DNA amplification device, and the movement of the sample solution between the two reaction cells 102 and 103 are performed by one transport means 123. To do. The conveying means 123 may be provided on either the sample introduction part side or the sample discharge part side. Further, the transport means is not particularly limited as long as the sample solution can be moved. For example, a peristaltic pump can be used. When a tube joint is installed on the lid plate 113, the sample introduction part 104 and the sample discharge part 105 are preferably circular with a diameter of 0.5 mm or more.

  The size of this DNA amplification device is arbitrary, and changes depending on the volume of the sample solution in which the DNA amplification reaction is performed in the reaction cells 102 and 103. For example, when the volume of the sample solution is 50 μl, the hexagonal reaction cell shown in the figure. In this case, one side is 7mm and the depth is about 0.4mm. In this case, when the depth is unified to 0.4 mm, the width of the flow path 106 that connects the sample introduction part 104 and the reaction cell 102 and the width of the flow path 108 that connects the reaction cell 103 and the sample discharge part 105 is about 0.1-1 mm. It is appropriate that the width of the flow path 107 communicating the reaction cells 102 and 103 is about 0.3-1 mm, and the sample solution naturally flows out due to the surface tension of the reaction cell wall by using such a dimension. Will disappear.

  Next, a procedure for a DNA amplification reaction by PCR using the apparatus of the first embodiment will be described.

  1) First, a sample including materials necessary for performing a DNA amplification reaction, that is, two or more primers, a heat-resistant DNA synthase, four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) and a specimen The solution is introduced only from the sample introduction unit 104 into the first reaction cell 102 using the conveying means 123.

  2) The sample solution introduced into the reaction cell 102 is repeatedly moved between the first reaction cell 102 and the second reaction cell 103 at a predetermined interval of about 1 second to 1 minute. Thereafter, this movement is continuously repeated until the DNA amplification reaction is completed.

  3) While measuring the temperature of the sample solution in the reaction cell with the temperature measuring units 109 and 110, the temperature is raised to 90-93 ° C. with the heating means 111 and 112, and this temperature is maintained for about 1 second to 1 minute. In this case, the maintenance temperature error should be kept within 0.1 ℃. The setting of the maintenance temperature is determined by the heat resistance of the enzyme used.

  4) Lower the temperature of the sample solution in the reaction cell to 37 ° C and maintain this temperature for 1 second to 3 minutes. At this time, keep the temperature error within 0.1 ℃.

  5) Raise the temperature of the sample solution in the reaction cell to 40-65 ° C and maintain this temperature for 1 second to 3 minutes. At this time, keep the temperature error within 0.1 ℃.

  6) Repeat the above 3) -5) cycle 2-1000 times. The number of repetitions depends on the concentration of DNA contained in the sample solution and the desired amplification factor.

  7) After repeating the predetermined number of times 3) -5), the movement of the sample solution between the two reaction cells is stopped, and the sample solution is discharged from the sample discharge unit 105 to the outside by the transfer means 123.

  This DNA amplification apparatus can heat a sample solution in a flat manner in a very shallow reaction cell. For this reason, it becomes difficult to generate a temperature gradient in the sample solution. Further, as described above, in this DNA amplification apparatus, the sample solution is continuously reciprocated between the two reaction cells during the DNA amplification reaction. As a result, the sample solution is constantly stirred, and the temperature of the sample solution and the components in the sample solution become more uniform.

  In addition, by constantly moving the sample solution, minute bubbles generated in the solution due to repeated heating and cooling of the sample solution can be largely collected. In addition, the fact that the width of the flow path connecting the two reaction cells is smaller than the width of the reaction cell also contributes to an increase in bubbles. Large bubbles can be removed much more easily than many small bubbles.

  Furthermore, by completely moving the sample solution to one of the reaction cells, the bubbles adsorbed on the emptied inner surface of the reaction cell can also be removed.

  Thus, by using this DNA amplification device, it is possible to prevent the temperature distribution in the sample solution from becoming non-uniform, and in addition, the sample solution which is one of the factors of the non-uniform temperature distribution. It is possible to easily remove minute bubbles generated by repeated heating and cooling. Therefore, the accuracy of the DNA amplification reaction can be increased.

  Further, since each of the two reaction cells includes a heating means and a temperature measuring unit, the internal temperature can be individually controlled to repeat the heating and cooling cycles of the DNA amplification reaction more efficiently. For example, while the sample solution is moved to one reaction cell and held at a predetermined temperature for a predetermined time, the internal temperature of the other reaction cell is adjusted to the temperature required for the next reaction, thereby allowing a DNA amplification reaction. Can be shortened. At that time, if the distance between the two reaction cells is short and it is difficult to control the temperature, a position between the two reaction cells that does not interfere with the flow path connecting the two reaction cells, for example, the inorganic substrate 101 It is also possible to insulate the two reaction cells by arranging grooves or pipes on the back surface or inside the cover plate 113 and flowing cooling water there.

  Next, a second embodiment of the DNA amplification apparatus according to the present invention will be described.

  6 is a plan view showing the upper surface of the second embodiment, and FIG. 7 is a cross-sectional view of the apparatus shown in FIG. As shown in the figure, the second embodiment has substantially the same configuration as the first embodiment. That is, two planar reaction cells 102 and 103 as well as a sample introduction unit 104 and a sample discharge unit 105 are formed on the top surface of the inorganic substrate 101. The sample introduction unit 104 and the reaction cell 102 are a flow channel 106, and a reaction cell. 102 and 103 communicate with each other through a channel 107, and the reaction cell 103 and the sample discharge unit 105 communicate with each other through a channel. Temperature measuring units 109 and 110 are provided on the bottom surfaces of the reaction cells 102 and 103, respectively, and heating means 111 and 112 are provided on the bottom surface of the inorganic substrate 101 at positions corresponding to the reaction cells 102 and 103, respectively (see FIG. 2). Further, a lid plate 113 is joined to the upper surface of the inorganic substrate 101, and the reaction cells 102 and 103, the sample introduction unit 104, the sample discharge unit 105, and the flow paths 106, 107, and 108 formed on the upper surface of the substrate 101 are connected. A space is formed by the cover plate and the bottom and side surfaces of each cover plate. In the second embodiment shown in the figure, a gas discharge hole 201 is further formed in a portion corresponding to the upper surface of the flow path 107 connecting the reaction cells 102 and 103 of the cover plate 113. The planar shape of the gas discharge hole 201 is not particularly limited, and may be, for example, circular or rectangular. When the planar shape is circular, the diameter is 1 to 100 μm, preferably 1 to 30 μm. Further, as shown in FIG. 7, the inner peripheral surface of the gas discharge hole 201 is tapered so that the opening area decreases from the flow path 107 side to the outside. Further, the inner peripheral surface is made of a hydrophobic substance or is given hydrophobicity by, for example, silane treatment.

  Next, a method for manufacturing the device of the second embodiment will be described.

  This apparatus can be manufactured by the same method as the apparatus of the first embodiment except that the gas discharge hole 201 is formed in advance in a portion corresponding to the flow path 107 of the lid plate 113.

  The gas discharge hole 201 can be formed by etching the inorganic substrate constituting the cover plate 113. The method for etching the inorganic substrate varies depending on the type of the substrate, and can be performed by a conventional method. For example, when the inorganic substrate is a silicon wafer, an oxide film or a nitride film is first formed on the silicon wafer by a conventional method, and then gas is produced using a commonly used photolithography technique and an oxide film or nitride film etching technique. A mask pattern for the discharge hole 201 is formed. At this time, the oxide film can be easily etched by, for example, buffer etching using a mixed solution of hydrofluoric acid and ammonium fluoride, and the nitride film can be etched by, for example, plasma etching using CF4 or RIE (Reactive Ion Etching). Can be done.

  Thereafter, the silicon wafer is anisotropically etched using this mask pattern to form gas discharge holes 201. Since anisotropic etching proceeds along the crystal plane <111> of single crystal silicon, by using a <100> plane silicon wafer, gas discharge having a tapered inner peripheral surface as shown in the figure. Holes 201 can be formed. The anisotropic etching is performed, for example, by immersing the masked silicon wafer in a 32% KOH solution heated to 80 ° C. In this case, anisotropic etching proceeds inward at an angle of 54.7 ° with respect to the <100> plane. Therefore, the size of the opening of the gas discharge hole 201 can be controlled by appropriately setting the size of the mask in consideration of the etching depth (substrate thickness). For example, in order to form a 10 μm square opening, a mask with an etching depth of d and a square with a side of (2d / tan (54.7 °) +10) μm may be used.

  After the gas discharge hole 201 is formed, the mask pattern is removed and an oxide film is formed on the entire surface of the silicon wafer. However, when the mask pattern is made of an oxide film, the mask pattern can be used as it is as an oxide film.

  Subsequently, the inner peripheral surface of the gas discharge hole 201 is hydrophobized by silane treatment as described below. First, a region to be imparted with hydrophobicity, that is, a region other than the inner peripheral surface of the gas discharge hole 201 is covered with a resist by a photolithography technique. Next, this silicon wafer is sealed in a sealed container, and a silane coupling agent, for example, toluene in which 2% of dichlorooctylmethylsilane is dissolved is bubbled with dry nitrogen gas and introduced into the sealed container. As a result, a hydrophobic silane-treated film is formed in the region not covered with the resist by a gas phase reaction. After the film is formed, the resist is removed from the silicon wafer.

  Note that pure single crystal silicon exhibits hydrophobicity. Therefore, when the mask pattern of the gas discharge hole is made of an oxide film, it can be used without performing a treatment for imparting hydrophobicity to the inner peripheral surface after the formation of the gas discharge hole is completed.

  When the inorganic substrate is a glass substrate, etching is usually performed using an excimer laser. An excimer laser is a powerful laser having a wavelength in the ultraviolet region, and the bond between molecules is broken by irradiation, and etching can be performed with a resolution of 10 μm or less. For etching of glass, an ArF gas laser (wavelength: 193 nm) is usually used in consideration of efficiency.

  In order to form a gas discharge hole in the glass substrate, laser light may be guided to the optical system and irradiated to the glass substrate through a mask having a desired pattern. The positioning at this time is performed on a precise positioning stage. The size of the opening of the gas discharge hole can be determined in consideration of the laser output, the irradiation time, the thickness of the glass substrate, and the like. For example, when a glass substrate is irradiated with an excimer pulse laser having an energy density of 3.1 J / cm @ 2, an etching rate of 100 .mu.m / 600 shots can be obtained. Therefore, if a glass substrate having a thickness of 0.5 mm is irradiated with a laser pulse at 100 Hz, a communication hole can be formed in 30 seconds. The inner peripheral surface of the gas discharge hole formed by laser irradiation in this way becomes a tapered shape in which the inner diameter gradually decreases from the laser entrance to the exit.

  After the gas discharge hole is formed in the glass substrate, the inner peripheral surface thereof is hydrophobized by silane treatment as described below. First, a region to be imparted with hydrophobicity, that is, a region other than the inner peripheral surface of the gas discharge hole is covered with a resist by a photolithography technique. Next, the glass substrate is sealed in a sealed container, and a silane coupling agent, for example, toluene in which 2% of dichlorooctylmethylsilane is dissolved is bubbled with dry nitrogen gas and introduced into the sealed container. As a result, a hydrophobic silane-treated film is formed in the region not covered with the resist by a gas phase reaction. After the film is formed, the resist is removed from the glass substrate.

  In the drawing, only one gas discharge hole 201 is shown in the flow path 107, but the number of gas discharge holes 201 is not limited to one, and a plurality of gas discharge holes 201 may be formed. Further, the manufacturing method is not limited to the above-described method, and any method can be used as long as equivalent processing can be performed by a technique well known to those skilled in the art. For example, although the reaction in the gas phase has been described as the silane treatment method, it is also possible to treat in the liquid phase using chloroform, toluene, or the like. Various silane treatment agents can be used as long as hydrophobic surface treatment is possible. For example, a reagent having one or more alkyl groups or phenyl groups as a hydrophobic group, or a reagent containing a chloro group, a methoxy group, an ethoxy group, or the like as a reactive group for glass can be used. Moreover, the titanate coupling agent which changed Si of the silane coupling agent to Ti can also be used. Furthermore, surface treatment can be performed by vapor deposition, sputtering, etc. For example, a hydrophobic material such as parylene may be vapor-deposited by masking other than the opening of the communication hole, or a hydrophobic material such as Teflon may be deposited by sputtering. It is also possible to adhere to a predetermined area.

  As described above, the second embodiment has the same configuration as the first embodiment except that the gas discharge hole 201 is formed in the cover plate 113. Therefore, the apparatus of the second embodiment can perform a DNA amplification reaction in the same procedure as that of the first embodiment, and similarly exhibits the effects exhibited by the first embodiment. In addition to this, the second embodiment has an effect due to the presence of the gas discharge hole 201. As described above, the sample solution is continuously moved between the two reaction cells 102 and 103, so that minute bubbles are largely collected. The large air bubbles move between the two reaction cells through the flow path 107 together with the sample solution. Since the inner peripheral surface of the gas discharge hole 201 provided on the upper surface of the flow path 107 is hydrophobic, the hydrophilic liquid is bounced and does not flow out to the outside. It easily adheres to the peripheral surface, passes through the gas discharge hole 201 as it is, and is discharged to the outside.

  As described above, according to the apparatus of the second embodiment, the bubble removing mechanism and the flow path can be integrally formed relatively easily, and it is not necessary to connect to an external bubble removing means. For this reason, the entire circuit can be made more compact, and the number of external connection portions can be reduced, so that the structure is simplified. Furthermore, since the wall surface of the flow path is mainly hydrophilic, it is possible to flow a water-soluble liquid with a small resistance. When the cover plate 113 is a silicon wafer, the gas discharge hole 201 can be formed by anisotropic etching, and more precise processing is possible. Further, the efficiency of removing bubbles through the gas discharge holes can be further improved by reducing the external pressure. In addition, even in such a case, since there are few connection parts with the outside, there is no fear of liquid leakage and the stability of the apparatus is improved.

  The form of the gas discharge hole is not limited to the form shown in FIG. 7, and for example, as shown in FIG. 8, a form in which a porous layer 203 is provided at the opening on the flow path 107 side of the gas discharge hole 202 is provided. But you can. The method for forming the porous layer 203 is not particularly limited, but when the cover plate 113 is a silicon wafer, it can be formed relatively easily by the following procedure.

  First, a mask pattern of the porous layer 203 is formed on a silicon wafer to be the cover plate 113 by using a commonly used photolithography technique and an oxide film or nitride film etching technique. At this time, the oxide film can be easily etched by, for example, buffer etching using a mixed solution of hydrofluoric acid and ammonium fluoride, and the nitride film can be etched by, for example, plasma etching using CF4 or RIE (Reactive Ion Etching). Can be done. After the mask pattern is formed, anodization is performed at a current density of 15 mA / cm @ 2 and a temperature of 30 DEG C., for example. Thereby, a porous layer is formed from the surface of the silicon wafer toward the inside. The thickness of the porous layer is not particularly limited, but 100 μm or less is usually sufficient. The thickness can be controlled by anodizing treatment conditions and treatment time.

  After the anodization is completed, a mask pattern of gas discharge holes 202 is formed on the back surface of the surface on which the porous layer 203 is formed, and the silicon wafer is etched to expose the porous layer 203. For this etching, anisotropic etching can be used in the same manner as the gas exhaust hole 201 shown in FIG. 7, but here it is not necessary to penetrate the silicon wafer, and the depth at which the porous layer 203 is exposed is not necessary. Etching is sufficient. For this reason, the size of the mask pattern of the gas discharge hole 202 need not be as strict as that of the gas discharge hole 201.

  After the gas exhaust hole 202 is formed, the mask pattern is removed, and an oxide film is formed on the entire surface of the silicon wafer including the surface of the porous layer 203. However, when the mask pattern is made of an oxide film, the mask pattern can be used as it is as an oxide film.

  Subsequently, the surface of the porous layer 203 on the channel 107 side is hydrophobized by silane treatment as described below. First, a region other than the region to be imparted with hydrophobicity, that is, the region other than the surface of the porous layer 203 on the flow path 107 side is covered with a resist by a photolithography technique. Next, this silicon wafer is sealed in a sealed container, and a silane coupling agent, for example, toluene in which 2% of dichlorooctylmethylsilane is dissolved is bubbled with dry nitrogen gas and introduced into the sealed container. As a result, a hydrophobic silane-treated film is formed in the region not covered with the resist by a gas phase reaction. After the film is formed, the resist is removed from the silicon wafer.

  This gas discharge hole 202 also has the same effect as the gas discharge hole 201 described above. That is, the hydrophilic liquid is bounced and does not flow outside, but the hydrophobic bubbles easily pass through the pores of the porous layer 203 and are discharged to the outside. In addition, as described above, the gas discharge hole 202 does not need to strictly define the etching depth and the mask pattern size as compared with the case of the gas discharge hole 201. Therefore, it can process more easily. Further, since the porous layer 203 is provided in the hole of the gas discharge hole 202, the gas discharge hole 202 can always be regarded as composed of a large number of micro holes. That is, regardless of the size of the gas discharge hole 202, it is possible to maintain the bubble removing function without causing liquid leakage at all times. Therefore, the opening can be made larger than the gas discharge hole 201, and an increase in bubble removal efficiency can be expected.

  In FIG. 8, the porous layer 203 is provided in the opening on the flow path 107 side of the gas discharge hole 202, but it can also be provided in the opening on the outside. In this case, since the inner peripheral surface of the gas discharge hole 202 formed by etching is on the flow path 107 side, the above-mentioned silane treatment is also applied to the inner peripheral surface to impart hydrophobicity so that gas discharge can be performed. The effect is improved.

  Next, a third embodiment of the DNA amplification device according to the present invention will be described.

  FIG. 9 is a plan view showing the upper surface of the third embodiment. As shown in FIG. 9, the third embodiment is obtained by adding two gas flow holes 301 and 302 to the configuration of the second embodiment shown in FIG. That is, the configuration of the third embodiment is as follows. Two planar reaction cells 102 and 103, a sample introduction unit 104 and a sample discharge unit 105 are formed on the top surface of the inorganic substrate 101, and the sample introduction unit 104 and the reaction cell 102 are a flow path 106, and the reaction cell 102 and 103 is connected to the flow path 107, and the reaction cell 103 and the sample discharge portion 105 are connected to each other via the flow path 108. Temperature measuring units 109 and 110 are provided on the bottom surfaces of the reaction cells 102 and 103, respectively, and heating means 111 and 112 are provided on the bottom surface of the inorganic substrate 101 at positions corresponding to the reaction cells 102 and 103, respectively (see FIG. 2). Further, a lid plate 113 is joined to the upper surface of the inorganic substrate 101, and the reaction cells 102 and 103, the sample introduction unit 104, the sample discharge unit 105, and the flow paths 106, 107, and 108 formed on the upper surface of the substrate 101 are connected. A space is formed by the cover plate and the bottom and side surfaces of each cover plate. Further, the gas discharge hole 201 corresponds to the upper surface of the flow path 106 that connects the sample introduction unit 104 and the reaction cell 102 in the portion corresponding to the upper surface of the flow path 107 that connects the reaction cells 102 and 103 of the cover plate 113. A gas discharge hole 301 is formed in the part, and a gas discharge hole 302 is formed in a part corresponding to the upper surface of the flow path 108 connecting the reaction cell 103 and the sample discharge part 105.

  Further, this apparatus can also be provided with valves between the sample introduction unit 104 and the reaction cell 102, and between the reaction cell 103 and the sample discharge unit 105 (not shown). In that case, the gas discharge holes 301 and 302 must each be located between the valve and the reaction cell.

  The gas discharge holes 301 and 302 are the same as the gas discharge holes in the second embodiment, and may be any of the gas discharge holes shown in FIGS. 7 and 8, and the manufacturing method thereof is also as described above. It is.

  As described above, during the PCR reaction, bubbles generated in the solution by heating and / or the solution itself expand to increase the pressure in the reaction cell, and the sample solution unexpectedly reacts with the other reaction. It may move to the cell or the joints may be damaged at high pressure. In the apparatus of the third embodiment, when the pressure in the reaction cell rises, air flows out from the gas discharge holes 301 or 302 formed between each reaction cell and the sample introduction part or the sample discharge part. Thus, the pressure in the reaction cell is lowered to prevent movement of the sample solution and damage to the apparatus due to high pressure. That is, the gas discharge holes 301 and 302 make it possible to always keep the pressure in the reaction cell constant.

  Next, a fourth embodiment of the DNA amplification device according to the present invention will be described.

  10 is a plan view showing the upper surface of the fourth embodiment, FIG. 11 is a plan view showing the lower surface of the apparatus shown in FIG. 10, and FIG. 12 is a cross-sectional view taken along the line CC of the apparatus shown in FIG. . As shown in the figure, in this apparatus, a planar reaction cell 402, a sample introduction part 403, and a sample discharge part 404 are formed on the upper surface of the inorganic substrate 401, and the sample introduction part 403 and the reaction cell 402 have a flow path 405. In addition, the reaction cell 402 and the sample discharge part 404 are communicated with each other through a flow path 406. A temperature measuring unit 407 is provided on the bottom surface of the reaction cell 402, and a heating means 408 is provided at a position corresponding to the reaction cell 402 on the bottom surface of the inorganic substrate 401. In addition, magnetic particles 409 are placed on the bottom surface of the reaction cell 402, and a magnetic stirrer 410 for controlling the movement of the magnetic particles 409 by magnetism is provided below the heating means 408. Further, a lid plate 411 is bonded to the upper surface of the inorganic substrate 401, and this lid plate is connected to the reaction cell 402, the sample introduction unit 403, the sample discharge unit 404, and the channels 405 and 406 formed on the upper surface of the substrate 401. A space is formed between 411 and each bottom and side surfaces. In the figure, the magnetic particle 409 is represented as an aggregate of a large number of particles. Hereinafter, the description regarding the flow diameter of the magnetic particles will be described for each of the magnetic particles, and the description regarding the movement of the magnetic particles will refer to the movement as an aggregate of the magnetic particles.

  In the fourth embodiment, the inorganic substrate 401, the planar reaction cell 402, the sample introduction unit 403, the sample discharge unit 404, the flow paths 405 and 406, the temperature measurement unit 407, the heating means 408 and the lid plate 411 are the same as those in the first embodiment. It can be made by the same materials and methods as the examples.

  Commercially available magnetic particles can be used as they are on the bottom surface of the reaction cell 402, but the diameter is preferably 0.1 μm to 100 μm, more preferably 1 μm to 50 μm. Further, by treating the surface of the magnetic particles with a hydrophilic polymer to impart hydrophilicity, it is possible to prevent components contained in the sample solution from adhering to the magnetic particles during the DNA amplification reaction.

  The magnetic stirrer 410 moves the magnetic particles 409 in the reaction cell 402 by magnetism and controls the movement thereof. The magnetic stirrer 410 is usually composed of a magnet, a motor, and a control circuit, and has a mechanism for rotating the magnet by the motor and controlling the rotation by the control circuit. In the fourth embodiment, the rotation speed of the magnet may be controlled in the range of 0 to 5000 rpm, preferably 0 to 2000 rpm.

  The procedure of DNA amplification reaction by PCR using the apparatus of the fourth embodiment is as follows.

  1) First, a sample including materials necessary for performing a DNA amplification reaction, that is, two or more primers, a heat-resistant DNA synthase, four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) and a specimen The solution is introduced into the reaction cell 402 from the sample introduction unit 403 using the same transport means as in the first embodiment.

  2) The magnetic stirrer 410 is operated, and the magnetic particles 409 in the reaction cell 402 are rotated. The number of rotations at this time is determined by the volume of the sample solution and the particle size of the magnetic particles.

  3) While measuring the temperature of the sample solution in the reaction cell with the temperature measuring unit 407, the temperature is raised to 90-93 ° C. with the heating means 408, and this temperature is maintained for about 1 second to 1 minute. In this case, the maintenance temperature error should be kept within 0.1 ℃. The setting of the maintenance temperature is determined by the heat resistance of the enzyme used.

  4) Lower the temperature of the sample solution in the reaction cell to 37 ° C and maintain this temperature for 1 second to 3 minutes. At this time, keep the temperature error within 0.1 ℃.

  5) Raise the temperature of the sample solution in the reaction cell to 40-65 ° C and maintain this temperature for 1 second to 3 minutes. At this time, keep the temperature error within 0.1 ℃.

  6) Repeat the above 3) -5) cycle 2-1000 times. The number of repetitions depends on the concentration of DNA contained in the sample solution and the desired amplification factor.

  7) After repeating the predetermined number of times 3) -5), the rotation of the magnetic particles is stopped, and the sample solution is discharged from the sample discharge portion 404 to the outside by the conveying means. At this time, the magnetic particles 409 are fixed in the reaction cell 402 by the magnetism of the magnetic stirrer 410 and are not discharged together with the sample solution.

  In the fourth embodiment, the sample solution is constantly stirred during the amplification reaction by rotating the magnetic particles in the reaction cell. For this reason, minute bubbles generated in the sample solution due to repeated heating and cooling during the amplification reaction are largely collected and easily removed. Therefore, it is possible to prevent the temperature from becoming non-uniform due to the presence of bubbles in the solution. In addition, since the sample solution can be sufficiently stirred, the uneven distribution of components in the solution can be eliminated.

  Next, a fifth embodiment of the DNA amplification apparatus according to the present invention will be described.

  FIG. 13 is a plan view showing the upper surface of the fifth embodiment, and FIG. 14 is a sectional view taken along the line DD of the apparatus shown in FIG. The fifth embodiment has the same configuration as that of the fourth embodiment except that a gas discharge hole 501 is formed in the cover plate 411 corresponding to the center of rotation of the magnetic particles 409. That is, the configuration of the fifth embodiment is as follows. A planar reaction cell 402, a sample introduction unit 403, and a sample discharge unit 404 are formed on the top surface of the inorganic substrate 401. The sample introduction unit 403 and the reaction cell 402 are a flow path 405, and the reaction cell 402 and the sample discharge unit 404. Are communicated with each other through a flow path 406. A temperature measuring unit 407 is provided on the bottom surface of the reaction cell 402, and a heating means 408 is provided at a position corresponding to the reaction cell 402 on the bottom surface of the inorganic substrate 401. In addition, magnetic particles 409 are placed on the bottom surface of the reaction cell 402, and a magnetic stirrer 410 for controlling the movement of the magnetic particles 409 by magnetism is provided below the heating means 408. A lid plate 411 is bonded to the upper surface of the inorganic substrate 401. The reaction cell 402, the sample introduction unit 403, the sample discharge unit 404, and the channels 405 and 406 formed on the upper surface of the substrate 401 are connected to the lid plate 411 and A space is formed between each bottom surface and side surfaces. A gas discharge hole 501 is formed in the cover plate 411 at a position corresponding to the rotation center of the magnetic particle 409.

  As shown in FIG. 14, the inner peripheral surface of the gas discharge hole 501 is tapered so that the opening area decreases from the reaction cell 402 side toward the outside. Further, the inner peripheral surface is made of a hydrophobic substance, or is given hydrophobicity by, for example, silane treatment. The form of the gas discharge hole 501 is not limited to the form shown in FIG. 14, but may be a form in which a porous layer 502 is provided in the opening on the reaction cell 402 side as shown in FIG. The porous layer 502 may be provided not in the opening on the reaction cell 402 side but in the opening on the outside. The gas discharge hole 501 can be produced by the same method as in the second embodiment.

  As described above, the fifth embodiment has the same configuration as the fourth embodiment except that the gas discharge hole 501 is formed in the lid plate 411. Therefore, the apparatus of the fifth embodiment can perform a DNA amplification reaction in the same procedure as that of the fourth embodiment, and similarly exhibits the effects exhibited by the fourth embodiment. In addition to this, the fifth embodiment has an effect due to the existence of the gas discharge holes 501. As described above, while the DNA amplification reaction is performed, the sample solution is stirred by rotating the magnetic particles 409, and minute bubbles generated in the solution are largely collected. The largely collected bubbles gather at the center of rotation of the magnetic particles 409 along the liquid flow generated as the magnetic particles 409 rotate. A gas discharge hole 501 is formed in the cover plate 411 at a position corresponding to the rotation center of the magnetic particle 409, and the hydrophilic liquid repels as described above, but the hydrophobic bubbles easily pass therethrough. For this reason, only the bubbles collected at the rotation center are discharged to the outside from the gas discharge hole 501.

  As described above, in the fifth embodiment, bubbles generated in the sample solution can be efficiently discharged to the outside by repeated heating and cooling during the amplification reaction, and the temperature distribution in the sample solution is uneven. Can be prevented.

  Next, a sixth embodiment of the DNA amplification device according to the present invention will be described.

  FIG. 16 is a plan view showing an upper surface of the sixth embodiment. As shown in the figure, the sixth embodiment is similar to the fifth embodiment except that a gas discharge hole is further formed in the flow path between the sample introduction part and the reaction cell and between the reaction cell and the sample discharge part. The configuration is the same as that of the embodiment. That is, the configuration of the sixth embodiment is as follows. A planar reaction cell 402, a sample introduction unit 403, and a sample discharge unit 404 are formed on the top surface of the inorganic substrate 401. The sample introduction unit 403 and the reaction cell 402 are a flow path 405, and the reaction cell 402 and the sample discharge unit 404. Are communicated with each other through a flow path 406. A temperature measuring unit 407 is provided on the bottom surface of the reaction cell 402, and heating means 408 is provided at a position corresponding to the reaction cell 402 on the bottom surface of the inorganic substrate 401 (see FIGS. 14 and 15). Also, magnetic particles 409 are placed on the bottom surface of the reaction cell 402, and a magnetic stirrer 410 for controlling the movement of the magnetic particles 409 by magnetism is provided below the heating means 408 (see FIGS. 14 and 15). . A lid plate 411 is bonded to the upper surface of the inorganic substrate 401. The reaction cell 402, the sample introduction unit 403, the sample discharge unit 404, and the channels 405 and 406 formed on the upper surface of the substrate 401 are connected to the lid plate 411 and A space is formed between each bottom surface and side surfaces. The lid plate 411 includes a position corresponding to the rotation center of the magnetic particle 409, a portion corresponding to the upper surface of the flow path 405 that communicates the reaction cell 402 and the sample introduction unit 403, and the reaction cell 402 and the sample discharge unit 404. Gas discharge holes 501, 601 and 602 are formed in portions corresponding to the upper surface of the communicating channel 406, respectively.

  Furthermore, in the apparatus shown in the figure, a valve can be provided in each of the flow paths 405 and 406 (not shown), but in that case, the gas discharge holes 601 and 602 are respectively connected to the reaction cell 402 and the respective valves. Must be located between.

  The form of the gas discharge holes 601 and 602 is exactly the same as that of the gas discharge hole 501, and may be the form shown in FIG. 14 or the form shown in FIG. The manufacturing method thereof is the same as that of the gas discharge hole 501, and can be manufactured in the same manner as in the second embodiment.

  As described above, the sixth embodiment has the same configuration as that of the fifth embodiment except that two gas discharge holes are further formed. Therefore, the same effect as that of the fifth embodiment is exhibited. Further, in the sixth embodiment, an effect due to the presence of two more gas discharge holes is added.

  During the PCR reaction, bubbles generated in the solution by heating and / or the solution itself expands to increase the pressure in the reaction cell, and the sample solution unexpectedly moves out of the reaction cell. As described above, the joint and the like may be damaged at high pressure. In the sixth embodiment, when the pressure in the reaction cell increases, air flows out from the gas discharge hole 601 or 602 formed between the reaction cell and the sample introduction part or the sample discharge part, and the reaction cell. The internal pressure is lowered to prevent movement of the sample solution and damage to the device due to high pressure. In other words, the gas discharge holes 601 and 602 can keep the pressure in the reaction cell constant.

  Next, a seventh embodiment of the DNA amplification device according to the present invention will be described.

  17 is a plan view showing the upper surface of the seventh embodiment, FIG. 18 is a plan view showing the lower surface of the apparatus shown in FIG. 17, FIG. 19 is a sectional view taken along line EE of the apparatus shown in FIG. It is the FF sectional view taken on the line of the apparatus shown by FIG. As shown in the figure, in the seventh embodiment, two planar reaction cells 702 and 703, a sample introduction unit 704 and a sample discharge unit 705 are formed on the top surface of the inorganic substrate 701. The reaction cell 702 communicates with the flow channel 706, the reaction cells 702 and 703 communicate with the flow channel 707, and the reaction cell 703 and the sample discharge unit 705 communicate with each other through the flow channel 708. Temperature measuring units 709 and 710 are provided on the bottom surfaces of the reaction cells 702 and 703, respectively, and heating means 711 and 712 are provided on the bottom surface of the inorganic substrate 701 at positions corresponding to the reaction cells 702 and 703, respectively. Further, a lid plate 713 is bonded to the upper surface of the inorganic substrate 701, and the reaction cells 702 and 703, the sample introduction unit 704, the sample discharge unit 705, and the flow paths 706, 707, and 708 formed on the upper surface of the substrate 701 are connected to each other. A space is formed by the cover plate 713 and the bottom and side surfaces of each. Further, at least one gas discharge hole 714 is formed in the cover plate 713 corresponding to the upper surface of the reaction cell 702. In addition, an ultrasonic wave is provided below the heating means 711 provided on the back surface of the reaction cell 702. A vibrator 715 is provided.

  In this seventh embodiment, inorganic substrate 701, reaction cells 702 and 703, sample introduction unit 704, sample discharge unit 705, flow paths 706, 707 and 708, temperature measurement units 709 and 710, heating means 711 and 712, and lid The plate 713 is made of the same material as that of the first embodiment and can be manufactured by the same method.

  As shown in FIG. 20, the inner peripheral surface of the gas discharge hole 714 is tapered so that the opening area decreases from the reaction cell 702 side toward the outside. Further, the inner peripheral surface is made of a hydrophobic substance, or is given hydrophobicity by, for example, silane treatment. The form of the gas discharge hole 714 is not limited to the form shown in FIG. 20, but may be a form in which a porous layer 716 is provided in the opening on the reaction cell 702 side as shown in FIG. Further, the porous layer 716 may be provided not in the opening on the reaction cell 702 side but in the opening on the outside. The gas discharge hole 714 can be manufactured by the same method as in the second embodiment.

  The ultrasonic vibrator 715 is an element that generates ultrasonic waves, and is usually controlled by a high-frequency power source (not shown) using a piezoelectric body. The output of the high frequency power source is determined depending on the size of the reaction cell 702. In the drawing, the ultrasonic vibrator 715 is provided below the heating means 711, but is not limited to such a configuration, and even between the inorganic substrate 701 and the heating means 711, or the lid It may be above the plate 713. In addition, it is not always necessary to be positioned below or above the reaction cell 702, as long as it is provided in contact with the inorganic substrate 701. Further, if it can be brought into contact with the reaction cell 702 during use, the inorganic substrate 701 is permanently installed. There is no need to be.

  The procedure of DNA amplification reaction by PCR using this seventh embodiment is as follows.

  1) First, a sample including materials necessary for performing a DNA amplification reaction, that is, two or more primers, a heat-resistant DNA synthase, four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) and a specimen The solution is introduced into the first reaction cell 702 from the sample introduction unit 704 using the same transport means as in the first embodiment.

  2) Activate the ultrasonic vibrator 715 and oscillate the ultrasonic wave for 1 second to 10 minutes.

  3) The sample solution is moved to the second reaction cell 703 again using the transport means.

  4) While the temperature of the sample solution in the second reaction cell 703 is measured by the temperature measuring unit 710, the temperature is raised to 90-93 ° C. by the heating means 712, and this temperature is maintained for about 1 second to 1 minute. In this case, the maintenance temperature error should be kept within 0.1 ℃. The setting of the maintenance temperature is determined by the heat resistance of the enzyme used.

  5) Lower the temperature of the sample solution in the second reaction cell 703 to 37 ° C., and maintain this temperature for about 1 second to 3 minutes. At this time, keep the temperature error within 0.1 ℃.

  6) The temperature of the sample solution in the second reaction cell 703 is raised to 40-65 ° C., and this temperature is maintained for about 1 second to 3 minutes. At this time, keep the temperature error within 0.1 ℃.

  7) Repeat the above 4) -6) cycle 2-1000 times. The number of repetitions depends on the concentration of DNA contained in the sample solution and the desired amplification factor.

  8) After repeating the predetermined number of times 4) -6), the sample solution is discharged from the sample discharge unit 705 to the outside by the conveying means.

  In the seventh embodiment, after applying ultrasonic vibration to the sample solution introduced into the first reaction cell, the DNA amplification reaction is performed in the second reaction cell. In this way, the sample solution is pretreated with ultrasonic waves prior to the DNA amplification reaction, so that dissolved air in the solution is generated as bubbles and discharged from the gas discharge holes formed on the upper surface of the first reaction cell. Can do. For this reason, generation | occurrence | production of the micro bubble by repetition of the heating in the DNA amplification reaction and cooling can be suppressed, and it can prevent that the temperature in a solution becomes non-uniform | heterogenous. In addition, when the sample solution is pretreated with ultrasonic waves, the sample solution can be heated by the heating means 711, whereby the dissolved air in the solution can be further reduced.

  In the description of the procedure of the DNA amplification reaction, the first reaction cell is used for pretreatment of the sample solution by ultrasonic waves, and the DNA amplification reaction is performed only in the second reaction cell. The reaction can also be performed in the same procedure as in the embodiment. That is, the sample solution may be moved between the first reaction cell and the second reaction cell, and the reaction may be sequentially performed. In this case, even if dissolved air that could not be discharged in the first ultrasonic pretreatment is generated as bubbles in the second reaction cell, the gas provided on the upper surface of the first reaction cell in the next step It can be quickly discharged from the discharge hole.

  In the seventh embodiment, two reaction cells are used. However, the same effect can be expected even with one reaction cell as in the fourth to sixth embodiments. In this case, for example, in the fourth embodiment, an ultrasonic vibrator is installed below the heating means 408, the sample solution is introduced into the reaction cell 402, and then the ultrasonic vibrator is first operated for 1 second to 10 minutes. The dissolved air in the solution is removed, and then the reaction is performed by rotating the magnetic particles.

  Further, by reducing the pressure around the apparatus of the seventh embodiment, the bubbles can be discharged from the gas discharge holes 714 more efficiently. However, in this case, it is necessary to adjust the degree of pressure reduction so that the sample solution does not flow out from the gas discharge hole 714.

  Next, an eighth embodiment of the DNA amplification device according to the present invention will be described.

  22 is a plan view showing the upper surface of the eighth embodiment, FIG. 23 is a plan view showing the lower surface of the apparatus shown in FIG. 22, and FIG. 24 is a sectional view taken along the line GG of the apparatus shown in FIG. The eighth embodiment has the same configuration as that of the seventh embodiment, except that a pressure reducing tube is connected to the cover plate and a valve is provided in two of the three flow paths. . That is, the configuration of the eighth embodiment is as follows. Two planar reaction cells 702 and 703, a sample introduction unit 704 and a sample discharge unit 705 are formed on the top surface of the inorganic substrate 701. The sample introduction unit 704 and the reaction cell 702 are a flow path 706, and the reaction cell 702 and Reference numeral 703 denotes a flow path 707, and the reaction cell 703 and the sample discharge unit 705 communicate with each other via a flow path 708. Temperature measuring units 709 and 710 are provided on the bottom surfaces of the reaction cells 702 and 703, respectively, and heating means 711 and 712 are provided on the bottom surface of the inorganic substrate 701 at positions corresponding to the reaction cells 702 and 703, respectively. Further, a lid plate 713 is bonded to the upper surface of the inorganic substrate 701, and the reaction cells 702 and 703, the sample introduction unit 704, the sample discharge unit 705, and the flow paths 706, 707, and 708 formed on the upper surface of the substrate 701 are connected to each other. A space is formed by the cover plate 713 and the bottom and side surfaces of each. Further, at least one gas discharge hole 714 is formed in the cover plate 713 corresponding to the upper surface of the reaction cell 702. In addition, an ultrasonic wave is provided below the heating means 711 provided on the back surface of the reaction cell 702. A vibrator 715 is provided. In addition, a pressure reducing pipe 802 is connected to the gas discharge hole 714 via a joint portion 801, and valves 803 and 804 are provided in the flow paths 706 and 707, respectively.

  In this eighth embodiment, inorganic substrate 701, reaction cells 702 and 703, sample introduction unit 704, sample discharge unit 705, flow paths 706, 707 and 708, temperature measurement units 709 and 710, heating means 711 and 712, and lid Similar to the seventh embodiment, the plate 713 can be made of the same material as that of the first embodiment and by the same method.

  Similarly to the seventh embodiment, the gas discharge hole 714 can be formed by the same method as in the first embodiment. As shown in FIG. 24, the opening area decreases from the reaction cell 702 side to the outside. Even in such a tapered form, a porous layer 716 may be provided in the opening on the reaction cell 702 side as shown in FIG. Further, the porous layer 716 may be provided not in the opening on the reaction cell 702 side but in the opening on the outside.

  The pressure reducing pipe 802 is connected to a gas discharge hole 714 formed in the lid plate 713 through a joint portion 801. The material and connection method of the joint portion 801 and the pressure reducing pipe 802 used here are not particularly limited, and any material can be used as long as the gas exhaust hole 714 and the pressure reducing pipe 802 can be connected in an airtight manner. The decompression pipe 802 is further connected to suction means such as a rotary pump (not shown).

  For example, the valves 803 and 804 provided in the flow paths 706 and 707 are each formed with a through hole in the cover plate 713 in advance by etching or drilling, and a minute valve is fixed to the through hole and then bonded to the inorganic substrate 701. Can be produced. As the minute valve used here, a commercially available product can be used. For example, a Fluistor of Redwood microsystems, Inc. can be suitably used. This Fluistor is a gas valve, but it can also be used for liquids. In addition, as a method for fixing the valve, an adhesion method by anodic bonding is usually used, but it is also possible to adhere using an adhesive or a pressure-sensitive adhesive.

  The procedure of DNA amplification reaction by PCR using this eighth embodiment is as follows.

  1) First, the valves 803 and 804 are opened, and materials necessary for performing a DNA amplification reaction, that is, two or more kinds of primers, a heat-resistant DNA synthase, and four kinds of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP) And dTTP) and the sample solution containing the specimen are introduced from the sample introduction unit 704 into the first reaction cell 702 using the same transport means as in the first embodiment.

  2) Close valves 803 and 804.

  3) The suction means connected to the decompression pipe 802 is operated to decompress the inside of the first reaction cell 702.

  4) Activate the ultrasonic vibrator 715 and oscillate the ultrasonic wave for 1 second to 10 minutes.

  5) Open valves 803 and 804.

  6) The sample solution is moved to the second reaction cell 703 again using the transport means.

  7) While the temperature of the sample solution in the second reaction cell 703 is measured by the temperature measuring unit 710, the temperature is raised to 90-93 ° C. by the heating means 712, and this temperature is maintained for about 1 second to 1 minute. In this case, the maintenance temperature error should be kept within 0.1 ℃. The setting of the maintenance temperature is determined by the heat resistance of the enzyme used.

  8) Lower the temperature of the sample solution in the second reaction cell 703 to 37 ° C., and maintain this temperature for about 1 second to 3 minutes. At this time, keep the temperature error within 0.1 ℃.

  9) The temperature of the sample solution in the second reaction cell 703 is raised to 40-65 ° C., and this temperature is maintained for about 1 second to 3 minutes. At this time, keep the temperature error within 0.1 ℃.

  10) Repeat the above 7) -9) cycle 2-1000 times. The number of repetitions depends on the concentration of DNA contained in the sample solution and the desired amplification factor.

  11) After repeating the cycle of the predetermined number of times 7) -9), the sample solution is discharged from the sample discharge portion 705 to the outside by the conveying means.

  In the eighth embodiment, similarly to the seventh embodiment, after introducing the sample solution into the first reaction cell, first, pretreatment is performed by ultrasonic waves to generate dissolved air in the solution as bubbles. At this time, since the inside of the reaction cell is maintained at a reduced pressure, dissolved air that cannot be removed at normal pressure is also generated as bubbles. Further, the generated bubbles are forcibly removed by suction means such as a rotary pump connected to the gas discharge hole via a pressure reducing tube. In this way, the dissolved air in the sample solution can be more efficiently removed, and the first reaction cell is completely sealed by closing the two valves, so that the sample solution is out of gas. There is little risk of escaping from the hole. Therefore, generation of minute bubbles due to repeated heating and cooling during the DNA amplification reaction in the second reaction cell can be suppressed, and the temperature in the solution can be prevented from becoming non-uniform.

  In the description of the procedure for the DNA amplification reaction, the first reaction cell is used for ultrasonic pretreatment of the sample solution, and the DNA amplification reaction is performed only in the second reaction cell. Similarly to the embodiment, the reaction can be performed in the same procedure as in the first to third embodiments. That is, the sample solution may be moved between the first reaction cell and the second reaction cell, and the reaction may be sequentially performed. In this case, even if dissolved air that could not be excluded by the first pressure reduction treatment and ultrasonic treatment was generated as bubbles in the second reaction cell, it was provided on the upper surface of the first reaction cell in the next step. The gas can be forcibly discharged from the gas discharge hole.

  In addition, in the eighth embodiment, two reaction cells are used. However, the same effect can be expected even with one reaction cell as in the fourth to sixth embodiments. The same as the seventh embodiment.

  The embodiments of the DNA amplification device according to the present invention have been described with reference to the drawings. However, the present invention is not limited to the above-described embodiments, and various modifications and applications are possible within the scope of the present invention. It is. Here, the gist of the present invention is summarized as follows.

(1) Corresponding to FIGS. 1 to 5:
First and second planar reaction cells formed on an inorganic substrate, a sample introduction part for introducing a sample solution into the first reaction cell, and discharging the sample solution from the second reaction cell A sample discharge section for connecting the sample reaction section to the sample reaction section, the first reaction cell, the first reaction cell and the second reaction cell, and the second reaction cell and the sample discharge section. A flow path, at least one temperature measurement unit provided on each of the bottom surfaces of the first and second reaction cells, a heating unit provided on the back surfaces of the first and second reaction cells of the inorganic substrate, and A DNA amplification apparatus comprising a sample solution transport means.

  In this apparatus, after the sample solution is introduced into the first reaction cell via the flow path from the sample introduction unit, the sample solution is brought to a predetermined temperature using the temperature measurement unit and heating means provided on the bottom surface of the reaction cell. The DNA amplification reaction is carried out by heating. At this time, the sample solution is alternately moved between the first reaction cell and the second reaction cell by using the transport means. After the reaction is completed, the movement of the sample solution is stopped, and the solution is discharged to the outside from the second reaction cell via the sample discharge unit.

  In this way, by moving the sample solution between the first reaction cell and the second reaction cell, the solution is agitated, and minute bubbles generated in the solution can be largely collected. Large bubbles can be removed more easily than many small bubbles. For this reason, bubbles in the solution can be efficiently removed, and temperature nonuniformity and damage to the apparatus due to the presence of bubbles can be prevented.

(2) Corresponding to FIGS. 6 to 8:
The DNA amplification apparatus according to (1), wherein a gas discharge hole is formed in a flow path that communicates the first reaction cell and the second reaction cell.

  In this apparatus, as in the apparatus (1), the sample solution is alternately moved between the first reaction cell and the second reaction cell during the amplification reaction. At this time, bubbles largely collected by stirring accompanying the movement of the solution are discharged to the outside through gas discharge holes formed in a flow path that connects the two reaction cells.

  As described in (1) above, by moving the sample solution between the first reaction cell and the second reaction cell, the minute bubbles generated by repeated heating and cooling in the amplification reaction are largely collected. That's right. The large air bubbles move between the two reaction cells together with the sample solution. However, when passing through the flow path connecting the two reaction cells, only the bubbles that show hydrophobicity are formed in the flow path. It passes through the discharge hole and is discharged to the outside. Therefore, it is possible to efficiently remove bubbles by simply moving the sample solution between the two reaction cells, and more efficiently preventing the temperature of the solution from becoming non-uniform due to the presence of bubbles. it can.

(3) Corresponding to FIGS. 10 to 12:
A planar reaction cell formed on an inorganic substrate, a sample introduction part for introducing a sample solution into the reaction cell, a sample discharge part for discharging the sample solution from the reaction cell, the sample introduction part and the reaction Two channels for communicating the cell and the reaction cell with the sample discharge unit, at least one temperature measurement unit provided on the bottom surface of the reaction cell, and heating provided on the back surface of the reaction cell of the inorganic substrate Means, a rotor placed on the bottom of the reaction cell, and a control means for controlling the rotation of the rotor.

  In this device, a rotor such as magnetic particles is placed on the bottom of the reaction cell, and after the sample solution is introduced into the reaction cell, the rotor is rotated by a control device such as a magnetic stirrer during the amplification reaction. Let With the rotation of the rotor, the sample solution in the reaction cell also circulates, and minute bubbles existing in the solution are carried along the liquid flow to the rotation center of the rotor and are largely collected.

  Bubbles gathered in this way can be easily removed, and the presence of bubbles can prevent the temperature distribution of the solution from becoming uneven. For this reason, the DNA amplification reaction can be performed more accurately and efficiently. In addition, since the sample solution is constantly stirred during the amplification reaction, it is possible to prevent the component distribution in the solution from becoming non-uniform, and the accuracy of the DNA amplification reaction can be improved. Further, when magnetic particles are used as the rotor, a simple structure and a low cost apparatus can be obtained.

  The present invention also includes the following aspects.

(4) Corresponding to FIGS. 7 to 9:
(1) or (2) in which gas discharge holes are respectively formed in a flow path communicating the sample introduction part and the first reaction cell and in a flow path communicating the second reaction cell and the sample discharge part. The DNA amplification apparatus described.

  The sample solution in the reaction cell expands or contracts due to heating or cooling during the amplification reaction. Similarly, the air around the sample solution in the reaction cell expands or contracts. Furthermore, when bubbles are generated in the solution, the bubbles also expand or contract. Thus, when performing an amplification reaction, the pressure in the reaction cell changes greatly. Therefore, if the reaction cell is sealed, particularly when the internal pressure rises, there is a risk that the cell junction or the like may be damaged, or the sample solution may move unexpectedly. In order to prevent these, it is necessary to open the reaction cell and adjust the pressure change. In this apparatus, the gas discharge holes formed in the respective channels serve to adjust the pressure in the reaction cell. That is, when the pressure in the reaction cell increases, air flows out from the gas discharge hole, and conversely, when the pressure in the reaction cell decreases, air flows in.

  As mentioned above, it is important to open the reaction cell, but if it is opened too much to the outside, moisture and highly volatile components in the sample solution will evaporate during heating and the component concentration will change. Resulting in. The gas discharge holes formed in each flow path of this apparatus are minute holes, and only the pressure can be adjusted without causing almost any change in the component concentration due to evaporation. Therefore, it is possible to prevent cell breakage due to pressure increase in the reaction cell and unexpected movement of the sample solution.

(5) Corresponding to FIGS. 13 to 15:
The DNA amplification apparatus according to (3), wherein a gas discharge hole is formed on the upper surface of the reaction cell.

  In this apparatus, as in the apparatus described in (3) above, the sample solution is agitated by rotating the rotor during the DNA amplification reaction. At this time, as described above, minute bubbles present in the solution are largely collected. In this apparatus, gas discharge holes are formed on the upper surface of the reaction cell, and it is possible to discharge the bubbles that are largely collected from the gas discharge holes. Since the bubbles gathered by the rotation of the rotor gather at the rotation center of the rotor, the gas discharge hole is preferably formed in a portion corresponding to the rotation center of the rotor on the upper surface of the reaction cell.

  In this way, this apparatus can efficiently eliminate bubbles present in the sample solution, suppress the non-uniform temperature distribution in the sample solution, and perform the DNA amplification reaction more accurately and efficiently. Can be done.

(6) Corresponding to FIG. 16:
The DNA amplification device according to (3) or (5), wherein a gas discharge hole is formed in a flow path that communicates the sample introduction part and the reaction cell and a flow path that communicates the reaction cell and the sample discharge part.

  The sample solution in the reaction cell expands or contracts due to heating or cooling during the amplification reaction. Similarly, the air around the sample solution in the reaction cell expands or contracts. Furthermore, when bubbles are generated in the solution, the bubbles also expand or contract. Thus, when performing an amplification reaction, the pressure in the reaction cell changes greatly. Therefore, if the reaction cell is sealed, particularly when the internal pressure rises, there is a risk that the cell junction or the like may be damaged, or the sample solution may move unexpectedly. In order to prevent these, it is necessary to open the reaction cell and adjust the pressure change. In this apparatus, the gas discharge holes formed in the respective channels serve to adjust the pressure in the reaction cell. That is, when the pressure in the reaction cell increases, air flows out from the gas discharge hole, and conversely, when the pressure in the reaction cell decreases, air flows in.

  As mentioned above, it is important to open the reaction cell, but if it is opened too much to the outside, moisture and highly volatile components in the sample solution will evaporate during heating and the component concentration will change. Resulting in. The gas discharge holes formed in each flow path of this apparatus are minute holes, and only the pressure can be adjusted without causing almost any change in the component concentration due to evaporation. Therefore, it is possible to prevent cell breakage due to pressure increase in the reaction cell and unexpected movement of the sample solution.

(7) Corresponding to FIGS. 17 to 21:
The DNA amplification device according to any one of (1) to (6), further comprising an ultrasonic transducer that applies ultrasonic vibration to the sample solution in the reaction cell.

  In this apparatus, after introducing the sample solution into the reaction cell and before performing the DNA amplification reaction, the ultrasonic vibrator is operated to apply ultrasonic waves to the sample solution, and the dissolved air in the sample solution is made into bubbles to remove from the solution. Kick out. The bubbles expelled from the solution can be discharged to the outside by using the function of each device.

  In this way, by expelling dissolved air in the sample solution from the solution before the DNA amplification reaction, it becomes possible to suppress the generation of bubbles due to repeated heating and cooling during the amplification reaction, and the temperature distribution of the solution Can be prevented from becoming uneven.

(8) Corresponding to FIGS. 22 to 25:
The DNA amplifying apparatus according to (7), wherein valves are provided in all the flow paths connected to the reaction cell that performs pretreatment of the sample solution with ultrasonic waves, and pressure reducing means for reducing the pressure in the reaction cell is provided.

  In the apparatus of (7) above, after introducing the sample solution into the reaction cell and before performing the DNA amplification reaction, the ultrasonic vibrator is operated to apply ultrasonic waves to the sample solution to drive out the dissolved air in the solution. What to do is as described above. In this apparatus, during the pretreatment of the sample solution by ultrasonic waves, all the valves provided in all the flow paths connected to the reaction cell are closed, and the inside of the reaction cell is depressurized using a depressurizing means. As a means for depressurization, a suction means such as a pump may be directly connected to a gas discharge hole formed in the upper surface of the reaction cell, or the periphery of the apparatus may be simply depressurized.

  In this way, in addition to sonication of the sample solution, by reducing the pressure in the reaction cell, dissolved air that could not be expelled by sonication alone can be expelled from the solution, and heating during the DNA amplification reaction Further, it is possible to further suppress the generation of bubbles due to repeated cooling. Therefore, it is possible to more effectively prevent the temperature distribution of the solution from becoming nonuniform during the DNA amplification reaction.

The top view which shows the upper surface of 1st Embodiment of the DNA amplification apparatus by this invention. The top view which shows the lower surface of embodiment shown by FIG. FIG. 2 is a sectional view taken along line AA of the embodiment shown in FIG. 1. The figure which shows the cross section of the diode-type temperature measurement part using a pn junction. The figure which shows typically an example of the connection with embodiment shown by FIG. 1, and the introduction or discharge | emission means of a sample solution. The top view which shows the upper surface of 2nd Embodiment of the DNA amplification apparatus by this invention. FIG. 7 is a cross-sectional view of the embodiment shown in FIG. The figure which shows the cross section of the modification of the gas exhaust hole formed in embodiment shown by FIG. The top view which shows the upper surface of 3rd Embodiment of the DNA amplification apparatus by this invention. The top view which shows the upper surface of 4th Embodiment of the DNA amplification apparatus by this invention. The top view which shows the lower surface of embodiment shown by FIG. CC sectional view taken on the line of the embodiment shown in FIG. The top view which shows the upper surface of 5th Embodiment of the DNA amplification apparatus by this invention. FIG. 14 is a sectional view taken along the line DD of the embodiment shown in FIG. 13. The figure which shows the cross section of the modification of the gas exhaust hole formed in embodiment shown by FIG. The top view which shows the upper surface of 6th Embodiment of the DNA amplification apparatus by this invention. The top view which shows the upper surface of 7th Embodiment of the DNA amplification apparatus by this invention. The top view which shows the lower surface of embodiment shown by FIG. EE sectional view taken on the line of embodiment shown by FIG. FF sectional view taken on the line of embodiment shown by FIG. The figure which shows the cross section of the modification of the gas exhaust hole formed in embodiment shown by FIG. The top view which shows the upper surface of 8th Embodiment of the DNA amplification apparatus by this invention. The top view which shows the lower surface of embodiment shown by FIG. GG sectional view of the embodiment shown in FIG. The figure which shows the cross section of the modification of the gas exhaust hole formed in embodiment shown by FIG.

Explanation of symbols

  101, 401, 701 ... inorganic substrate, 102, 103, 402, 702, 703 ... reaction cell, 104, 403, 704 ... sample introduction part, 105, 404, 705 ... sample discharge part, 106, 107, 108, 405, 406, 706, 707, 708 ... flow path, 109, 110, 407, 709, 710 ... temperature measuring unit, 111, 112, 408, 711, 712 ... heating means, 113, 411, 713 ... lid plate, 114, 115 , 116 ... well, 117, 118 ... insulating layer, 119, 120 ... metal wiring, 123 ... transport means, 201, 301, 302, 501, 601, 602, 714 ... gas exhaust holes, 202, 502, 716 ... porous Layer, 409 ... rotor, 410 ... control means, 715 ... ultrasonic transducer, 801 ... joint part, 802 ... pressure reducing pipe, 803, 804 ... valve

Claims (2)

A reaction cell formed on a thermally conductive substrate,
A sample introduction part for introducing a sample solution into the reaction cell;
A DNA amplifying apparatus comprising: a sample discharging unit for discharging a sample solution from the reaction cell; and a heating means for heating the reaction cell,
Magnetic particles with hydrophilicity provided on the surface, disposed in the reaction cell to stir the sample solution in the reaction cell; and
A DNA amplifying apparatus comprising driving means for driving the magnetic particles from outside the reaction cell . The DNA amplification device according to claim 1 ,
A gas discharge hole for discharging bubbles generated in the sample solution in the reaction cell to the outside ;
The DNA amplification apparatus , wherein the gas discharge hole is formed at a position corresponding to a rotation center of the magnetic particle on a cover plate which is disposed on the substrate and forms an upper surface of the reaction cell .

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