WO2000067893A1

WO2000067893A1 - Chemical reactor

Info

Publication number: WO2000067893A1
Application number: PCT/JP2000/002999
Authority: WO
Inventors: Michifumi Tanga; Kojiro Takahashi
Original assignee: Toyo Kohan Co., Ltd.
Priority date: 1999-05-10
Filing date: 2000-05-10
Publication date: 2000-11-16
Also published as: AU4430600A

Abstract

The invention provides a chemical reactor capable of increasing the rate of reaction within a reaction pool. The reactor comprises a substrate (10) with a reaction pool (11) formed in its surface, a high-thermal-conductivity diamond layer (12) forming at least the bottom of the reaction pool (11), a Peltier device (13) attached on the back of the diamond layer (12), and temperature control means (15) for controlling the Peltier device (13) to periodically changing the temperature of the buffer in the reaction pool (11).

Description

Description Chemical reaction vessel ζ Technical field

The present invention relates to a chemical reaction vessel for a general chemical reaction. In particular, it relates to containers for microchemical reactions used for biochemical reactions, enzymatic reactions, etc., and particularly to containers for chemical reactions used for DNA amplification reactions.

10 Background technology

In recent years, the polymerase chain reaction (hereinafter referred to as PCR) has been widely used as a means for amplifying a small amount of DNA, and an example of a DNA amplification apparatus used for that purpose is disclosed in Japanese Patent Application Laid-Open No. 9-94086. ing.

The configuration of this DNA amplification device will be described with reference to FIGS. 11 and 12.

15 ", a reaction pool section 102 and flow paths 103 and 104 are formed on the upper surface of the substrate 101, and a transparent lid 105 is joined to the upper surface thereof. The flow paths 103 and 104 have valves 106 and 104, respectively. A heating element 108 is provided in contact with the back surface of the surface of the substrate 101 on which the reaction pool section 102 is formed, and a temperature sensor 109 is provided at the bottom of the reaction pool section 102. , 110 are buried.

The procedure of DNA amplification using this DNA amplification apparatus will be described. First, the valve 106 and the valve 107 are opened, and a target nucleic acid, two or more types of primers, After introducing a buffer containing an enzyme and four types of deoxyribonucleoside triphosphates (dCTP, dGTP, dTTP, dATP) into the reaction pool section 102, the valves 106 and 107 are closed.

Next, the heating element 108 is operated to heat the buffer in the reaction pool section 102. Cycles are added to amplify the target nucleic acid. Specifically, first, the heating element 108 is heated to heat the buffer 1 to a temperature (90 to 94 "C) at which the nucleic acid in the buffer is dissociated, and this temperature is maintained for a predetermined time. Next, the operation of the heating element 108 is stopped, and the buffer is cooled to a temperature (at about 54) at which the dissociated nucleic acid and the primer are bound. Then, the exothermic cooling body 108 is operated again, and the buffer is heated to a temperature (50 to 75) at which the activity of the thermostable DNA synthase is maximized and maintained for a predetermined time. Elongates and the nucleic acid is amplified.

After the extension of the primer is completed, the heating element 108 is operated again to heat the buffer 1 to a temperature at which the nucleic acid is dissociated, and the generated double-stranded nucleic acid is dissociated. Hereinafter, a nucleic acid amplification reaction is performed by repeating this cycle a required number of times.

Here, in the DNA amplification apparatus described above, since the shape of the reaction pool section 102 is on the planar surface, the heat generated from the heating element 108 is transmitted to the buffer in the reaction pool section 102 to a certain degree of efficiency. I can increase the efficiency of heating and cooling.

However, the above-described DNA amplification apparatus still has the following problems to be solved. In other words, since the substrate 101 is mainly formed of a silicon wafer substrate or a glass substrate having low thermal conductivity, a sufficiently high reaction rate cannot be secured in practice, and the efficiency of the amplification reaction of the buffer is not increased. There were certain restrictions on raising

20 The present invention can solve the above-described problems as the DNA amplification device, and can improve the chemical reaction efficiency in the reaction pool by forming the reaction pool forming portion of the substrate from diamond. It is intended to provide a container for use.

Further, the present invention aims to solve not only the above-described problems as a DNA amplification device but also problems as general XT biochemistry and enzyme reaction devices. By forming, in the reaction pool part An object of the present invention is to provide a container for chemical reaction that can increase the efficiency of biochemical reaction and enzyme reaction. Disclosure of the invention

In order to achieve the above object, a chemical reaction vessel according to the first invention comprises: a substrate having a reaction pool formed on a surface thereof; and a diamond heat transfer layer forming at least a bottom surface of the reaction pool. It is characterized by comprising a heat-generating cooling body attached to the back surface of the heat transfer layer, and a temperature adjusting means for periodically changing the temperature of the buffer in the reaction pool by the heat-generating cooling body.

! 0 In order to achieve the above object, the chemical reaction vessel according to the second invention is composed of a plurality of chemical reaction vessels arranged in series, and each of the plurality of chemical reaction vessels reacts on the surface. A substrate formed with a pool portion, a heat transfer layer made of diamond forming at least a bottom surface of the reaction pool portion, and an exothermic cooling body attached to the back surface of the heat transfer layer. It is characterized in that the temperatures of the buffers in the reaction pool by the respective heat-generating cooling bodies are different from each other.

In order to achieve the above object, the chemical reaction vessel according to the third invention is composed of a plurality of chemical reaction vessels connected in series, and each of the plurality of chemical reaction vessels is internally provided with a reaction pool section. A diamond-made cylinder having: a heat-generating cooling element attached to the outer periphery of the cylinder; and a power supply connected to the heat-generating cooling element. It is characterized in that the temperatures of the buffers in the pool are different from each other. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a cross-sectional view of the chemical reaction vessel according to the first embodiment of the present invention. FIG. 2 is a perspective view of the same. FIG. 3 is a graph showing a change in one temperature of a buffer using the chemical reaction vessel according to the first embodiment of the present invention. FIG. 4 shows the second embodiment of the present invention. It is a cross-sectional view of the container for chemical reactions which concerns on embodiment. FIG. 5 is a cross-sectional view of a chemical reaction vessel according to a modification of the second embodiment of the present invention. FIG. 6 is a cross-sectional view of a chemical reaction vessel according to a modification of the second embodiment of the present invention. FIG. 7 is a cross-sectional view of a chemical reaction vessel according to a modification of the second embodiment of the present invention. FIG. 8 is a perspective view of a chemical reaction container according to the third embodiment of the present invention. FIG. 9 is a cross-sectional view of a container for chemical reaction according to the fourth embodiment of the present invention. FIG. 10 is an explanatory diagram of a manufacturing procedure of the chemical reaction container according to the fourth embodiment of the present invention. FIG. 11 is a plan view of a conventional chemical reaction vessel. FIG. 12 is a cross-sectional view taken along the line I-I of FIG. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be specifically described with reference to an embodiment shown in the accompanying drawings. (First Embodiment)

First, the overall configuration of a chemical reaction vessel A according to a first embodiment of the present invention will be described with reference to FIGS.

As shown in the figure, a substrate 10 composed of a silicon wafer substrate forming the main body of the chemical reaction vessel A is a rectangular plate having a width and a length of several cm each and a thickness of sub / zm to several hundreds // m. Consists of A reaction pool 11 is formed on one surface of the substrate 10.

A heat transfer layer 12 made of diamond is formed on the bottom surface of the reaction pool section 11, and a substrate 10, which forms the back side of the heat transfer layer 12, has a pel, which is an example of a heat-generating cooling body. The Che element 13 is buried. A power supply 14 is connected to the Peltier element 13, and a temperature adjustment device 15, which is an example of temperature adjustment means, is connected to the power supply 14. Note that, as the heat-generating cooling body, one other than the Peltier element 13, for example, a heat transfer heater can be used.

Also, as shown in FIGS. 1 and 2, in the present embodiment, A transparent lid 16 is joined, and a temperature detection sensor 17 for detecting the temperature of the buffer in the reaction pool 11 is embedded on one side thereof.

In the above configuration, the diamond heat transfer layer 12 is formed only on the bottom surface of the reaction pool 11, but may it be formed over the entire surface of the reaction pool 11? it can.

Further, the temperature adjusting device 15 is provided with a timer, and by changing the power supply voltage every time a predetermined time elapses, the temperature of the buffer 1 in the reaction pool section 11 is controlled in several steps as described later. Can be adjusted.

Next, a DNA amplification method using the chemical reaction vessel A having the above configuration will be described.

First, using a delivery means (not shown), the target nucleic acid, two or more types of primers, a thermostable DNA synthase, and four types of deoxyribonucleoside triphosphates (dCTP, dGTP, dTTP, dATP) Is introduced into the reaction pool part 11.

I Next, a predetermined voltage is applied to the Peltier element 13 by the power supply 14 to operate the Peltier element 13, and a heat cycle is applied to the buffer in the reaction pool section 11 to remove the nucleic acid of interest. Amplify. Specifically, as shown in FIG. 3, first, the Peltier element 13 is heated to heat the buffer to a temperature (94) at which the nucleic acid in the buffer is dissociated, and this temperature is maintained for a predetermined time. Next, the operation of the Peltier element 13 is stopped, and the buffer is cooled to a temperature (at about 54) at which the dissociated nucleic acid and the primer bind. After the dissociated nucleic acid binds to the primer, the Peltier element 13 is activated again, and the buffer is heated to a temperature (74 T :) at which the activity of the heat-resistant DNA synthase is maximized and maintained for a predetermined time. . This extends the primer and amplifies the nucleic acid.

After the primer extension is completed, the buffer is heated to the temperature (at 94) at which the nucleic acid dissociates by operating the Peltier element 13 again to dissociate the generated double-stranded nucleic acid. 0

Let Hereinafter, the nucleic acid amplification reaction is performed by repeating this cycle a required number of times.

At this time, since the heat transfer layer 12 formed on the bottom surface of the reaction pool 11 is made of diamond having extremely high thermal conductivity, the heat generated by the Peltier element 13 is instantaneously passed through the heat transfer layer 5 12 to the reaction pool. The heat is transferred to the buffer in (11) and the buffer is rapidly heated or cooled, so that the efficiency of DNA amplification by the chemical reaction container (2) can be increased.

(Second embodiment)

FIG. 4 shows the configuration of the chemical reaction vessel A1 according to the present embodiment.

^As shown in the figure, the chemical reaction vessel A1 is composed of a plurality of chemical reaction vessels 20, 21, 22 arranged in series in the horizontal direction.

Each of the containers 20, 21, and 22 has substantially the same configuration as the chemical reaction container A according to the first embodiment. That is, each of the containers 20, 21, and 22 includes a substrate 10 having a reaction pool portion 11 formed on the surface thereof, a diamond heat transfer layer 12 forming at least a bottom surface of the reaction pool portion 11 ", and a heat transfer layer. It has a Peltier device 13 which is an example of a heat-generating cooling body attached to the back surface of the layer 12. A power source 14 is connected to the Bertier device 13 and a temperature adjusting device 15 is connected to the power source 14. .

However, in the present embodiment, the temperature controller 15 of each of the containers 20, 21, and 22 adjusts the buffer in each of the reaction pool sections 11 to different heating temperatures (for example, 94T :, 54 :, 74t). Heat with :).

Next, a DNA amplification method using the chemical reaction container A1 having the above configuration will be described.

First, the target nucleic acid, two or more primers, thermostable DNA synthase, and four types of deoxyribonucleoside triphosphates (dCTP, dGTP, dTTP, dATP) are contained using a delivery means (not shown). Buffer to be placed in the first container 20 It is introduced into the reaction pool section 11.

Next, a predetermined voltage is applied to the Peltier element 13 by the power source 14 to operate the Peltier element 13, and the temperature at which the nucleic acid in the buffer 1 in the reaction pool 11 is dissociated (at 94) The temperature of the buffer is maintained for a predetermined time. Next, the buffer 5 is removed from the reaction pool section 11 and introduced into the reaction pool section 11 of the second container 21. At the same time, the temperature at which the dissociated nucleic acid binds to the primer (about 54: Cool the buffer until). After the dissociated nucleic acid binds to the primer, the buffer is removed from the reaction pool section 11 and introduced into the reaction pool section 11 of the third container 22. The buffer is heated to a temperature (74 :) at which the activity of the DNA synthase is highest, and maintained for a predetermined time. As a result, the primer is extended, and the nucleic acid is amplified.

After the extension of the primer is completed, the buffer is again taken out of the reaction pool section 11 and introduced into the reaction pool section 11 of the first container 20, and at the same time, the Peltier element 13 is activated to remove the nucleic acid. Heat the buffer to the dissociation temperature (at 94) to dissociate the generated double-stranded nucleic acids. Hereinafter, a nucleic acid amplification reaction is performed by repeating this cycle a required number of times.

Also in the present embodiment, since the heat transfer layer 12 formed on the bottom surface of the reaction pool section 11 is made of extremely high thermal conductivity diamond, the heat generated by the Peltier element 13 is It is transmitted to the buffer in the reaction pool section 11 through 2 and quickly heats or cools the buffer, thereby increasing the efficiency of DNA amplification by the chemical reaction vessel A.

5 to 7 show the configurations of the chemical reaction vessels A2 to A4 according to the modified examples of the above-described second embodiment.

The chemical reaction vessel A3 according to the modified example shown in FIG. 5 is configured by integrating the first to third vessels 20 to 22 in the chemical reaction vessel A1 according to the second embodiment into one. In addition to the substrate 23, the first to third containers 20 to 22 are partitioned by partition walls 24, 25. It is characterized by forming by forming. In this modification,

The reaction pool portions 11 of the first to third vessels 20 to 22 are mutually connected by communication grooves 26 and 27 formed in partition walls 24 and 25, respectively. 26 and 27 are provided with on-off valves 28 and 29, respectively.

The chemical reaction vessel A4 according to the modification shown in FIG. 6 is different from the first to third vessels 20 to 22 in the chemical reaction vessel A1 according to the second embodiment in that the first to third vessels 20 to 22 are arranged vertically instead of horizontally. The reaction pools 11 of the first to third vessels 20 to 22 are connected to each other by vertical communication holes 30 and 31, and the vertical communication holes 30 and 31 Are provided with on-off valves 32 and 33, respectively.

1 The chemical reaction vessel A 4 according to the modification shown in FIG. 7 is a reaction pool part 1 1 of the first to third vessels 20 to 22 in the chemical reaction vessel A 1 according to the embodiment of FIG. Is divided into a plurality of divided reaction pool sections 10b by partition walls 10a, respectively, and as shown in FIG. 7, the divided reaction pool section 10b used once is not used again. It is characterized in that the buffer is circulated a plurality of times to carry out a chemical reaction.

Also in the above-described modification, the heat transfer layer 12 formed on the bottom surface of the reaction pool portion 11 is made of diamond having extremely high thermal conductivity, so that the heat generated by the Peltier element 13 is To the buffer in the reaction pool section 11 and quickly heat or cool the buffer to increase the reaction efficiency of the chemical reaction vessel A.

20

(Third embodiment)

FIG. 8 shows the configuration of the chemical reaction container A5 according to the present embodiment.

As shown in the figure, the chemical reaction vessel A5 is composed of first to third vessels 40 to 42 connected in series. Each of the vessels 40, 41, and 42 has a diamond cylindrical body 43 having a reaction pool therein, and a heat-generating cooling body attached to the outer periphery of the cylindrical body 43. Line 4 4 and Heater Line 4 4 The power supply 45 is connected. The containers 40, 41, and 42 are connected to each other by communication pipes 46, 47.

In the present embodiment, since the cylinder 43 itself forming the reaction pool portion itself is made of diamond having a very high thermal conductivity, the heat generated by the heater wire 44 passes through the reaction pool through the cylinder 43. The reaction is transferred to the buffer inside the unit, and the buffer is quickly heated or cooled, so that the reaction efficiency of the chemical reaction vessel A can be increased.

(Fourth embodiment)

FIG. 9 shows the configuration of the chemical reaction container A6 according to the present embodiment.

As shown, the chemical reaction vessel A 6 according to the present embodiment has substantially the same configuration as the chemical reaction vessel A according to the first embodiment except for the following points. Therefore, the same components are denoted by the same reference numerals.

That is, in the present embodiment, the heat transfer layer 12 is formed not only on the bottom surface of the reaction pool 11 but also on the entire surface of the substrate 10 including the entire surface of the reaction pool 11. The Peltier element 13 is attached to the outer peripheral surface of the substrate 10.

Also in the present embodiment, since the heat transfer layer 12 formed on the entire surface of the reaction pool section 11 is made of extremely high thermal conductivity diamond, the heat generated by the Peltier element 13 is The reaction is transferred to the buffer in the reaction pool 11 through 2 and the buffer can be quickly heated or cooled to increase the reaction rate. Therefore, the efficiency of DNA amplification by the chemical reaction container A can be increased.

In the above-described embodiment, the reaction pool section 11 can be formed and processed by melting using a diamond laser or the like. However, as described below with reference to FIG. The chemical reaction pool portion 11 having a clean cross section can be formed by the method of transferring the chemical reaction pool onto the zero. Note that this method is for the case of forming the reaction pool section 11 according to the fourth embodiment.

That is, as shown in FIG. 10A, the silicon oxide film 51 is formed on the surface of the silicon 50. Grow. As shown in FIG. 10B, patterning is performed using a photoresist 50a. After that, isotropic etching using hydrofluoric acid (HF) is performed to remove the silicon oxide film 51. As shown in FIG. 10 (c), silicon 50 is anisotropically etched using a tetramethylammonium hydroxide ((CH ₃ ) ₄ NOH) solution.

As shown in FIG. 10 (d), the trapezoidal silicon substrate 52 obtained is shaped into a triangle, and diamond 53 is grown by hot filament CVD. After the growth of the diamond 53, a conductive epoxy 53a is applied to the growth surface and baked and fixed on a platinum 54 plate. As shown in FIG. 10 (e), a silicon group in France nitrate (HF + HN_〇 ₃₎

The ID plate 52 is removed to form a chemical reaction pool 11.

In addition, instead of growing the silicon oxide film 51 on the surface of the silicon 50 described in FIG. 10A on the silicon substrate, There is also a method of patterning using such a method. Further, instead of etching using hydrofluoric acid (HF) in the explanation of FIG. 10B, silicon hexafluoride is used as an etching gas by RIE (Reactive Ion Etching). There is also a method of etching the surface. Then, as shown in FIG. 10 (d), the process leads to a step of growing diamond 53 by thermal filament CVD.

^> Industrial applicability

As described above, in the present invention, since the heat transfer layer formed on the bottom surface of the reaction pool portion or the substrate itself on which the reaction pool portion is formed is made of diamond having extremely high thermal conductivity, it corresponds to the reaction pool portion. By attaching a heat-generating cooling body such as a Peltier element at the location where the chemical reaction occurs, the chemical reaction rate can be increased.

Claims

The scope of the claims

1. A substrate having a reaction pool portion formed on the surface thereof, a heat transfer layer made of diamond forming at least a bottom surface of the reaction pool portion, and heat generation attached to the back surface of the heat transfer layer.

5 A chemical reaction container comprising: a cooling body; and a temperature adjusting means for periodically changing a temperature of a buffer in the reaction pool by the heat-generating cooling body.

2. Consisting of a plurality of chemical reaction vessels connected in series, each of the plurality of chemical reaction vessels forming a substrate having a reaction pool on the surface thereof and at least a bottom surface of the reaction pool. A heat transfer layer made of diamond and a backside of the heat transfer layer

(A heat-generating cooling body attached to the plurality of chemical reaction vessels, wherein the temperature of the buffer in the reaction pool by the heat-generating cooling bodies of the plurality of chemical reaction vessels is different from each other. Container.

3. Consisting of a plurality of chemical reaction vessels connected in series, each of the plurality of chemical reaction vessels being attached to a diamond cylinder having a reaction pool therein, and an outer periphery of the cylinder. A heat-generating cooling body; and a power supply connected to the heat-generating cooling body, wherein the temperatures of the buffers in the reaction pool part by the respective heat-generating coolers of the plurality of chemical reaction vessels are different from each other. Characteristic vessel for chemical reaction.