JP2003107094A - Instrument and method for chemical analysis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a micro-reactor (chemical analyzer) that can perform complicated chemical reactions, etc., and can be reduced in running cost and can be used for many various purposes. SOLUTION: This chemical analyzer (nucleic acid amplifier) is constituted at least of a container-like member 12 in which a plurality of sample wells 9 used as sample containers are arranged, a cover member 11 which covers the sample injecting surface of the member 12, base-side wells 10 attachably/ detachably holding the bottom sections of the wells 9, and a base 14 having heat generating sections 35 which can individually heat the base-side wells 10. After the amplification reaction of a nucleic acid (DNA) ends, only the container-like member 12 can be disposed and the base 14, etc., can be used repeatedly.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a miniaturization / integration technique for chemical / biochemical related devices, and more particularly to a structure of a microreactor (chemical analysis device) and an analysis method.

[0002]

2. Description of the Related Art In recent years, μ-TAS (Micro Total Analys
There is proposed a chemical / biochemical analysis integrated system called is System) in which functional parts such as liquid feeding, mixing, reaction, and analysis are integrated on a chip such as glass or silicon of several cm square. FIG. 18 shows one of conventional examples of μ-TAS. As shown in FIG. 18, in the μ-TAS, the sample channel 51 is formed in the glass chip 80, and the sample (reagent) is mixed, reacted, and detected in the sample channel 51,
Conduct preliminary experiments such as drug discovery and medical diagnosis. When the sample is injected from the sample injection section 62, the sample passes through the sample flow path 51 and is mixed in the mixing section 61a. Then, the chemical reaction of the sample occurs in the reaction section 61b, and the separated sample is separated in the separation section 61c. The sample after the reaction is detected by the detection unit 61d, and the unnecessary sample is collected by the waste liquid unit 60. Since the μ-TAS can significantly reduce the amount of samples and samples compared to the conventional integrated analysis system, it is expected to reduce the throughput time and waste liquid.

A PCR method known as a method for efficiently amplifying a very small amount of nucleic acid (DNA) (US Pat. No. 4,684)
3,202), for example, DNA at around 95 ° C.
Is repeated, the step of annealing at about 55 ° C., and the step of performing replication at about 70 ° C. are repeated. FIG. 19 shows one of conventional examples of a nucleic acid amplifier which is an apparatus for automatically performing the PCR method. FIG. 19
Then, the reaction tube 81 containing the sample is inserted into the well 10b provided in the metal block (such as aluminum) 82, and the heater 1
5 and the cooler 83 change the temperature of the metal block 82 to control the temperature of the reaction tube 81 to cause the reaction in the reaction tube 81. A heat pump such as a Peltier device may be used to change the temperature. Furthermore, a microreactor (chemical analysis device) has been proposed, in which device functions such as a nucleic acid amplifier can be created in a micro space such as a microchip, and a plurality of more complicated chemical reactions, drug discovery, etc. can be performed at one time. . For example, "" Development of a microreactor for PCR "in T. IEE Japan Vol.119-E,
No. 10, '99 pp. 448-453 ”describes the use of a microreactor for the PCR method.

[0004]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
It is desirable that the AS is disposable in order to prevent contamination and misjudgment when it is used in the field of drug discovery or medical diagnosis. Also, μ-
It will be necessary in the future to build a heat generating part such as a micro heater, a valve and a pump for liquid transfer into the TAS. However, it has been difficult to dispose of the μ-TAS having a complicated structure including the heat generating portion, the valve, etc., from the viewpoint of cost increase. In addition, if a heat generating part, valve, etc. are built in the μ-TAS, it will be used for other purposes.
There was a problem that you could not use.

Further, in order to analyze a large number of pathogenic bacteria in gene diagnosis and the like, it is essential to develop a nucleic acid amplifier capable of applying different temperature / reaction control to a large number of samples at once. However, to do this requires current devices to be integrated or chipped, as well as disposable. However, when integrated, it is particularly difficult to control individual temperature, which has been an obstacle to development. Also, to make it disposable, μ-T
Similar to AS, there was a problem of increased running cost.

[0006] In a microreactor (chemical analysis device) having the above-mentioned functions, more complicated functions are to be included in the chip. Therefore, the running cost associated with the disposal is increased, the chip is applied to multipurpose purposes, or the chip is used. Obviously, it becomes difficult to control the individual temperature of each well.

In view of the above problems, the present invention provides a microreactor (chemical analysis device) capable of performing a complicated chemical reaction and the like, which contributes to reduction of running cost and versatility of use. To aim.

[0008]

In order to achieve the above object, the first feature of the present invention is that (a) a container-shaped member having a plurality of sample wells arranged therein, and (b) a sample of the container-shaped member is injected. A gist of the chemical analysis device is to include a cover member that covers the surface, and (c) a base-side well that detachably holds the bottom of the sample well, and a base on which a container member can be placed.

Here, the "chemical analysis device" refers to a microreactor in which various chemical analysis functions are integrated. This chemical analyzer also has functions of μ-TAS and nucleic acid amplifier.

According to the chemical analysis device of the first feature,
Since the container-shaped member that holds the sample can be disposable, it is possible to prevent contamination and misjudgment, and the parts that require manufacturing costs such as the base can be reused repeatedly, which leads to a reduction in running costs and the base It is possible to contribute to the multipurpose use.

Further, the container-like member of the chemical analysis device according to the first feature further has a sample flow path for allowing movement of the sample, and the base has a sample flow path for detachably holding the sample flow path. You may further have a recess for use. According to this chemical analyzer, it becomes possible to perform chemical analysis such as mixing, reaction, separation and detection of the sample on the sample flow path.

Further, the base of the chemical analysis apparatus according to the first feature may further have a heating portion capable of heating the sample individually for each sample well in each of the base side wells. According to this chemical analysis device, it is possible to individually heat each sample by controlling each heat generating part.

The chemical analyzer according to the first feature may further include a microvalve provided on the base and capable of pushing up the bottom of the sample channel to close the sample channel. Here, the "microvalve" is a minute fluid control element required for a microchemical analysis system, and controls the flow of a sample flow path by means of minute valves and rollers. Further, the function of the micro pump may be provided by the vertical movement and the rotational movement of the valve and the roller. According to this chemical analysis device, the flow of the sample on the container-shaped member can be stopped or moved.

A second feature of the present invention is (a) a container-shaped member having a plurality of sample wells arranged therein, (b) a cover member for covering the sample-injecting surface of the container-shaped member, and (c) a container-shaped member. A base having a through hole that is adjacent to the through hole and that is separable from the container-like member and that detachably fits the bottom of the sample well; (d) a heat transfer member selectively disposed adjacent to the through hole;
An insulating member adjacent to the heat transfer member, a heat generating portion adjacent to the (e) insulating member and selectively arranged to heat the through hole, and a heat transfer member for a cold plate adjacent to the (e) heat generating portion. And (h) a cold plate adjacent to the cold plate heat transfer member.

According to the chemical analysis device of the second feature,
The container-like member, the metal thin film on the inner wall of the through hole, the heat transfer member, the heat generating part, the heat transfer member for the cold plate, the cold plate, etc. are in thermal communication, so if the temperature of the cold plate is determined, the heat generating part is turned on / off. This enables temperature control above the cold plate temperature of the container-shaped member. or,
Similar to the chemical analysis device according to the first feature, the container-shaped member in which the sample is placed can be disposable, so that it is possible to prevent contamination and misjudgment, and a portion such as a base that requires a high manufacturing cost can be repeatedly used. Therefore, it is possible to reduce the running cost and contribute to the multipurpose use of the base and the like.

A third feature of the present invention is (a) a step of dispensing a sample into a sample well on a container-shaped member molded by using a convex mold for molding and a concave mold for molding, and (b) a sample well. The gist of the analysis method is to include at least a step of fitting each of the bottom portions of the above into the inside of a plurality of base-side wells arranged on the base, and (c) a step of analyzing the sample.

According to the analysis method of the third feature, since the container-shaped member in which the sample is placed can be disposable, it is possible to prevent contamination and misjudgment, and the portion such as the base, which is costly to manufacture, can be repeatedly used. Therefore, the running cost can be reduced and the base can be used for various purposes.

Further, in the dispensing step in the analysis method according to the third feature, after the cover member is adhered to the surface of the container-shaped member on which the sample is injected, the dispenser is pierced through the cover member from above the cover member. Is inserted and the sample is injected into the sample well, and the step of closing the hole of the cover member made by the dispenser with a sealant may be included. According to this analysis method, since the sample is injected after the container-shaped member and the cover member are adhered to each other, there is no problem that the sample does not yet enter at the time of adhering and the sample does not leak at the time of adhering.

The fourth feature of the present invention is (a) a step of injecting a sample into the sample well of the container-shaped member by a pipetting device, and (b) a cover member on the surface of the container-shaped member on which the sample is injected. And (c) fitting each of the bottoms of the sample wells into a plurality of base-side wells arranged on the base, and (d) analyzing the sample. That is the summary.

According to the analysis method of the fourth feature, since the sample is injected into the sample well of the container-shaped member by the dispenser before the cover member and the container-shaped member are bonded together,
There is no need to use a sealant or the like. Further, like the analysis method according to the third feature, since the container-shaped member for storing the sample can be disposable, it is possible to prevent contamination and misjudgment, and the part such as the base, which is costly to manufacture, can be repeatedly used. Therefore, the running cost can be reduced and the base can be used for various purposes.

The analysis method according to the third and fourth characteristics may further include a step of controlling the temperature of the sample well. According to this analysis method, it becomes possible to control the temperature for each sample well, and for example, the same effect as the analysis using a nucleic acid amplifier can be obtained.

Further, in the analyzing methods according to the third and fourth characteristics, the container-shaped member has a sample flow path connecting the sample wells, and the step of sending the sample through the sample flow path is performed. You may also have. According to this analysis method, the reaction of the sample can be promoted, and, for example, it has the same effect as the analysis using μ-TAS.

In the analysis method according to the third and fourth characteristics, when the container-shaped member and the cover member are bonded together,
An adhesive may be used. According to this method, the container-shaped member and the cover member can be easily bonded.

In the analysis method according to the third and fourth characteristics, when the container-shaped member and the cover member are bonded,
A method of sandwiching the container-shaped member and the cover member in the base using a cross beam may be used. According to this method, the container-shaped member, the cover member, and the base can be easily separated by simply removing the cross beam.

Further, although the heating portion described in the first and second characteristics performs contact type heating, the heating at the time of analysis may be contact type or non-contact type. Examples of the light source used for non-contact heating include visible light, infrared rays, lasers, electromagnetic waves, and the like. In this case, an electric shutter is arranged between the light source and the sample, and it is possible to control heating of each sample by opening and closing the electric shutter. When the light source is visible light, it is not an electric shutter but a micromirror array (optical semiconductor).
Alternatively, the individual sample may be heated.

Further, a thermocouple, a temperature-sensitive liquid crystal, a black body or the like may be installed on or near the sample of the chemical analyzer or in the base, and the temperature may be measured for each sample to control the temperature.

BEST MODE FOR CARRYING OUT THE INVENTION Next, first and second embodiments of the present invention will be described with reference to the drawings. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic,
It should be noted that the relationship between the thickness and the plane dimension, the thickness ratio of each layer, and the like are different from the actual ones. Therefore, the specific thickness and dimensions should be determined in consideration of the following description. Further, it is needless to say that the drawings include some parts having different dimensional relationships and ratios.

(First Embodiment) An example in which a chemical analyzer (microreactor) is used as a nucleic acid amplifier in the first embodiment will be described. As shown in FIG. 1A, the chemical analyzer (nucleic acid amplifier) according to the first embodiment includes a container-shaped member 12 in which a plurality of sample wells 9 used as containers for containing a sample are arranged, and a container-shaped member 12. The cover member 11 that covers the surface of the member 12 into which the sample is injected, the base-side well 10 that detachably holds the bottom of the sample well 9, and the heat-generating portion 35 that can individually heat the base-side well 10. And a base 14 having The container-shaped member 12 is made of a resin having a strong chemical reaction, such as polypropylene having a thickness of 10 to 50 μm. The cover member 11 is made of a material such as resin and is adhered to the container-shaped member 12 with an adhesive 25. The base 14 is made of a material having low thermal conductivity. For example, it is made of glass epoxy resin, fluororesin such as polytetrafluoroethylene (PTFE), metal or the like. Here, the metal is limited to one having a low thermal conductivity. The base 14 includes a base-side well 10 for arranging the container-shaped member 12 and a heating portion 35 provided for each of the base-side wells 10. As the heat generating portion 35, an electrothermal heater made of a metal wire having a high electric resistance such as a nichrome wire can be considered. A thermocouple (not shown) is provided as a temperature sensor in contact with the lower portion of the well 10 on the base side, and the heat generating portion (heater) 35 and the thermocouple are connected to each other.
It is connected to a control device (not shown). The control device receives the measurement result from the thermocouple, generates a signal that is a current value to be applied to the heat generating portion (heater) 35 based on the input value, and outputs this signal to the heat generating portion (heater) 35. ing. As the heat generating portion 35, an optical absorber such as a carbon film molded in the shape of each base side well 10 is provided, and this optical absorber is heated optically by a halogen lamp (infrared lamp), laser, or the like. Is also good. Alternatively, the sample well 9 may be heated directly with an infrared lamp. Further, the heat generating portion 35 is provided in each well 10 on the base side.
It is also possible to use an RF induction heating method in which an RF coil is provided to heat the RF coil with high-frequency power, and an electromagnetic wave absorber such as a metal film accommodating each sample well 9 is used as the heat generating portion 35, and 50 MHz to 2.5 MHz. A method of irradiating a high frequency electromagnetic wave of GHz may be used. If the base-side wells 10 are insulated from each other, the base-side wells 10 can be controlled by controlling the temperature of each heat generating portion 35 or by individually applying light energy to each of the base-side wells 10.
Can be heated individually.

FIG. 1B is a view of the chemical analyzer (nucleic acid amplifier) according to the first embodiment as seen from above with the cover member 11 removed. Chemical analyzer (nucleic acid amplifier)
L1 is, for example, 10 to 30 mm, and the diameter d of the sample well 9 is preferably 1 μm to 5 mm. The diameter may be 5 mm or more, but the integration density is reduced and the function as a microreactor is reduced. If processing technology allows, 1
A diameter d of less than μm is possible, but it is not realistic from the viewpoint of operability and analytical sensitivity in performing mixing, reaction, and analysis.
Therefore, considering the integration density as a microreactor, the analysis sensitivity, and the ease of manufacturing, the thickness may be about 20 μm to 1 mm. More preferably, if it is about 0.1 to 1 mm, the integration density is kept high and the operability is good.

In the chemical analyzer (nucleic acid amplifier) according to the first embodiment, as shown in FIG. 1A, a container-shaped member 12 made of a film for containing a sample and a base 14 are separable from each other. It has become. Therefore, the container-shaped member 12 for containing the sample can be disposable, and the base 14 and the heat generating portion 35, which are costly to manufacture, can be repeatedly used and contribute to the versatility of the use of the base 14 and the like.

FIG. 2 shows a modification of the structure of the chemical analyzer (nucleic acid amplifier) according to the first embodiment. 2A is an exploded sectional view showing details of a part of the chemical analyzer (nucleic acid amplifier), and FIG. 2B is a sectional view in which those parts are assembled.

The chemical analyzer (nucleic acid amplifier) shown in FIG.
Is a container-shaped member 12 in which a plurality of sample wells 9 are arranged, a cover member 11 that covers the surface of the container-shaped member into which the sample is injected,
A through hole 30 that is adjacent to the container-shaped member 12, is separable from the container-shaped member 12, and detachably fits the bottom of the sample well 9
Which has a base 14, a heat transfer member 33 selectively disposed adjacent to the through hole 30, an insulating member 34 adjacent to the heat transfer member 33, and an insulating member 34 adjacent to and heats the through hole 30. Therefore, at least a heat generating portion 35 selectively disposed for that purpose, a cold plate heat transfer member 36 adjacent to the heat generating portion 35, and a cold plate 37 adjacent to the cold plate heat transfer member 36 are configured.

The cover member 11 is bonded to a container-shaped member 12 made of a resin having a strong chemical reaction such as polypropylene having a thickness of 10 to 50 μm. The container-shaped member 12 containing the sample 50 is fitted into the through hole 30 formed in the base 14. The thickness L12 of the base 14 is 0.1 to
The inner radius r2 of the through hole is about 250 μm at 1.0 mm. The inner wall of the through hole 30 is covered with the metal thin film 31. The thickness L22 of the metal thin film 31 is about 25 μm. The metal thin film 31 forms a flange-shaped land under the through hole 30, and the outer radius r3 of the land is 500 μm. Further, the contact thermal resistance between the metal thin film 31 and the container-shaped member 12 is reduced by silicone oil, heat conductive grease or the like. A heat transfer member 33 made of copper or the like having high heat conductivity is coated or fitted in a lower portion of the through hole 30 via a connecting member 32 of solder or the like having an outer diameter substantially the same as that of the land. The thickness L13 of the coupling member 32 and the thickness L14 of the heat transfer member 33 are each several tens of μm. After all, the coupling member 32 and the heat transfer member 33 also have an outer radius r3 = 500 μm which is substantially the same as the land, and the diameter becomes 1 mm. An insulating member 34 is fitted between the heat generating portion 35 and the heat transfer member 33. In the chemical analyzer (nucleic acid amplifier) according to the first embodiment, if the heat generating portion 35 is a heater, it is necessary to interpose an insulating member 34 such as polyimide having a small thickness in order to transfer heat to the heat transfer member 33. Is. The thickness L15 of the insulating member is several tens of μm. Electric power is supplied to the heat generating parts 35 in a matrix form by using two-layer wiring using a metal thin film of copper (Cu), aluminum (Al) or the like using the upper surface and the lower surface of the insulating member 34. To be done. The two-layer wiring can be easily formed by the photolithography technique or the screen printing technique used for semiconductor integrated circuits. Further, since the heat generating part 35 is thermally connected to the cold plate 37 located therebelow via the cold plate heat transfer member 36 having a thickness L16 = several hundred μm, the power of the heat generating part (heater) 35 is cut off. For example, the heat of the container-shaped member 12 is released to the cold plate 37. The cold plate 37 is always 50 ° C., for example.
It is kept at a constant temperature. Or sample 50 is D
If it is other than NA, it may be maintained at a constant temperature of about 10 ° C., which is lower than room temperature, by electronic cooling or the like.

In this chemical analyzer (nucleic acid amplifier), the container-shaped member 12, the metal thin film 31 on the inner wall of the through hole, and the heat transfer member 3 are used.
3, the heat generating portion 35, the cold plate heat transfer member 36, and the cold plate 37 are in thermal communication with each other. Therefore, if the temperature of the cold plate 37 is determined, the heat generating portion 35 is turned on / off to turn the cold plate of the container-shaped member 12 on and off. It is possible to control the temperature above the temperature. Further, in this chemical analysis device (nucleic acid amplifier), the individual through holes 30 are insulated from each other by resin, air or the like, so that individual thermal control is possible.

Further, since only the container-shaped member 12 can be detached from the base 14 and made disposable, it is possible to prevent contamination and erroneous judgment. Since the parts such as the heat generating part 35 and the cold plate 37, which are costly to manufacture, can be repeatedly used, the running cost can be reduced and the base 14 and the like can be used for multiple purposes.

Next, a method for amplifying DNA using the chemical analyzer (nucleic acid amplifier) according to the first embodiment will be described with reference to FIGS.

(A) First, as shown in FIG. 3A, the film 5 is placed between the molding convex mold 20 and the molding concave mold 21. The molding convex mold 20 and the molding concave mold 21 are adapted to the position and shape of the base-side well 10 or the through hole 30 above the base 14. The molding concave mold 21 may be used as the base 14. Next, as shown in FIG. 3B, heat or pressure is applied from the upper part of the convex mold 20 for molding or the lower part of the concave mold for molding, or both to mold the film. As a result, the container-shaped member 12 having the sample well 9 as shown in FIG. 3C is produced.

(B) Next, as shown in FIG. 4, the molded container-shaped member 12 and the cover member 11 are bonded together.
As the bonding method, heat, ultrasonic vibration, or the like may be used instead of using an adhesive.

(C) Next, the sample 50 is dispensed into the container-shaped member 12. When the cover member 11 and the container member 12 are bonded with an adhesive, the container member 12 and the cover member 11 are bonded with the adhesive 25, as shown in FIG. Next, as shown in FIG. 5B, the dispenser 23 is attached to the cover member 11 and the adhesive 2 from the top of the cover member 11.
5 is inserted so as to penetrate, and the sample 50 is injected into each of the sample wells 9 of the container-shaped member 12. After withdrawing the dispenser 23, as shown in FIG. 5C, there is a hole formed by the dispenser 23 in the upper part of the cover member 11. This hole of the cover member 11 is closed by a sealant 24 such as an adhesive dropped from the dispenser 23. FIG. 5D is a diagram in which the holes of the cover member 11 are closed with the sealant 24. This dispenser 23 has an injection needle-shaped capillary 27 at its tip, and the inner diameter of the capillary 27 is, for example, about 0.1 mm,
The outer diameter is about 0.3 mm. Basic dimension d of sample well 9
Is about 1 μm, a microcapillary having a fine structure similar to the probe of the scanning tunneling microscope may be used. The dispenser 23 includes the sample well 9 of the container-shaped member 12.
The plurality of capillaries 27 are provided so as to match the depressions of the sample 5 and the sample 5 is placed in the sample wells 9 of the plurality of container-shaped members 12 by one dispensing.
0 can be injected. Further, since the sample 50 is injected after the container-shaped member 12 and the cover member 11 are adhered to each other, the sample 50 is not yet contained in the case of adhering, and there is no problem that the sample leaks at the time of adhering.

(D) Next, as shown in FIGS. 1A and 2, the container-shaped member 12 containing the sample 50 in each sample well 9.
Are placed on the base 14. Then, the sample 50 on the base 14 is heated by the heat generating portion 35 to perform analysis. Since the sample 50 is a nucleic acid (DNA), for example, DNA is dissociated at around 95 ° C. and annealed at around 55 ° C.
Replicate and amplify at around 70 ° C.

(E) After the analysis, the container-shaped member 12 containing the sample 50 is removed from the base 14, and only the container-shaped member 12 and the cover member 11 that are in direct contact with the sample 50 are discarded. The base 14 and the heat generating portion 35 are repeatedly used.

In addition to the above-described bonding method shown in FIG. 4, FIG.
As shown in FIG. 5, a method of sandwiching the container-shaped member 12 and the cover member 11 in the base 14 by the cross beam 22 may be used. This method will be described in detail. First, as shown in FIG. 7A, a well girder 22 is prepared. The cross beam 22 is preferably made of a heat-resistant synthetic resin or the like. Well 22
The lower portion of is shaped to fit the groove 16 of the base 14. Next, as shown in FIG. 7B, the base 14 in which the groove 16 is arranged around the base side well 10 is prepared. This groove 1
6 has a shape that matches the shape of the lower part of the cross girder 22.
The container-shaped member 12 containing the sample 50 is arranged on the base 14, and the cover member 11 is arranged thereon. Next, FIG.
As shown in (c), the cross girder 22 is aligned with the recess on the base 14 from the upper part of the cover member 11 overlaid on the container-shaped member 12 and fitted. That is, the cross 22 and the base 1
4, the film 12 on the container and the cover member 11 are sandwiched. According to this bonding method, it is not necessary to use an adhesive or the like, and the container-shaped member 12 and the cover member 11 can be easily separated only by removing the cross girder 22 after the analysis is completed.

In addition to the dispensing method shown in FIG. 5 described above,
After injecting the sample 50 into the container-shaped member 12, the cover member 1
The method of bonding 1 may be used. After injecting the sample 50 from the dispenser 23 into each of the sample wells 9 of the container-shaped member 12 as shown in FIG. 8A, the cover member 11 is adhered as shown in FIG. 8B. The state shown in FIG. When this method is used, dispensing is performed after the container-shaped member 12 and the cover member 11 are bonded. This method has an advantage that it is not necessary to use the sealant 24 or the like. As a method of heating the sample, a method of heating in a non-contact manner other than the contact method described in FIGS. 9 to 11 are schematic views of the non-contact heating of the chemical analysis device according to the first embodiment.

FIG. 9 shows a light source (radiation source) 44 and a constant temperature bath 41.
It is a schematic diagram of the heating method which has arrange | positioned the electric shutter 42 between them. A plurality of sample wells 9 for containing the sample 50 are formed in the constant temperature bath 41, and the sample 50 is placed therein. For example, an optical window may be provided at each of the through holes 30 of the cold plate 37 shown in FIG. The sample well 9 and the optical window are individually insulated. An electric shutter 42 made of liquid crystal or the like is arranged under the constant temperature bath 41, and a light source (radiation source) 44 is arranged under the electric shutter 42.
Are arranged. As the light source (radiation source) 44, a halogen lamp (infrared lamp) or a laser can be used. The sample well 9 arranged in each optical window is heated by light energy (more generally, electromagnetic wave energy) from a light source (radiation source) 44 passing through the electric shutter 42. Therefore, the sample 5 in the sample well 9 designated by controlling the opening / closing of the electrical shutter 42 is used.
It becomes possible to control the temperature of only 0. Constant temperature bath 41
If the material is optically opaque and has a good heat insulating property, the accuracy of temperature control can be improved by heating only the sample 50 without heating the extra portion other than the optical window. The optical window may be made of an optical material transparent to the wavelength of the heating light source, or may be a through hole.

FIG. 10 is a schematic diagram of a heating method using the micromirror array 45 instead of the electric shutter. The light from the light source (radiation source) 44 is projected onto the targets 46 arranged in a matrix using the micromirror array 45. The target 46 has a structure in which optical windows are provided at the positions of the through holes of the cold plate 37 in FIG. Micromirror array 45 is about 16μ
It is made up of 500 to 1.3 million m square mirrors. By moving the angle of each mirror by about ± 10 °, it is possible to change the optical path and project onto individual targets 46. Sample 5 is placed in the sample well 9 of the target 46.
By arranging 0 and controlling the angle of the mirrors of the micro mirror array 45, it becomes possible to supply light energy to a specific optical window and heat only a specific sample 50.

Further, FIG. 11 is a schematic view of a heating method in which an electric shutter 42 is arranged between the laser 47 and the constant temperature bath 41. Specific examples of the laser 47 include a semiconductor laser. The wavelength of a commercially available semiconductor laser is 600 to 10
About 00 μm, output over 3 mW, recently 100 mW
High-power semiconductor lasers that exceed this range have also been developed. Since a semiconductor laser emits light with high efficiency with a small crystal, it is suitable as a light source for a small device such as a chemical analyzer. An electric shutter 42 is arranged below the constant temperature bath 41 having a structure as shown in FIG. 2, and a laser 47 is arranged below the electric shutter 42. The laser light from the laser 47 is
The lens 49 is used to uniformly hit the entire surface of the electrical shutter 42. In the constant temperature bath 41, the sample 50 is placed in each sample well 9 as in FIG. By opening and closing the electrical shutter 42, it is possible to control the laser light and heat only the sample 50 in the specific sample well 9.

As a method for controlling the temperature of the sample 50 on the container-shaped member 12, a control method as shown in FIGS. 12 and 13 can be considered.

FIG. 12 is a schematic diagram for controlling the temperature of the sample 50 on the container-shaped member 12. Similar to FIG. 2, a plurality of sample wells 9 are formed in the constant temperature bath 41, and the sample 50 is placed therein.
Put. An electric shutter 42 made of liquid crystal or the like is arranged under the constant temperature bath 41, and a light source (radiation source) 44 is arranged under the electric shutter 42. A thermocouple, a temperature-sensitive liquid crystal, a black body, or the like is installed on or near the sample 50 or in the constant temperature bath 41, and the temperature is measured. In FIG. 12, an infrared camera 48 for detecting temperature is provided above the sample 50.
Are arranged.

A procedure for controlling the temperature of the sample 50 on the container 12 will be described with reference to FIG.

(A) In step S 101, the temperature of the sample 50 in each sample well 9 on the thermostat 41 is measured by the infrared camera 48 for each sample 50.

(B) Next, in step S102, with respect to the sample 50 in the sample well 9 whose measured temperature is higher than the target temperature, the process proceeds to step S103 and is located in the optical path of the light source (radiation source) 44 with respect to the sample 50. The electric shutter 42 is closed. Then, the process proceeds to step S101 again to measure the temperature.

(C) In step S102, if the measured temperature is not higher than the target temperature, the process proceeds to step S104. Then, for the sample 50 in the sample well 9 whose measured temperature is lower than the target temperature, the process proceeds to step S105,
The electrical shutter 42 located in the optical path of the light source (radiation source) 44 for the sample 50 is opened. Then, the process proceeds to step S101 again to measure the temperature.

(D) In step S104, if the measured temperature is not lower than the target temperature, it is just the target temperature, so nothing is done and the process proceeds to step S101 again to measure the temperature.

Each sample 50 can be maintained at an appropriate temperature by repeating the steps (a) to (d) by optically scanning each sample well 9 at regular intervals.

(Second Embodiment) The second embodiment is a chemical analyzer (microreactor) according to the present invention.
Is an example of using as a μ-TAS. In the chemical analysis device according to the second embodiment, the container for containing the sample is made of a film, and the container-shaped member is separable from the base and the like, as in the first embodiment. 14
As shown in (a), a sample well 9 used as a container for containing the sample 50 and a container-shaped member 12 in which a plurality of sample flow paths 51 are arranged, and a cover member 11 for covering the surface of the container-shaped member 12 into which the sample is injected. And at least the base side well 10 that detachably holds the sample well 9 and the sample flow channel 51, and the base 14 having the sample flow channel recess 52.
As in the first embodiment, the container-shaped member 12 has 10 to 10
It is made of a chemically resistant resin such as polypropylene having a thickness of 50 μm. The cover member 11 is made of resin or the like, and is adhered to the container-shaped member 12 with an adhesive 25. Base 1
4 is made of a material having a low thermal conductivity. For example, it is made of glass epoxy resin, fluororesin, metal or the like. Here, the metal is limited to one having a low thermal conductivity.

FIG. 14B is a view of the chemical analyzer (μ-TAS) according to the second embodiment as seen from above. The chemical analyzer (μ-TAS) is composed of a sample injection section 62 including a sample well 9 into which the sample 50 is injected, a mixed reaction separation / detection section 61 to analyze the sample, and a sample well 9 into which the sample 50 after the reaction is collected. It has a waste liquid portion 60. The mixed reaction separation detection unit 61 includes a mixing unit 61a for mixing the sample 50, a reaction unit 61b for reacting the sample 50, a separation unit 61c for separating the sample 50, and a detection unit 61d for detecting the sample 50. The length L31 of one side of the base 14 of the chemical analyzer (μ-TAS) is 10 to 30 mm, and the diameter d of the sample well 9 is 1 μm to 5 mm, preferably 20 μm to 2 mm.
It is about 0.1 to 1 mm, more preferably about 0.1 to 1 mm. The width w of the sample channel 51 is d ≧ w> (1/10) d, for example, about 100
μm.

Although not shown in FIG. 14A, a mechanism such as a heat generating portion 35 and a micro valve, which is necessary for a chemical reaction, is provided in the base 14 or under the base 14. FIGS. 15 to 17 show a chemical analyzer (μ which moves up and down or rotates by electromagnetic force and pushes up the sample 50 from under the container-shaped member 12 to close the flow path of the sample 50 (μ -TAS) is a cross-sectional view.
These microvalves are provided inside the base 14 or below the base 14 and can be separated from the container-shaped member 12.

In the microvalve shown in FIG. 15, a valve 70 for crushing the sample flow path 51 by vertical movement is used as the base 1.
4, the fluid sample 50 is shut off. Further, the function of the micro pump may be provided by the vertical movement of the valve 70.
The valve 70 uses an electromagnetic valve 70 that opens and closes by the force of an electromagnet (solenoid). In FIG. 15A, the solenoid valve 70 operates upward with respect to the sample flow channel 51, and in FIG.
The solenoid valve 70 shuts off the sample flow path 51. FIG. 16 shows a micropump that sends the sample 50 in the sample flow path 51 in the container-shaped member 12 to the right by rotating the eccentric roller 71 by an electromagnetic force. When the eccentric roller 71 rotates in the direction of the arrow, the sample 50 flows to the right.
In the micropump shown in FIG. 17, the three solenoid valves 70a, 70b, 70c provided with the base 14 move up and down to crush the sample 50 in the sample flow path 51 formed in the container-shaped member 12 in order. The sample 50 is transported by going down. Further, if one of these valves is used, it can be used as a microvalve for fluid. As a specific movement of the pump, in FIG. 17A, the electromagnetic valve 70c on the right side is raised to close the sample flow channel 51. Next, FIG.
In (b), the electromagnetic valve 70a on the left side also rises, closing the sample flow path 51. Next, in FIG. 17C, the electromagnetic valve 70c on the right side is lowered, so that the sample 50 flows to the right. Next, in FIG. 17D, the central solenoid valve 70
By increasing b, the sample 50 further flows to the right. Next, in FIG. 17 (e), the solenoid valve 70c on the right side
Also, the flow of the sample 50 to the right is further strengthened. Next, in Fig. 17 (f), the sample 50 flows in from the left by lowering the left solenoid valve 70a. Thereafter, the solenoid valve 70b at the center is lowered to return to the state of FIG. 17 (a). By repeating the states of FIGS. 17A to 17F, the sample 50 flows from left to right. These micropumps can stop or move the flow of the sample 50 on the container-shaped member 12. Figure 15-
The microvalve (micropump) shown in FIG.
Although it is operated by electromagnetic force, other power may be used. For example, a tube through which air is passed may be provided in a part of the sample channel recess 52, and the sample channel 51 may be moved up and down by air pressure.

In addition, the small-sized analyzer (μ-TAS) according to the second embodiment has a heating portion capable of individually heating each of the base side wells 10 as described in the first embodiment. Or an optical window may be further provided. In the chemical analyzer (μ-TAS) according to the second embodiment, the sample 50 is supplied from a part of the sample wells 9 and moves while causing heating and cooling or a chemical reaction, and the waste liquid portion 60 is repeatedly mixed and separated. Flow to. After the reaction is completed, only the container-shaped member 12 can be disposable, and the base 14 and the solenoid valves 70, 70, which are expensive to manufacture, are expensive.
The a, 70b, 70c, the eccentric roller 71 and the like can be repeatedly used.

Next, using the chemical analyzer (μ-TAS) according to the second embodiment, liquid feeding, mixing, reaction of the sample,
A method of performing analysis and the like will be described with reference to FIG.

(B) Similar to the method for amplifying DNA according to the first embodiment, the container-shaped member 12 as shown in FIG.
Molding, the container-shaped member 12 as shown in FIGS. 4 and 6 to 7.
Then, the cover member 11 is bonded. Since these methods have been described in the first embodiment, description thereof will be omitted here.

(B) Next, the container-shaped member 12 is attached to the base 14
The sample 50 is placed on the upper side, and the sample 50 is dispensed into the sample wells 9a and 9b of the sample injection unit 62. Here, as an example, the sample A is injected into the sample well 9a and the sample B is injected into the sample well 9b. The sample A and the sample B respectively pass through the sample flow path 51 and flow into the mixing section 61a where they are mixed. At this time, the heat generating part 35 may be provided below the mixing part 61a to accelerate the reaction by heat. The mixed sample A and sample B cause a chemical reaction while passing through the reaction section 61b, and, for example, sample C and sample D are synthesized. A micro valve is provided below the reaction section 61b, and the electromagnetic valve 70 and the eccentric roller 71 are used to make the sample C,
The solution of D may be sent. Next, the separation unit 61c separates the sample C and the sample D. This separation is, for example,
By tilting the miniaturized chemical analyzer (μ-TAS), the difference in specific gravity between the sample C and the sample D may be used. The separated sample C and sample D are respectively detected by the detection unit 6
Detected in 1d, analysis and the like are performed. Thereafter, unnecessary substances flow into the sample wells 9c and 9d of the waste liquid portion 60 and are collected.

(C) After the analysis of the series of samples 50 is completed, only the container-shaped member 12 and the cover member 11 that are in direct contact with the samples 50 are discarded. The base 14, the heat generating portion 35, the micro valve, etc. are repeatedly used.

(Other Embodiments) Although the present invention has been described by the above-mentioned first and second embodiments, it is understood that the discussion and drawings forming a part of this disclosure limit the present invention. should not do. From this disclosure, various alternative embodiments, examples and operational techniques will be apparent to those skilled in the art.

For example, in the analysis method according to the first embodiment of the present invention, the container-shaped member 12 is arranged on the base 14 after the sample 50 is dispensed. However, the container-shaped member 12 is arranged on the base 14. After that, dispense the sample 50 into the base 14
You can go above.

Further, in the small analytical chemistry apparatus according to the first and second embodiments of the present invention, the heat generating portion 35 and the solenoid valve 7 are provided.
A mechanism such as 0 may be built in the base 14 and integrated with the base, or may be disposed under the base 14 and separable from the base. Similarly, the electric shutter 42 and the like may be built in the base 14 or may be arranged below the base 14.

Further, in the small analytical chemistry apparatus according to the first and second embodiments of the present invention, the present invention was used as a nucleic acid amplifier or μ-TAS, but the present invention is not limited to these, and the present invention can be applied to a small reaction apparatus. May be used.

Further, in the small analytical chemistry apparatus according to the first and second embodiments of the present invention, the base 14 is provided with the heat generating portion 35, and the micro valve mechanism such as the electromagnetic valve 70 is provided. However, the base 14 may include both of them at the same time, or may include another mechanism for tilting or vibrating the base 14. As described above, it goes without saying that the present invention includes various embodiments and the like not described here. Therefore, the technical scope of the present invention is defined only by the matters specifying the invention according to the scope of claims reasonable from the above description.

[0068]

Industrial Applicability According to the present invention, it is possible to provide a microreactor (chemical analysis device) which can carry out complicated chemical reactions and can contribute to reduction of running cost and versatility of use.

[Brief description of drawings]

FIG. 1A is a cross-sectional view of a chemical analyzer according to a first embodiment. (B) is the figure which looked at the chemical-analysis apparatus which concerns on 1st Embodiment from the top.

FIG. 2 is a cross-sectional view of a part of another chemical analyzer according to the first embodiment, (a) is a component diagram, and (b) is an assembly diagram.

FIG. 3 is a cross-sectional view when molding a container-shaped member of the chemical analysis device according to the first embodiment.

FIG. 4 is a method of adhering a container-shaped member and a cover member of the chemical analysis device according to the first embodiment.

FIG. 5 is a method for dispensing a sample into a container-shaped member of the chemical analysis device according to the first embodiment.

FIG. 6 is another method for adhering the container-shaped member and the cover member of the chemical analysis device according to the first embodiment.

FIG. 7 is a diagram showing a procedure of the bonding method shown in FIG.

FIG. 8 is another method for dispensing a sample into the container-shaped member of the chemical analysis device according to the first embodiment.

FIG. 9 is a non-contact heating method for the chemical analyzer according to the first embodiment (No. 1).

FIG. 10 is a non-contact heating method for the chemical analyzer according to the first embodiment (No. 2).

FIG. 11 is a non-contact heating method for the chemical analyzer according to the first embodiment (No. 3).

FIG. 12 is a schematic diagram of a temperature control method for the chemical analyzer according to the first embodiment.

FIG. 13 is a flowchart of a temperature control method for the chemical analysis device according to the first embodiment.

FIG. 14A is a cross-sectional view of a chemical analysis device according to a second embodiment. (B) is the figure which looked at the chemical analysis device concerning a 2nd embodiment from the top.

FIG. 15 is a cross-sectional view (part 1) of a chemical analyzer having a microvalve function according to a second embodiment.

FIG. 16 is a cross-sectional view of a chemical analysis device having a microvalve function according to a second embodiment (No. 2).

FIG. 17 is a cross-sectional view of the chemical analysis device having the microvalve function according to the second embodiment (No. 3).

FIG. 18 is a perspective view of a conventional μ-TAS.

FIG. 19 is a sectional view of a conventional nucleic acid amplifier.

[Explanation of symbols]

1 Chemical analyzer (nucleic acid amplifier) 5 films 9, 9a, 9b, 9c, 9d Sample well 10 Base well 10a, 10b, ..., 10e wells 11 Cover member 12 Container-shaped member 14 base 15 heater 16 grooves 20 Convex type for molding 21 Recessed mold 22 double digits 23 dispenser 24 Sealant 25 adhesive 27 capillaries 30 through holes 31 metal thin film 32 coupling members 33 Heat transfer member 34 Insulation member 35 Heat generating part 36 Heat transfer member for cold plate 37 cold plate 41 constant temperature bath 42 electrical shutter 44 Light source (radiation source) 45 micro mirror array 46 Target 47 laser 48 infrared camera 49 lenses 50 samples 51 Sample flow path 52 Sample channel recess 60 waste 61 Mixed reaction separation detector 61a mixing section 61b Reaction part 61c Separation part 61d detector 62 Sample injection part 70, 70a, 70b, 70c Solenoid valve 71 Eccentric roller 80 glass chips 81 reaction tube 82 Metal block 83 Cooler

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/68 G01N 31/20 4B063 G01N 1/28 35/00 B 4G075 31/20 37/00 101 35 / 00 C12N 15/00 A // G01N 37/00 101 G01N 1/28 K (72) Inventor Yoshinori Motomiya 1 Komukai Toshiba-cho, Saiwai-ku, Kawasaki-shi, Kanagawa Incorporated Toshiba Research and Development Center (72) Inventor Kaname Miyazaki, 1 Komukai Toshiba-cho, Sachi-ku, Kawasaki-shi, Kanagawa Within the Toshiba Research and Development Center, Inc. (72) Inventor Masataka Shirato 1 Komu-shi, Toshiba-cho, Sai-ku, Kawasaki, Kanagawa 72) Inventor Takeshi Morino 1 Komukai Toshiba-cho, Sachi-ku, Kawasaki-shi, Kanagawa Prefectural Research & Development Center, Toshiba Corporation (72) Inventor Masahiro Kuwata Kanagawa Komukai-shi, Takasaki-cho, Saki-shi, F-term, Toshiba Research and Development Center, stock company (reference) 2G042 AA01 BD12 CB03 GA01 HA02 HA03 HA05 HA07 HA10 2G052 AA28 AB20 AD06 AD26 AD46 CA02 DA06 DA09 EB11 HC22 2G058 AA01 BB26 CA01 CA02 CA04 DA07 GE01 GE05 4B024 AA11 AA19 CA01 CA11 CA20 HA11 4B029 AA07 AA23 BB20 CC01 FA12 FA15 4B063 QA01 QA13 QA17 QQ41 QR08 QR31 QR38 QR42 QR55 QR62 QS16 QS25 QS34 QX02 4G075 AA30 FC11 FA01 FA10 FA02 FC15 FA01

Claims (10)

[Claims] 1. A container-shaped member having a plurality of sample wells arranged therein, a cover member for covering a surface of the container-shaped member for injecting a sample, and a base-side well for detachably holding a bottom portion of the sample well, A chemical analyzer comprising: a base on which the container member can be placed. 2. The container-shaped member further arranges a sample channel for enabling movement of the sample, and the base further has a sample channel recess for detachably holding the sample channel. The chemical analyzer according to claim 1. 3. The base according to claim 1, further comprising a heating portion capable of individually heating the sample in each of the sample wells in each of the base-side wells. Chemical analyzer. 4. The chemistry according to claim 2, further comprising a microvalve provided on the base and capable of pushing up a bottom portion of the sample channel to close the sample channel. Analysis equipment. 5. A container-shaped member having a plurality of sample wells arranged therein, a cover member for covering a surface of the container-shaped member for injecting a sample, a sample adjacent to the container-shaped member and separable from the container-shaped member. A base having a through hole into which the bottom of the well is removably fitted, a heat transfer member selectively disposed adjacent to the through hole, an insulating member adjacent to the heat transfer member, and an insulating member adjacent to the insulating member. A heat generating part selectively arranged to heat the through hole; a heat transfer member for a cold plate adjacent to the heat generating part; and a cold plate adjacent to the heat transfer member for a cold plate. A chemical analyzer characterized by. 6. A step of dispensing a sample into a sample well on a container-shaped member molded using a molding convex mold and a molding concave mold, and a plurality of bottom parts of the sample well arranged on a base. And a step of analyzing the sample, the method comprising: 7. The sample dispensing step comprises bonding a cover member to a sample-injecting surface of the container-shaped member, and then inserting a pipetting instrument through the cover member from above the cover member, 7. The analysis method according to claim 6, further comprising a step of injecting the sample into the inside of a well, and further including a step of closing a hole of the cover member formed by the dispenser with a sealant. 8. A step of injecting a sample into a sample well of a container-like member by a pipetting device, a step of adhering a cover member to a surface of the container-like member into which the sample is injected, and a bottom portion of the sample well. An analysis method comprising at least the step of fitting each of the above into a plurality of base-side wells arranged on a base, and the step of analyzing the sample. 9. The analysis method according to claim 6, further comprising the step of controlling the temperature of each of the sample wells. 10. The container-shaped member has a sample flow path connecting between the sample wells, and further comprises a step of sending the sample through the sample flow path. 9. The analysis method according to any one of 9 above.