JP2003190772A - Chemical reaction cartridge - Google Patents

Abstract

(57) [Problem] To provide a chemical reaction cartridge for performing a chemical reaction quickly and simply. SOLUTION: An airtight flat member made of an elastic material is provided.
It is alternately sandwiched and pressed by a plate member for a substrate made of a material having lower elasticity and higher hardness than the elastic material to form a laminated structure of three or more layers, and provided on the plate member for the substrate or the plate member for airtightness. Channels, reservoirs, etc.
Using a chemical reaction cartridge constituted by a reaction tank or the like, a chemical reaction is performed with a liquid or gaseous sample or reagent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a chemical reaction cartridge having a novel structure. More specifically, at least one airtight flat plate member made of an elastic material is sandwiched by at least two flat plate members for a substrate, which are made of a material having a lower elasticity and a higher hardness than the elastic material, and are crimped. The present invention relates to a chemical reaction cartridge having a laminated structure of three or more layers.

In the cartridge, a liquid or gaseous sample or reagent, or a mixed fluid of the sample and the reagent is caused to flow from at least one reservoir or reaction tank into at least one flow path to obtain the sample. A cartridge for applying a chemical reaction to a predetermined component in a medium, a reagent, or the mixed fluid. The channel inside the cartridge includes a groove and / or a hole provided in the flat plate member for a substrate, a groove and / or a hole provided in the flat plate member for airtightness, the sample, the reagent, and / or the It is composed of at least one selected from the group consisting of voids formed by deforming the portion of the airtight flat plate member by the pressure of the mixed fluid. The reservoir and the reaction tank inside the cartridge are constituted by grooves and holes provided in the airtight flat plate member and / or the substrate flat plate member. Further, a valve for liquid or gas can be formed in the cartridge.

The present invention facilitates making cartridges that perform chemical reactions. By using this cartridge and a simple machine for controlling the reaction on the cartridge, it becomes possible to easily, quickly and safely carry out a chemical reaction for diagnosis in the medical field and analysis in various fields.

[0004]

2. Description of the Related Art In recent years, in clinical examination sites in medicine, so-called "point-of-point", in which specimens collected from patients are tested quickly and simply in clinical situations and the results are judged and immediately used for medical examination. There is a need for a diagnostic method called the "Kare test". In addition, for the analysis of environmental fields such as analysis of harmful substances in rivers, oceans and wastes, and the analysis of food fields such as inspection of foods for bacteria and poisons, analysis and measurement will be performed at each site or near the site. ” "Point of field test" is attracting attention. Hereafter, these inspections are referred to as "simple and quick inspections".
Enter.

In the simple and quick inspection, it is required to reduce the manual operation in the shortest possible time. That is, it is desirable that the steps from the processing of the sample to the detection are completed in one device called a "cartridge". Further, it is desirable that the cartridge be designed to be inexpensive to manufacture and "disposable" or simply "reusable." The fine processing technology has been developed, and the technology for completing such medical diagnosis on a cartridge has been developed. Micro-total analysis system: μTAS (micro total analysis syste)
m) ”technology has been studied for application to simple and quick tests. With μTAS, in order to minimize the amount of sample, a groove is made on the surface of glass or silicon, and a reagent solution or sample is placed in the groove. A small amount of sample is analyzed by flowing, separating, and reacting (JP-A-2-245655, JP-A-3-22).
6666, JP-A-8-233778, Analytical Che
m. 69, 2626-2630 (1997) Aclara Biosciences). The applicant of the present application also discloses “analyzer” WO99-6484.
6, "Analytical cartridge and liquid transfer control device" WO01
Patent applications have been filed for μTAS-related inventions such as -13127.
In these application specifications, it is described that a resin microchip is used as the cartridge.

However, in producing these cartridges, it is necessary to bond the finely processed flow paths so as not to block them. This bonding technique requires a high level of technology, which is one of the reasons for increasing the cost of the cartridge. Further, in the case of a cartridge produced by pasting together using such an adhesive, it is not easy to form a valve inside the cartridge. In addition, when pasted with an adhesive or the like, since such adhesion is generally irreversible, the cartridge once processed is
Cannot be disassembled. Therefore, when the cartridge is reused, it is practically impossible to wash the inside of the flow path while the fine flow path is formed.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to construct a technology for producing a cartridge at a low cost with a simple structure in a system for carrying out a chemical reaction with a cartridge utilizing the above-mentioned fine processing technology. To aim.

[0008]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies in order to solve these problems, and as a result, completed the present invention. That is, the present invention provides (1) a liquid or gaseous sample having at least one reservoir and / or reaction tank, and at least one flow path,
A reagent, or a mixed fluid of the sample and the reagent, is caused to flow from the at least one reservoir and / or the reaction tank into the at least one flow path so that the sample, the reagent, or the mixed fluid is A cartridge for applying a chemical reaction to a predetermined component, comprising at least one airtight flat plate member made of an elastic material, and at least two flat plates for substrates made of a material having elasticity and hardness lower than the elastic material. Has a laminated structure of three or more layers formed by alternately sandwiching and crimping the plate-shaped members, and the flow path has (i) a groove and / or a hole provided in the plate-shaped member for a substrate, and (ii) Grooves and / or holes provided in the airtight plate member, or
(Iii) At least one selected from the group consisting of the sample, the reagent, and / or the void formed by deforming the portion of the airtight flat plate member by the pressure of the mixed fluid, and the reservoir. And / or the reaction tank is constituted by a groove and / or a hole provided in the airtight flat plate member and / or the substrate flat plate member, and relates to a chemical reaction cartridge.

In one embodiment of the present invention, in the void, the portion of the airtight flat plate member is pressure-bonded to the airtight flat plate member by the pressure of the sample, the reagent and / or the mixed fluid. The airtight plate-like member and the one plate-like plate member opposite to each other, which are formed by deforming in the direction of the groove and / or hole different from the flow path provided in the one plate member for the substrate, are opposite to each other. It is a space formed between the flat plate member for the substrate on the side of.

Elastic materials used for the airtight plate member (hereinafter referred to as airtight plate) include natural rubber, butadiene rubber, styrene rubber, butyl rubber, ethylene / propylene rubber, nitrile rubber, acrylic rubber, urethane rubber, and silicone. Rubbers such as rubber and fluororubber, soft vinyl chloride resin, polyethylene resin, polypropylene resin,
Examples thereof include polyvinylidene chloride resin, polyurethane resin, fluororesin, nylon resin and the like.

The material used for the flat plate member for the substrate (hereinafter referred to as the substrate) is a hard vinyl chloride resin, polystyrene resin, ABS resin, polyethylene resin, polypropylene resin, nylon resin, acrylic resin, fluororesin, polycarbonate. Resins, plastics such as methylpentene resin, polyurethane resin, phenol resin, melamine resin, and epoxy resin, metals such as aluminum, brass, iron, copper, stainless steel, titanium alloy, magnesium alloy, duralumin, glass, and ceramics. .

In the present invention, it is important that the airtight plate has a higher elastic modulus than the substrate and the substrate has a higher hardness than the airtight plate. For example, a combination of silicon rubber for the airtight plate, fluororesin for the substrate, or fluororesin for the airtight plate, and stainless steel for the substrate may be used. In addition, it is essential that both the airtight plate and the substrate are materials that do not affect the intended chemical reaction. Further, in the case of carrying out a reaction in a liquid, a material that is substantially impermeable to liquid during the reaction is used. It must be a non-permeable material.

The flow path in the cartridge may be formed by processing an airtight plate or a substrate. As for the size of the flow path, it is necessary to have a width and a depth such that even if the airtight plate and the substrate are pressure-bonded, the flow path is not blocked by the deformation of the airtight plate and the substrate due to the pressure. The width and depth of the produced flow path vary depending on the elastic modulus of the material used for the airtight plate and the substrate and the pressure of pressing when the airtight plate and the substrate are laminated. Substantially, a groove having a depth of 10 μm and a width of 10 μm or more is required to function as the flow path of the cartridge. To complete a series of reactions within one cartridge, too large a flow path would lose the advantages of μTAS. The upper limit of the practical size of the flow path is about 5 mm in width and 1 cm in depth. It should be noted that it is also possible to use a groove or hole deeper and wider than this as the reservoir portion for storing the reaction liquid in the cartridge. The upper limit of the size of the reservoir for storing the reaction solution is about 10 cm in width and 10 cm in depth.

According to the present invention, it becomes possible to prepare a cartridge which can easily carry out a chemical reaction by using a substrate or an airtight plate in which grooves or holes are processed, without using an advanced technique such as bonding. Further, since the cartridge does not require irreversible processing such as adhesion, it is possible to disassemble the once processed cartridge, wash it, and reuse it. In the present invention, in order to make a laminated structure consisting of the substrate and the airtight plate,
The pressure bonding method must not interfere with the laminated structure forming the flow path, the reservoir, the reaction tank, and the valve. Therefore, as the method, in the portion that does not interfere with the laminated structure forming the flow path, the reservoir, the reaction tank, the valve, a through hole is drilled with a screw and a nut, or a through hole is drilled with a rivet, or Examples of the method include crimping a hole not formed with a screw, a spring from the outside of the laminated structure, or adhesion of an outer portion of the laminated structure, but the method is not limited to these.

The present invention also relates to (2) the chemical reaction cartridge according to the above (1), which comprises at least one valve for controlling opening / closing of the flow path. Further, the present invention provides (3) at least one of the valves has a rod-shaped member, the rod-shaped member being movable with respect to a flow path on the cartridge, and the rod-shaped member having an open section and a closed section. The open section has a structure in which the projected area on a plane perpendicular to the movement direction is smaller than that of the closed section, and the flow path on the cartridge and the open section of the rod-shaped member are moved by the movement of the rod-shaped member. The communication opens the valve, and the movement of the rod member closes the flow passage on the cartridge by the closed section of the rod member, thereby closing the valve and thereby the flow passage on the cartridge. The present invention relates to the chemical reaction cartridge, which functions as a valve that controls opening and closing of the cartridge.

Here, "the open section has a structure in which the projected area on a plane perpendicular to the moving direction is smaller than that of the closed section" means, for example, a notch, a groove, or a groove in the rod member.
A hole or the like is added to form the open section of the rod-shaped member. Further, the invention provides (4) at least one of said valves
Function as a valve for controlling the opening and closing of the flow path by controlling the formation of voids formed by the deformation of the airtight flat plate member due to the pressure of the sample, the reagent or the mixed fluid. The present invention relates to the above chemical reaction cartridge.

The above-mentioned valve is provided, for example, on a flat plate member for a substrate in which a portion of the flat plate member for airtightness is pressure-bonded to the flat plate member for airtightness by the pressure of the sample, the reagent or the mixed fluid. The air-tight plate-like member and the one plate-like plate member for the substrate, which is formed by deforming in a direction of a groove and / or a hole different from that of the formed flow path. In a flow path constituted by a gap between the flow path and a groove, and / or a hole different from the flow path, a pressing force is applied to the portion of the airtight flat plate-like member to suppress the formation of the gap. The flow path may be closed by the above, or the pressing force may be reduced to release the suppression to open the flow path, thereby functioning as a valve for controlling the opening and closing of the flow path. It is not particularly limited. Further, the present invention relates to (5) the above chemical reaction cartridge, which is equipped with a control device for controlling at least one opening / closing of the valve by an actuator.

According to the inventions of (2) to (5) above, it becomes possible to incorporate a valve having a simple structure into the chemical reaction cartridge. Therefore, when carrying out a chemical reaction, it becomes possible to prevent mixing of substances which should be avoided by a valve when handling liquid or gas, and to prevent backflow of liquid or gas, etc. It becomes possible to control the chemical reaction in.

In the present invention, (6) the chemical reaction involves the step of releasing nucleic acid from the material containing the nucleic acid, the step of adsorbing the released nucleic acid on the nucleic acid-binding carrier, and the step of washing the nucleic acid-binding carrier on which the nucleic acid is adsorbed. The present invention relates to the above-mentioned chemical reaction cartridge, which comprises a nucleic acid isolation reaction comprising the steps of: eluting the nucleic acid from the nucleic acid-binding carrier to which the nucleic acid is adsorbed.

Further, the present invention provides (7) a release liquid for releasing nucleic acid from a material containing nucleic acid, a nucleic acid-binding carrier, an adsorption liquid for adsorbing the released nucleic acid on the nucleic acid-binding carrier, and a nucleic acid-binding property on which nucleic acid is adsorbed. The above-mentioned chemical reaction cartridge is provided with at least one of a washing solution for washing the carrier and an eluent for eluting the nucleic acid from the nucleic acid-binding carrier on which the nucleic acid is adsorbed.

Further, the present invention provides (8) a nucleic acid-binding carrier, a free adsorbing liquid for releasing nucleic acid from a nucleic acid-containing material and adsorbing the released nucleic acid on the nucleic acid-binding carrier, and a nucleic acid-binding carrier on which the nucleic acid is adsorbed. The above-mentioned chemical reaction cartridge is provided with at least one of a washing solution for washing the solution and an eluate for eluting the nucleic acid from the nucleic acid-binding carrier to which the nucleic acid is adsorbed.

Furthermore, the present invention is characterized in that (9) the nucleic acid-binding carrier is made of silica or a silica derivative, and the step of adsorbing the liberated nucleic acid to the nucleic acid-binding carrier uses a solution containing chaotropic ions. The present invention relates to a cartridge for chemical reaction. The present invention also relates to (10) the cartridge for chemical reaction, wherein the nucleic acid-binding carrier is made of silica or a silica derivative, and the adsorbing liquid contains chaotropic ions.

The present invention also relates to (11) the above cartridge for chemical reaction, wherein the nucleic acid-binding carrier is made of silica or a silica derivative, and the free adsorption liquid contains chaotropic ions. Furthermore, the present invention provides (1
2) The above-mentioned cartridge for chemical reaction, wherein the nucleic acid-binding carrier is a nucleic acid-binding magnetic substance-containing particle.

Furthermore, the present invention relates to (13) the above cartridge for chemical reaction, wherein the nucleic acid-binding carrier is a film made of silica or a silica derivative. According to the inventions of (6) to (13) above, it becomes possible to extract and purify nucleic acid from a sample containing nucleic acid on a chemical reaction cartridge. That is, it is possible to prepare a cartridge that easily achieves isolation of nucleic acid.

The present invention also relates to (14) the chemical reaction cartridge, wherein the chemical reaction includes a nucleic acid amplification reaction. Further, the present invention relates to (15) the above-mentioned chemical reaction cartridge, wherein the chemical reaction further comprises a nucleic acid amplification reaction for amplifying the nucleic acid isolated by the nucleic acid isolation reaction. Furthermore, the present invention relates to (16) the above-mentioned chemical reaction cartridge, wherein the nucleic acid amplification reaction is a polymerase chain reaction (PCR).

According to the above inventions (14) to (16),
A nucleic acid amplification reaction can be performed on the chemical reaction cartridge. Further, according to the invention of the above (15), it becomes possible to carry out the amplification reaction of the nucleic acid in the chemical reaction cartridge while being eluted with the eluent on the chemical reaction cartridge, and to detect the nucleic acid more quickly. Will be possible. Furthermore, according to the invention of (16) above,
The nucleic acid polymerase /
The chain reaction can be carried out, and the nucleic acid can be detected more quickly.

Further, the present invention relates to (17) the chemical reaction cartridge described above, wherein the temperature is controlled by heating or cooling at least a part of the cartridge. Further, the present invention provides
(18) The chemical reaction cartridge described above, wherein at least two locations of the cartridge are heated and / or cooled to control different temperatures. The means for controlling the temperature is not particularly limited, and for example, a water bath having a set temperature may be used, or various heating devices and cooling devices may be used.

Examples of the material containing nucleic acid to be subjected to nucleic acid isolation reaction or nucleic acid amplification reaction using the chemical reaction cartridge of the present invention include, for example, sputum, saliva, which are clinical specimens used for diagnosis of human diseases, Urine, feces, semen, blood, tissues,
Examples thereof include organs and other body fluids, fractions of these body fluids, foods used for inspection of microbial contamination, drinking water, soil, drainage, river water, seawater, wiping solution, wiping cotton and the like.
It is also possible to use a bacterial suspension such as E. coli.

As the nucleic acid-binding carrier used in the chemical reaction cartridge of the present invention, any carrier commonly used in the art can be preferably used. Since the amount of the carrier can be freely changed according to the amount and elution with a small amount of eluent is possible, silica particles or silica derivative particles, and magnetic silica particles or magnetic silica derivative particles are particularly preferable. . Examples of the silica particles used in the present invention include SiO ₂ manufactured by Sigma, amorphous silicon oxide, glass powder and the like, and silica derivative particles include alkyl silica, aluminum silicate, activated silica having —NH ₂ , latex particles and the like. Can be mentioned. As magnetic silica particles or magnetic silica derivative particles used in the present invention, magnetic beads (hereinafter referred to as MagExtractor ^TM ) included in the MagExtractor ^TM kit manufactured by Toyobo Co., Ltd.
Examples include magnetic particles contained in the MagNA Pure LC DNA Isolation Kit. When magnetic silica particles or magnetic silica derivative particles are used as the nucleic acid-binding carrier, the carrier can be easily recovered using a magnet. Also,
A membrane made of silica or a silica derivative may be used as the nucleic acid-binding carrier.

The nucleic acid-releasing / adsorbing liquid used in the present invention may be any one commonly used in the art, and is not particularly limited, but a solution containing a chaotropic ion is particularly preferable. Examples of chaotropic ions include guanidine ion, thiocyanate ion, and iodine ion.

The step of adsorbing the nucleic acid onto the nucleic acid-binding carrier can be carried out by heating. At this time, heating is 60
C. to 130.degree. C., preferably 80.degree. C. to 110.degree. C., more preferably 90.degree. C. to 100.degree. The washing of the nucleic acid-binding carrier bound with nucleic acid is preferably carried out in a solution containing ethanol, and it is particularly preferable to use a solution containing 70% or more of ethanol. Further, after washing, the nucleic acid-binding carrier to which the nucleic acid is bound is dried to remove factors such as ethanol used for washing that have an inhibitory effect on the subsequent amplification reaction.

Elution of the nucleic acid from the nucleic acid-binding carrier can be carried out using water or an elution buffer, as is commonly done in the art. Nucleic acid amplification reactions that can be carried out using the chemical reaction cartridge of the present invention include polymerase chain reaction and ligase chain reaction (LC
R: Science, 241: 1077-1080, 1988), RNA-specific amplification method (NASBA method: Nature, 350: 91-92, 1991),
SDA method (Proc. Natl. Acad. Sci. USA, 89: 392-396, 199)
2) etc., but the polymerase chain reaction, which is widely used in the world, is particularly preferable because it has excellent amplification efficiency for a very small amount of nucleic acid and can obtain reliable results in a short time. . The polymerase chain reaction is a nucleic acid amplification means commonly used in the art, and the procedure is described in, for example, Science, 230: 1350-1354, 1985.
Etc., and those skilled in the art can appropriately change the experimental conditions and the like based on the description of the above documents and the like. The amplification product can be confirmed by, for example, subjecting the amplification reaction product to electrophoresis under appropriate conditions, and detecting by, for example, ethidium bromide treatment and then irradiating with ultraviolet light, but the detection means is not particularly limited.

According to the above invention, it becomes possible to carry out a chemical reaction requiring heating or cooling on the chemical reaction cartridge, and the range of chemical reactions applicable on the cartridge is expanded. In the present invention, when heating is performed,
The material to be heated must have a mechanical strength and a liquid and / or gas flow path at a set temperature.

[0034]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. The numerical values such as the length, the depth, and the diameter described in the examples and the drawings are merely examples, and the present invention is not limited to these numerical values.

Example 1 A substrate (1) shown in FIG.
It was made of a polycarbonate sheet having a thickness of 5 mm. As shown in FIG. 1, the substrate comprises a 6 mm female screw (101) and a rod-shaped member (5) into which a joining part (made by Pharmacia (currently Amersham Bioscience)) of a tube used for liquid chromatography can be inserted. ) Is inserted and a hole (102) with a diameter of 3 mm having a movable clearance and a hole (103) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can be penetrated are machined.

The substrate (2) shown in FIG. 2 has a length of 50 mm,
It was made of a polycarbonate sheet having a width of 50 mm and a thickness of 2 mm. The substrate, as shown in FIG.
1mm at the position corresponding to the female screw hole (101) of 6mm
mm hole (201), hole (202) with a diameter of 3 mm, which has a clearance into which the rod-shaped member (5) can be inserted and moved.
A groove (204) having a depth of 1 mm and a width of 1 mm connecting these holes is formed as a flow path, and a hole (203) having a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for pressure bonding can be penetrated is processed.

The airtight plate (3) shown in FIG. 3 has a length of 50 m.
It was made of a silicon rubber plate having m, a width of 50 mm, and a thickness of 1 mm. As shown in FIG. 3, the airtight plate is configured so that a hole (301) having a diameter of 1 mm and a rod-shaped member (5) can be moved to a position corresponding to a female screw hole (101) of 6 mm of the substrate 1 as a flow path. 2mm diameter hole (302) with a clearance to maintain airtightness, and a stainless steel 2mm pan head screw used for crimping when the rod-shaped member has a notch-free portion. A hole (303) with a diameter of 2 mm is machined so that

The airtight plate (4) shown in FIG. 4 has a length of 50 m.
It was made of a silicon rubber plate having m, a width of 50 mm, and a thickness of 1 mm. The airtight plate is, as shown in FIG. 4, a rod-shaped member (5).
When the rod-shaped member is movably inserted and the notch-free portion of the rod-shaped member is inserted, a hole (40 mm in diameter having a clearance that maintains airtightness)
2) A hole (403) having a diameter of 2 mm is formed so that a 2 mm pan head screw made of stainless steel used for crimping can be penetrated.

The rod-shaped member (5) shown in FIG. 5 has a diameter of 3 m.
It was made from a Teflon (registered trademark) rod having a length of m and a length of 20 mm. As shown in the figure, the rod-shaped member has a notch (501) having a width of 2 mm and a depth of 1 mm near the center of the rod.
Is being processed. Assemble these parts with a stainless steel pan head screw (diameter 2 mm, length 20 mm) and nut (6)
Then, they were pressure-bonded in a laminated form to prepare the cartridge shown in FIG.

Teflon tubes (inner diameter: 0.9 mm, outer diameter: 1.7 mm) were connected to both sides of the cartridge by using a joint part (manufactured by Pharmacia) of a tube used for liquid chromatography, and further, to this cartridge. One end of the tube was connected to an air pump. Air whose maximum pressure was adjusted to 0.1 atm was supplied to this cartridge at a flow rate of 10 ml / min. As a result, no air leakage was confirmed from the flow path of this cartridge, and the movement of the rod-shaped member (5) achieved the open and closed states of the flow path inside the cartridge. That is, the cutout portion (501) of the rod-shaped member is
When the rod-shaped member is moved to be positioned so as to communicate with the flow path formed by one airtight plate 4, the valve is opened and the cutout portion (501) has two When the substrate 2 was moved to the substrate 1 so as not to communicate the flow path formed by the single airtight plate 4, the valve was closed.

Therefore, it was confirmed that this cartridge functions as a flow path provided with a valve that can be opened and closed. Since the airtight plates (3, 4) have elasticity as compared with the substrates (1, 2), the airtight plates (3, 4) are mainly affected by the pressure of the screws and the nuts (6). Airtight plate because it deforms a little
It is possible to adjust the degree of close contact between the substrates (3, 4) and the substrates (1, 2) and the degree of clearance between the rod-shaped member (5) and the airtight plate (3, 4). By this adjustment, it is possible to adjust the airtightness of the flow path and the valve portion on the cartridge.

Example 2 The substrate (7) shown in FIG.
It was made of a polycarbonate sheet having a thickness of 5 mm. As shown in FIG. 7, the substrate is penetrated by a 6 mm female screw (701) into which a joining part (made by Pharmacia) of a tube used for liquid chromatography is inserted, and a 2 mm pan head screw made of stainless steel used for crimping. Diameter 2 as possible
mm holes (702) are machined.

The substrate (8) shown in FIG. 8 has a length of 50 mm,
It was made of a polycarbonate sheet having a width of 50 mm and a thickness of 2 mm. As shown in FIG. 8, the substrate serves as a substrate 9 as a channel.
1 mm diameter at the position corresponding to the female screw hole (901) of 6 mm
mm hole (801), 2m made of stainless steel used for crimping
hole with a diameter of 2 mm (80 mm
2), two holes (803) with a diameter of 1 mm as flow paths,
And two channels with a depth of 1 mm and a width of 1 mm (8
04) has been processed.

The substrate (9) shown in FIG. 9 has a length of 50 mm,
It was made of a polycarbonate sheet having a width of 50 mm and a thickness of 5 mm. As shown in FIG. 9, the substrate is penetrated by a 6 mm female screw (901) into which a joining part (made by Pharmacia) of a tube used for liquid chromatography can be inserted and a 2 mm pan head screw made of stainless steel used for crimping. A hole (902) having a diameter of 2 mm, a rod-shaped member (12) having a movable clearance, and a hole (903) having a diameter of 3 mm and a groove (904) having a width of 3 mm and a depth of 2 mm connected to the hole. Is being processed.

The airtight plate (10) shown in FIG.
A silicon rubber plate having a size of 50 mm, a width of 50 mm, and a thickness of 1 mm was used. As shown in FIG. 10, 6 m of the substrate 7 is used as a flow path.
m hole (1001) having a diameter of 1 mm at a position corresponding to the female screw hole (701) of m, and a hole having a diameter of 2 mm (100 mm) through which a 2 mm pan head screw made of stainless steel used for crimping can pass.
2) is processed.

The airtight plate (11) shown in FIG.
A silicon rubber plate having a size of 50 mm, a width of 50 mm, and a thickness of 1 mm was used. As shown in FIG. 11, the airtight plate has a hole (1101) with a diameter of 1 mm at a position corresponding to a female screw hole (901) of 6 mm as a flow path, and a 2 mm pan head screw made of stainless steel used for crimping. A hole (1102) having a diameter of 2 mm is machined so that it can penetrate therethrough.

The rod-shaped member (12) shown in FIG. 12 was made of a Teflon rod having a diameter of 3 mm and a length of 20 mm. These parts are made of stainless steel pan head screw (diameter 2 mm, length 2
(0 mm) and nuts (13) are crimped in a laminated shape,
The cartridge shown in No. 3 was prepared. Teflon tubes (inner diameter: 0.9 mm, outer diameter: 1.7 mm) were connected to both sides of the cartridge by using a joining part of a tube used in liquid chromatography (Pharmacia), and further, to the cartridge. One tip was connected to an air pump. Air whose maximum pressure was adjusted to 0.1 atm was supplied to this cartridge at a flow rate of 10 ml / min. As a result, no air leakage was confirmed in the flow path of this cartridge, and the open and closed states of the flow path in the cartridge were achieved depending on the presence or absence of the force that pushes the rod-shaped member (12) toward the flow path. . Therefore, it was confirmed that this cartridge functions as a flow path provided with a valve that can be opened and closed. That is, in the case where this valve has no pushing force against the rod-shaped member, the portion of the airtight plate 11 that is in contact with the groove (904) of the substrate 9 is directed in the direction of the groove by the pressure of air flowing in the flow path. When the rod-shaped member is pushed in the flow direction, the air-tight plate 11 is pressed against the substrate 8 to secure the flow path. It was closed and closed. Further, since the airtight plates (10, 11) have elasticity as compared with the substrates (7, 8, 9), mainly the airtight plates (10, 11) are adjusted by adjusting the pressure force applied by the screws and the nuts (13). ) Is slightly deformed, it is possible to adjust the degree of close contact between the airtight plate (10, 11) and the substrate (7, 8, 9). By this adjustment, it is possible to adjust the airtightness of the flow path and the valve portion on the cartridge.

Example 3 The substrate (14) shown in FIG.
It was made of a polycarbonate sheet having a thickness of 5 mm and a thickness of 5 mm. As shown in FIG. 14, the substrate is penetrated by a 6 mm female screw (1401) into which a joining part (made by Pharmacia) of a tube used for liquid chromatography can be inserted and a 2 mm pan head screw made of stainless steel used for crimping. A hole with a diameter of 2 mm (1402) that can be inserted, a hole with a diameter of 4 mm that allows a plastic syringe (made by Terumo) to be inserted (1
403), a hole (1404) with a diameter of 2 mm having a movable clearance into which the rod-shaped member (21) is inserted, and an elliptical hole (2) with which two cylindrical magnets with a diameter of 5 mm and a length of 4 mm for trapping can be arranged side by side ( 1405) is processed.

The substrate (15) shown in FIG.
mm, width 150 mm, thickness 2 mm polycarbonate plate. As shown in FIG. 15, the substrate has a hole (1502) having a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can pass, and a hole having a diameter of 2 mm having a movable clearance into which a rod-shaped member (21) is inserted. (15
04), as a flow channel, a groove (15 mm in width and 1 mm in depth)
03), a hole (1501) having a diameter of 2 mm is formed as a flow path, and an elliptical groove (1505) having a depth of 1 mm is formed as a trap portion.

The substrate (16) shown in FIG.
mm, width 150 mm, thickness 10 mm made of a polycarbonate sheet. As shown in FIG. 16, the substrate has a hole (1602) having a diameter of 2 mm and a rod-like member (21) through which a 2 mm pan head screw made of stainless steel used for crimping can pass.
2mm in diameter (1604) having a clearance that allows the insertion of a hole, a channel having a width of 1mm and a depth of 1mm (1603), and a channel having a diameter of 2mm (1604).
1), pentagonal hole that functions as a reaction tank (160
6), a hole (1607) having a diameter of 5 mm is processed as a sample introduction port to the reaction tank. Furthermore, a female screw of 6 mm is processed at the open portion of the sample introduction port.

The substrate (17) shown in FIG.
mm, width 150 mm, thickness 2 mm polycarbonate plate. As shown in FIG. 17, the substrate has a hole (1702) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can pass, and a diameter of 2 mm with a movable clearance into which a rod-shaped member (21) is inserted. The hole (1704) and the groove (1703) having a width of 1 mm and a depth of 1 mm are processed as the flow path.

The airtight plate (18) shown in FIG.
It was made of a silicon rubber plate having a thickness of 150 mm, a width of 150 mm, and a thickness of 1 mm. As shown in FIG. 18, the airtight plate has a hole (1801) with a diameter of 2 mm as a flow path, and a diameter of 2 mm so that a 2 mm pan head screw made of stainless steel used for crimping can be penetrated.
The hole (1802) and the rod-shaped member (21) have a movable clearance, but when the portion of the rod-shaped member having a diameter of 2 mm is inserted, a hole having a diameter of 2 mm ( 1804), an elliptical hole (1805) into which two cylindrical magnets having a diameter of 5 mm and a length of 4 mm can be arranged and inserted.

The airtight plate (19) shown in FIG.
It was made of a silicon rubber plate having a thickness of 0 mm, a width of 150 mm, and a thickness of 1 mm. As shown in FIG. 19, the airtight plate has a hole (1901) having a diameter of 2 mm as a flow path and a diameter of 2 m so that a 2 mm pan head screw made of stainless steel used for pressure bonding can be penetrated.
A hole (1902) having a diameter of 2 mm and a rod-shaped member (21) having a movable clearance are inserted, but a hole having a diameter of 2 mm is kept airtight when a portion having a diameter of 2 mm of the rod-shaped member is inserted. (1904), the substrate 1 as a trap portion
An elliptical hole (1905) and a pentagonal hole (1906) functioning as a reaction tank are formed at positions corresponding to the positions of the elliptical groove (1505) of No. 5 in FIG.

The airtight plate (20) shown in FIG.
It was made of a silicon rubber plate having a thickness of 0 mm, a width of 150 mm, and a thickness of 1 mm. As shown in FIG. 20, the airtight plate has a hole (2002) having a diameter of 2 mm and a rod member (21) into which a stainless steel 2 mm pan head screw used for crimping can penetrate, and has a movable clearance. In the case where a 2 mm diameter portion of the rod-shaped member is inserted, a 2 mm diameter hole (2004) that maintains airtightness and a pentagonal hole (2006) that functions as a reaction tank are processed.

The rod-shaped member (21) shown in FIG. 21 was made of a stainless rod having a diameter of 2 mm and a length of 27 mm. As shown in the figure, the rod-shaped member has a central portion (210
1) is processed to have a diameter of 1 mm over a width of 2 mm in the length direction. These parts were pressure-bonded in a laminated form with a set of a stainless steel pan head screw (diameter: 2 mm, length: 20 mm) and a nut to produce the cartridge shown in FIG.

Reservoir 1 (R
As 1) 900 μl of free adsorption solution (20 mM EDT
A, a plastic syringe containing 50 mM Tris-HCl buffer containing 5.25 M guanidine thiocyanate (pH 6.4) containing 1.3% Triton X-100 was used as a reservoir 2 (R2). (70% ethanol) was injected into a plastic injection cylinder as a reservoir 3 (R3), and 1000 μl of a washing solution (99% ethanol) was injected into a plastic injection cylinder, and as a reservoir 4 (R4), 500 μl of an eluate (distilled water). ) Was injected into each of the plastic syringes. In the cartridge, the rod-shaped member (21) installed in each valve (V1 to V11) is moved by the pushing force from the outside, and the opening / closing operation of the flow path of the valve portion is possible. Further, it is possible to install and remove magnets (two cylindrical magnets having a diameter of 5 mm and a length of 4 mm) in the trap portions (M1 to M3). Teflon tubes (inner diameter 0.9 mm, outer diameter 1.7 mm) were used for joining ports (P1, P2, P3) with the pump part, using joining parts (made by Pharmacia) of tubes used in liquid chromatography. Is further joined, and further, an air pump using a syringe pump (P-6000, Pharmacia Co., Ltd.) is further joined, and liquid feeding by this air feeding is possible. Further, the lower portion of the cartridge including the free adsorption reaction tank (R5) can be heated by a heating device (H1) using a hot water bath.

Using this cartridge, the following procedure
The bacterial nucleic acid was extracted and purified from Escherichia coli. First of all,
The valves (V1 to V11) are closed, and the trap portion (M1 to
A magnet was installed in M3). Next, the free adsorption reaction tank (R
In 5), the number of bacterial cells is 10 ⁷Escherichia coli (Escheric
hia coli) and magnetic silica particles (Toyobo) suspension 4
0 μl was added. After introduction of bacterial cells, free adsorption reaction tank (R5)
The sample inlet (S1) at the top of the
Joining parts (fabs) for tubes used in
(Lumasia). Free adsorption reaction tank (R5)
The lower part (H1) of the cartridge containing
Heated. After opening valve 3 (V3),
1 (R1) free adsorbent is led to the reaction tank (R5)
It was After 3 minutes, turn valves 1, 4, 6 (V1, V4, V6)
Open, close valve 3 (V3), and connect to pump 1
Air is sent from (P1) and the free adsorption reaction tank (R5)
The liquid was discharged to the waste liquid port 1 (W1). Tiger at this stage
Magnetic silica particles were accumulated in the area of the top 1 (M1).
Valve 1 (V1), valve 6 (V6) are closed, valve 2
(V2), valve 7 (V7), valve 8 (V8), valve
Open the valve 10 (V10) and trap 1 (M1)
After removing the magnet of the above, wash the reservoir 2 (R2)
The liquid was poured. Furthermore, open the valve 2 (V2) and connect with the pump.
Air is sent from the joint port 2 (P2) of the
The liquid remaining in the tank (R5) is drained to the drain port 2 (W2).
At the same time, disperse and wash the magnetic silica particles.
It was integrated again in the part of the top 2 (M2). Valve 4 (V
4) Close and remove the magnet in the part of trap 2 (M2)
I was there. Flow the cleaning liquid from the reservoir 3 (R3) to the waste liquid outlet 2
Disperse the magnetic silica particles at the same time as waste liquid in (W2).
And wash, and then collect again on the trap 3 (M3) part.
It was Close valve 8 (V8) and open valve 5 (V5)
50 ml from the air pump joint (P3) for 10 minutes
/ Min of air is sent and remains on the magnetic silica particles
The ethanol was dried. Valve 5 (V5), valve 10
(V10) is closed, valve 9 (V9), valve 11 (V
11) Open and remove the magnet in the trap 3 (M3) part.
I removed it. Flow the eluate from reservoir 4 (R4) to make it magnetic.
While dispersing the silica particles, melt through the recovery port 3 (W3).
The effluent was collected.

Next, D derived from E. coli was added to the recovered eluate.
It was confirmed that NA was purified by the following method.
5 μl of the collected eluate was added to a polymerase chain reaction microtube (Multi Ultra PCR Tube, Sorenson
BioScience) and two kinds of oligonucleic acid (a gene encoding the Escherichia coli ribosomal protein L25) from the nucleotide sequence 449th c to the 472nd t (SEQ ID NO: 1) and the nucleotide sequence 628 of the same gene. 20 for the sequence of the complementary strand from the a-th to the 650-th a (SEQ ID NO: 2): chemically synthesized by requesting Sigma)
1 μl each of μM solution, substrate (dNTP mixture: TAK)
ARA, dATP, dTTP, dGTP and dCTP each containing 2.5 mM) 2 μl, DNA polymerase solution (Z-Taq, 2.5 U / μl, TAKARA) 0.5
μl, buffer (300 mM Tris-HCl, 17.5 mM
5 μl of magnesium chloride, pH 9.5) was added, and distilled water was further added to make the total volume 25 μl. When the polymerase chain reaction is performed using the oligonucleic acid of SEQ ID NO: 1 and SEQ ID NO: 2 as a primer, an amplified product of about 200 bp will be obtained in a sample containing purified E. coli-derived DNA. .

20 μl of mineral oil (manufactured by Sigma) was overlaid, and a polymerase chain reaction of 40 cycles of 98 ° C. for 20 seconds and 65 ° C. for 30 seconds was performed in a thermocycler (RoboCycler: Stratagene). After that, 1 μl of sample treatment solution (10 × Loading Buffer: TAKARA) was added to 10 μl of polymerase chain reaction solution, which was added to 3% agarose gel (NuSieve3: 1 agarose).
Was subjected to electrophoresis (Mupid: manufactured by ADVANCE) by TAE (Tris / acetic acid / EDTA) buffer solution and solidified. After electrophoresis, the agarose gel was immersed in an ethidium bromide aqueous solution (1 μg / ml) for 15 minutes. Next, the agarose gel was taken out, exposed to ultraviolet rays, and electrophoresed DNA.
Was detected. As a result, as shown in FIG. 23, the DNA amplified by the polymerase chain reaction at the same position as 200 bp of the marker DNA (22).
Was detected as a band (23) having a size expected from the nucleotide sequence. Therefore, it was shown that the E. coli DNA was isolated by the cartridge of the present invention.

Example 4 The substrate (24) shown in FIG.
It was made of a polycarbonate sheet having a thickness of 5 mm and a thickness of 5 mm. As shown in FIG. 24, the substrate is penetrated by a 6 mm female screw (2401) into which a tube joint part (Pharmacia) used for liquid chromatography is inserted, and a stainless steel 2 mm pan head screw used for crimping. 2mm diameter hole (2402) that can be used, 4mm diameter hole (2 that can be inserted with a plastic syringe (made by Terumo))
403), a hole (2404) with a diameter of 2 mm having a movable clearance into which the rod-shaped member (21) is inserted, the substrate 2
Rectangular holes (2405) having clearances into which 8, 29 and airtight plates 33, 34 can be inserted are machined.

The substrate (25) shown in FIG.
mm, width 150 mm, thickness 2 mm polycarbonate plate. As shown in FIG. 25, the substrate has a hole (2502) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can penetrate, and a diameter of 2 m as a flow path.
m hole (2501), width of flow passage is 1mm, depth is 1mm
Groove (2503), a hole (2504) having a diameter of 2 mm and having a movable clearance into which the rod-shaped member (21) is inserted,
Rectangular holes (2505) having a clearance into which the substrates 28 and 29 and the airtight plates 33 and 34 can be inserted are processed.

The substrate (26) shown in FIG.
mm, width 150 mm, thickness 10 mm made of a polycarbonate sheet. As shown in FIG. 26, the substrate has a hole (2602) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can penetrate, and a diameter of 2 mm as a flow path.
mm hole (2601), width of flow path is 1 mm, depth is 1 m
m groove (2603), a rod-shaped member (21) is inserted, and a hole (260) with a diameter of 2 mm having a movable clearance is provided.
4), a pentagonal hole (260 that functions as a reaction tank)
6), a hole (2607) having a diameter of 5 mm is formed as a sample introduction port to the reaction tank. Furthermore, a female screw of 6 mm is processed at the open portion of the sample introduction port.

The substrate (27) shown in FIG.
mm, width 150 mm, thickness 2 mm polycarbonate plate. As shown in FIG. 27, the substrate has a hole (2702) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can pass, and a diameter of 2 m as a flow path.
m hole (2701), width 1mm, depth 1mm
The groove (2703) and the rod-shaped member (21) are inserted to form a hole (2704) having a diameter of 2 mm and having a movable clearance.

The substrate (28) shown in FIG.
It was made of a polycarbonate sheet of m, width 30 mm, and thickness 5 mm. As shown in FIG. 28, the substrate has a hole (2802) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can pass, a hole (2801) with a diameter of 2 mm as a flow path, and a width of 1 mm as a flow path. , A groove (2803) having a depth of 1 mm is processed.

The substrate (29) shown in FIG. 29 has a length of 20 m.
It was made of a polycarbonate sheet of m, width 30 mm, and thickness 2 mm. As shown in FIG. 29, the substrate has a hole (2902) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can pass, a hole (2901) with a diameter of 2 mm as a flow path, and a silica film as a trap portion. Has a hole (2905) with a diameter of 8 mm that can be installed.

The airtight plate (30) shown in FIG.
It was made of a silicon rubber plate having a thickness of 150 mm, a width of 150 mm, and a thickness of 1 mm. As shown in FIG. 30, the airtight plate has a movable clearance in which a hole (3002) having a diameter of 2 mm and a rod-shaped member (21) through which a 2 mm pan head screw made of stainless steel used for crimping can pass can be inserted. , A hole (3004) having a diameter of 2 mm so that airtightness is maintained when a portion having a diameter of 2 mm of the rod-shaped member is inserted, the substrates 28, 2
9, a rectangular hole (3005) having a clearance into which the airtight plates 33 and 34 can be inserted, and a hole (3001) having a diameter of 2 mm is formed as a flow path.

The airtight plate (31) shown in FIG.
It was made of a silicon rubber plate having a thickness of 0 mm, a width of 150 mm, and a thickness of 1 mm. As shown in FIG. 31, the airtight plate has a hole (3102) having a diameter of 2 mm and a rod-shaped member (21) into which a stainless steel 2 mm pan head screw used for crimping can penetrate, and has a movable clearance. , A hole (3104) having a diameter of 2 mm, which is kept airtight when a portion having a diameter of 2 mm of the rod-shaped member is inserted, the substrates 28, 2
9, and a rectangular hole (3105) having a clearance into which the airtight plates 33 and 34 can be inserted, a pentagonal hole (3106) functioning as a reaction tank, and a hole (3101) having a diameter of 2 mm as a flow path. .

The airtight plate (32) shown in FIG.
It was made of a silicon rubber plate having a thickness of 0 mm, a width of 150 mm, and a thickness of 1 mm. As shown in FIG. 32, the airtight plate has a hole (3202) having a diameter of 2 mm through which a pan head screw of 2 mm made of stainless steel used for crimping and a rod-shaped member (21) are inserted and has a movable clearance. When a rod-shaped member having a diameter of 2 mm is inserted, a hole having a diameter of 2 mm (3204) that maintains airtightness, a pentagonal hole (3206) that functions as a reaction tank, and a diameter of 2 as a flow path
mm holes (3201) are machined.

The airtight plate (33) shown in FIG.
It was made of a silicon rubber plate having a size of 30 mm, a width of 30 mm, and a thickness of 1 mm. As shown in FIG. 33, the airtight plate has a hole (3302) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can pass, and a hole (3301) with a diameter of 2 mm is formed as a flow path. .

The airtight plate (34) shown in FIG.
It was made of a silicon rubber plate having a size of 30 mm, a width of 30 mm, and a thickness of 1 mm. As shown in FIG. 34, the airtight plate has a hole (3402) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can pass, a hole (3401) with a diameter of 2 mm as a flow path, and silica as a trap portion. A 6 mm diameter hole (3405) is machined so that the membrane can be installed and the flow path can be kept airtight. The same rod-shaped member (21) as those used in Example 3 and Example 3, a stainless steel pan head screw (diameter 2 mm, length 25 mm) and a set of nuts were assembled into a cartridge shown in FIG.

In the above cartridge, the reservoir 11 (R
11) 900 μl of free adsorbent (20 mM ED)
TA, 1.3% Triton X-100, 5.25M
Of 50 mM Tris-hydrochloric acid buffer containing guanidine thiocyanate, pH 6.4), and a plastic syringe into which 1000 μl of a washing solution (70% ethanol) was injected as a reservoir 12 (R12). 1000μ as the reservoir 13 (R13)
500 μl of plastic injection cylinder filled with 1 liter of cleaning solution (99% ethanol) as reservoir 14 (R14)
Plastic injection cylinders filled with 1 l of eluate (distilled water) were attached. In addition, a silica film (made by Qiagen, DNeasyR) is provided on the trap (M11).
Used from the Tissue Kit for use, diameter of about 8
mm, thickness about 2 mm). The cartridge has a valve (V21 to V32) that is pushed by an external force.
The rod-shaped member (21) installed at the position moves to enable the opening / closing operation of the flow path of the valve portion. In addition, a joint part of a tube used for liquid chromatography (manufactured by Pharmacia) at the joint port (P11, P12) with the pump part
Teflon tube (inner diameter 0.9m)
m, outer diameter 1.7 mm) are joined, and further an air pump using a syringe pump (P-6000, Pharmacia Co., Ltd.) is joined to the end of the joint to enable liquid feeding by this air feeding. Further, the lower part of the cartridge including the free adsorption reaction tank (R15) can be heated by a heating device (H11) using a hot water bath.

Using this cartridge, the following procedure
The bacterial nucleic acid was extracted and purified from Escherichia coli. First, all the valves (V21 to V32) were closed. Next, in the free adsorption reaction tank (R15), 10 ⁷ E. coli (Escherichia coli) was placed as the number of bacterial cells, and the sample introduction port (S11) at the upper part of the free adsorption reaction tank had a diameter of 6 mm and a length of 10 mm. Closed using the nut. The lower part (H11) of the cartridge containing the free adsorption reactor (R15) was heated to 98 ° C in a water bath. After opening the valves 22 and 26 (V22 and 26), the free adsorbed liquid in the reservoir 11 (R11) was introduced into the reaction tank (R15). After 3 minutes, valves 21, 2
Open valves 7, 32 (V21, V27, V32) and open valve 2
6 (V26) is closed and the connection port 11 (P11) with the pump
The air in the reaction tank (R15) was discharged to the waste liquid port 11 (W11) by supplying air from the above. At this stage, the nucleic acid released from E. coli was adsorbed on the silica membrane of the trap (M11). The valve 22 (V22) is closed and the valve 24 (V2
4) was opened and the washing liquid for the reservoir 12 (R12) was flown. Further, the valve 23 (V23) was opened, air was supplied from P11, and the liquid remaining in the reaction tank (R15) was discharged to the waste liquid port 11 (W11). Valve 27 (V2
7) was closed, the valve 28 (V28) was opened, and the cleaning solution for the reservoir 13 (R13) was flown. In addition, the valve 25
(V25) was opened, and air was sent from P11 to drain the liquid in the flow path to the waste liquid port 11 (W11). Valve 28
(V28) was closed, the valve 30 (V30) was opened, and 50 ml / min of air was supplied from the air pump joint port 12 (P12) for 10 minutes to dry the ethanol remaining on the silica film. The valves 30, 32 (V30, 32) are closed, the valves 29, 31 (V29, 31) are opened, and the eluate of the reservoir 14 (R14) is caused to flow to collect the recovery port 12 (W1.
The eluate was collected from 2).

Next, in the recovered eluate, E. coli-derived D
It was confirmed that NA was purified by the same method as in Example 3. As a result, as shown in FIG. 36, the DNA amplified by the polymerase chain reaction at the same position as 200 bp of the marker DNA (35).
Was detected as a band (36) of the expected size from the nucleotide sequence. Therefore, it was shown that the E. coli DNA was isolated by the cartridge of the present invention.

Example 5 The substrate (37) shown in FIG. 37 was used in a length of 20 mm and a width of 20 m.
It was made of a polycarbonate sheet having a thickness of 5 mm and a thickness of 5 mm. As shown in FIG. 37, the substrate is a joint part of a tube used in liquid chromatography (manufactured by Pharmacia).
A 6 mm female screw (3701) into which a hole is inserted and a 2 mm diameter hole (3702) through which a stainless steel 2 mm pan head screw used for crimping can pass are machined.

The substrate (38) shown in FIG. 38 has a length of 150.
mm, width 155 mm, thickness 2 mm made of polycarbonate sheet. As shown in FIG. 38, the substrate has a hole (3802) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can penetrate, and a 1.6 mm pan head screw made of stainless steel used for crimping can penetrate. A hole (3803) with a diameter of 1.6 mm and a width of 0.5 m as a flow path
m, depth 1 mm groove (3804), diameter 1 m as a flow path
m holes (3805) are machined.

The substrate (39) shown in FIG. 39 has a length of 150.
mm, width 155 mm, thickness 2 mm made of polycarbonate sheet. As shown in FIG. 39, the substrate has a hole (3902) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can be penetrated, and a 1.6 mm pan head screw made of stainless steel used for crimping can be penetrated. Hole (3903) with a diameter of 1.6 mm, width of 0.5 m as a flow path
m, depth 1 mm groove (3904), diameter 1 m as a flow path
m holes (3905) are machined.

The airtight plate (40) shown in FIG.
A Teflon plate having a size of 20 mm, a width of 20 mm, and a thickness of 1 mm was used. As shown in FIG. 40, the airtight plate has a hole (4002) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can pass, and a hole (4001) with a diameter of 2 mm is formed as a flow path. .

The airtight plate (41) shown in FIG.
It was made from a Teflon plate having a thickness of 0 mm, a width of 155 mm, and a thickness of 1 mm. As shown in FIG. 41, the airtight plate has a hole (4102) with a diameter of 2 mm through which a 2 mm pan head screw made of stainless steel used for crimping can penetrate, and a 1.6 mm pan head screw made of stainless steel used for crimping can penetrate. Diameter 1.6
mm hole (4103), 1 mm diameter hole (4
101) is processed.

These parts and screws (diameter 2 mm, length 2
0 mm, diameter 1.6 mm, length 8 mm) and nut (M
2, M1.6) was used to assemble the cartridge shown in FIG. Next, two cartridges shown in FIG. 42 are combined so that the reciprocating reaction between the two temperatures in the polymerase chain reaction can be performed 40 times, and a joining part of a tube used in liquid chromatography (Pharmacia) ( 42), Teflon tube (inner diameter 0.5 mm, outer diameter 1.7 mm) (43), two-way conversion valve (Pharmacia) (44), pump (P-50)
No. 0, manufactured by Pharmacia Co., Ltd. (45) and an injection cylinder (46) were joined together to produce the device shown in FIG. The cartridge part is heated to 92 ° C and 6 ° C by the heating device (47, 48).
It is designed to be heated to two temperatures of 5 ° C.

Of the sample obtained by purifying the Escherichia coli-derived nucleic acid according to Example 4, 50 μl was added to two 20 μM oligonucleic acids (the gene encoding the Escherichia coli ribosomal protein L25, the nucleotide sequence from the 449th nucleotide to the 472nd nucleotide sequence). Sequence up to t (SEQ ID NO: 1), base sequence 62 of the same gene
Sequence of complementary strand from 8th a to 650th a (SEQ ID NO: 2): 10 μl each for chemically synthesized by requesting from Sigma), substrate (dNTP mixture: T)
AKARA, dATP, dTTP, dGTP, dC
20 μl each containing 2.5 mM TP), DNA polymerase solution (Z-Taq, 2.5 U / μl, TAKAR
A, 25 μl, buffer (2.5 W / V% BSA,
0.5% Triton-X100, 300 mM Tris-
Hydrochloric acid, 17.5 mM magnesium chloride, pH 9.5) 5
Add 0 μl, and then add distilled water to bring the total volume to 250 μl.
set to l. This sample was injected into the syringe barrel (46).

Using the pump (45), the flow path of the cartridge was previously filled with mineral oil (manufactured by Sigma).
After rotating the valve (44), the sample of the injection cylinder (46) was introduced into the cartridge, and then the polymerase chain reaction was carried out using the oligonucleic acids of SEQ ID NO: 1 and SEQ ID NO: 2 as primers. If a reaction occurs, about 2
An amplification product of 00 bp will be obtained.

Next, the valve (44) was rotated, the mineral oil was fed from the pump (45) into the cartridge at a flow rate of 9 ml / hour, and the reaction liquid was recovered from the recovery port (49).
Agarose gel electrophoresis was performed according to Example 4, and the amplified nucleic acid in the reaction solution was detected. As a result, FIG.
As shown in, 200b of the marker DNA (50)
The DNA amplified by this polymerase chain reaction was detected as a band (51) having a size expected from the nucleotide sequence at approximately the same position as p. Therefore, it was found that the cartridge of the present invention achieved the nucleic acid amplification reaction.

[0083]

The chemical reaction cartridge according to the present invention can be manufactured at low cost. In addition, a chemical reaction can be performed therein, and medical diagnosis, chemical analysis, food analysis, etc. can be easily achieved. Particularly in the field of medical diagnosis, it is possible to quickly and easily achieve a chemical reaction in a cartridge, and to perform dangerous substances that may be present in a medical sample manually as in normal diagnosis. It is useful because it eliminates the need and reduces the risk of infection. Further, irreversible processing due to adhesion can be avoided, and by disassembling and washing, it can be reused and waste can be reduced.

[0084]

[Sequence list] <110> Asahi Kasei Corporation <120> A Cartridge for Chemical Reaction <130> X13-1109 <160> 2 <210> 1 <211> 24 <212> DNA <213> Escherichia Coli <400> 1 ctaacaagtt cccggcaatc atct 24 <210> 2 <211> 23 <212> DNA <213> Escherichia Coli <400> 2 tcgatgtgct gcagcttcgg ttt 23

[Brief description of drawings]

FIG. 1 shows a plan view and a sectional view of a substrate (1) which constitutes a cartridge of the present invention.

FIG. 2 shows a plan view and a sectional view of a substrate (2) which constitutes a cartridge of the present invention.

FIG. 3 is an airtight plate (3) constituting the cartridge of the present invention.
The top view and sectional drawing of are shown.

FIG. 4 is an airtight plate (4) constituting the cartridge of the present invention.
The top view and sectional drawing of are shown.

FIG. 5 shows a plan view and a sectional view of a rod-shaped member (5) constituting the cartridge of the present invention.

FIG. 6 shows a plan view and a sectional view of a cartridge of the present invention.

FIG. 7 shows a plan view and a sectional view of a substrate (7) which constitutes the cartridge of the present invention.

FIG. 8 shows a plan view and a sectional view of a substrate (8) constituting the cartridge of the present invention.

FIG. 9 shows a plan view and a sectional view of a substrate (9) constituting the cartridge of the present invention.

FIG. 10 is an airtight plate (1 which constitutes the cartridge of the present invention.
0) shows a plan view and a sectional view.

FIG. 11 is an airtight plate (1 which constitutes the cartridge of the present invention.
The top view and sectional drawing of 1) are shown.

FIG. 12 shows a plan view and a sectional view of a rod-shaped member (12) constituting the cartridge of the present invention.

FIG. 13 shows a plan view and a sectional view of a cartridge of the present invention.

FIG. 14 shows a plan view and a sectional view of a substrate (14) which constitutes a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 15 shows a plan view and a sectional view of a substrate (15) which constitutes a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 16 shows a plan view and a cross-sectional view of a substrate (16) constituting a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 17 shows a plan view and a cross-sectional view of a substrate (17) constituting a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 18 shows a plan view and a sectional view of an airtight plate (18) which constitutes a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 19 shows a plan view and a cross-sectional view of an airtight plate (19) constituting a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 20 shows a plan view and a cross-sectional view of an airtight plate (20) constituting a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 21 shows a plan view and a side view of a rod-shaped member (21) constituting a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 22 shows a plan view and a cross-sectional view of a chemical reaction cartridge for nucleic acid isolation of the present invention.

23 shows a photographic print of agarose gel electrophoresis showing the results of Example 3. FIG.

FIG. 24 shows a plan view and a cross-sectional view of a substrate (24) which constitutes a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 25 shows a plan view and a cross-sectional view of a substrate (25) constituting a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 26 shows a plan view and a cross-sectional view of a substrate (26) which constitutes a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 27 shows a plan view and a sectional view of a substrate (27) which constitutes a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 28 shows a plan view and a cross-sectional view of a substrate (28) constituting a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 29 shows a plan view and a sectional view of a substrate (29) constituting a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 30 shows a plan view and a sectional view of an airtight plate (30) which constitutes a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 31 shows a plan view and a sectional view of an airtight plate (31) which constitutes a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 32 shows a plan view and a cross-sectional view of an airtight plate (32) constituting a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 33 shows a plan view and a sectional view of an airtight plate (33) which constitutes a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 34 shows a plan view and a cross-sectional view of an airtight plate (34) which constitutes the chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 35 shows a plan view and a sectional view of a chemical reaction cartridge for nucleic acid isolation of the present invention.

FIG. 36 shows the results of agarose gel electrophoresis in Example 4.

FIG. 37 shows a plan view and a sectional view of a substrate (37) which constitutes a chemical reaction cartridge for carrying out a nucleic acid amplification reaction of the present invention.

FIG. 38 shows a plan view and a sectional view of a substrate (38) constituting a chemical reaction cartridge for carrying out a nucleic acid amplification reaction of the present invention.

FIG. 39 shows a plan view and a sectional view of a substrate (39) constituting a chemical reaction cartridge for carrying out a nucleic acid amplification reaction of the present invention.

FIG. 40 shows a plan view and a cross-sectional view of an airtight plate (40) constituting a chemical reaction cartridge for carrying out a nucleic acid amplification reaction of the present invention.

FIG. 41 shows a plan view and a cross-sectional view of an airtight plate (41) constituting a chemical reaction cartridge for carrying out a nucleic acid amplification reaction of the present invention.

FIG. 42 shows a plan view and a sectional view of a chemical reaction cartridge for carrying out a nucleic acid amplification reaction of the present invention.

FIG. 43 is a conceptual diagram showing the configuration of an apparatus for carrying out an amplification reaction using a chemical reaction cartridge for carrying out a nucleic acid amplification reaction of the present invention.

FIG. 44 shows the results of agarose gel electrophoresis in Example 5.

[Explanation of symbols]

1: substrate, 2: substrate, 3: airtight plate, 4: airtight plate, 5: rod-shaped member, 6: set of screw and nut 7: substrate, 8: substrate, 9: substrate, 10: airtight plate, 11:
Airtight plate, 12: bar-shaped member, 13: set of screw and nut 14: substrate, 15: substrate, 16: substrate, 17: substrate, 1
8: Airtight plate, 19: Airtight plate, 20: Airtight plate, 21: Rod-shaped member, 22: DNA molecular weight marker, 23: Amplified DNA fragment, 24: Substrate, 25: Substrate, 26: Substrate, 2
7: substrate, 28: substrate, 29: substrate, 30: airtight plate, 3
1: Airtight plate, 32: Airtight plate, 33: Airtight plate, 34: Airtight plate, 35: DNA molecular weight marker, 36: Amplified D
NA fragment, 37: substrate, 38: substrate, 39: substrate, 4
0: Airtight plate, 41: Airtight plate, 42: Joined parts, 43: Teflon tube, 44: Valve, 45: Pump, 46:
Syringe, 47: Heating device, 48: Heating device, 49: Collection port, 50: DNA molecular weight marker, 51: Amplified D
NA fragment, 101: female screw, 102: hole, 103: hole,
201: hole, 202: hole, 203: hole, 204: groove, 3
01: hole, 302: hole, 303: hole, 402: hole, 40
3: hole, 501: notch, 701: female screw, 702:
Hole, 801: hole, 802: hole, 803: hole, 804:
Groove, 901: internal thread, 902: hole, 903: hole, 90
4: groove, 1001: hole, 1002: hole, 1101: hole,
1102: hole, 1401: female screw, 1402: hole, 14
03: hole, 1404: hole, 1405: hole, 1501:
Hole, 1502: hole, 1503: groove, 1504: hole, 15
05: groove, 1601: hole, 1602: hole, 1603:
Groove, 1604: hole, 1606: hole, 1607: hole, 17
02: hole, 1703: groove, 1704: hole, 1801:
Hole, 1802: hole, 1804: hole, 1805: hole, 19
01: hole, 1902: hole, 1904: hole, 1905:
Hole, 1906: hole, 2002: hole, 2004: hole, 20
06: hole, 2101: central part, 2401: female screw, 2
402: hole, 2403: hole, 2404: hole, 2405:
Hole, 2501: hole, 2502: hole, 2503: groove, 25
04: hole, 2505: hole, 2601: hole, 2602:
Hole, 2603: groove, 2604: hole, 2606: hole, 26
07: hole, 2701: hole, 2702: hole, 2703:
Groove, 2704: hole, 2801: hole, 2802: hole, 28
03: groove, 2901: hole, 2902: hole, 2905:
Hole, 3001: hole, 3002: hole, 3004: hole, 30
05: hole, 3101: hole, 3102: hole, 3104:
Hole, 3105: hole, 3106: hole, 3201: hole, 32
02: hole, 3204: hole, 3206: hole, 3301:
Hole, 3302: hole, 3401: hole, 3402: hole, 34
05: hole, 3701: female screw, 3702: hole, 380
2: hole, 3803: hole, 3804: groove, 3805: hole,
3902: hole, 3903: hole, 3904: groove, 390
5: hole, 4001: hole, 4002: hole, 4101: hole,
4102: hole, 4103: hole, V1: valve 1, V2:
Valve 2, V3: Valve 3, V4: Valve 4, V5: Valve 5, V6: Valve 6, V7: Valve 7, V8: Valve 8, V9: Valve 9, V10: Valve 10, V11:
Valve 11, M1: Trap 1, M2: Trap 2, M
3: Trap 3, P1: Pump junction 1, P2: Pump junction 2, P3: Pump junction 3, R1: Reservoir 1, R2: Reservoir 2, R3: Reservoir 3, R4:
Reservoir 4, R5: free adsorption reaction tank, W1: discharge port 1, W2: discharge port 2, W3: recovery port 3, S1: sample introduction port, H1: heating device V21: valve 21, V22: valve 22, V23: Valve 23, V24: valve 24, V25: valve 25,
V26: valve 26, V27: valve 27, V28: valve 28, V29: valve 29, V30: valve 30,
V31: Valve 31, V32: Valve 32, M11: Trap, P11: Air pump joint port 11, P12: Air pump joint port 12, R11: Reservoir 11, R12:
Reservoir 12, R13: Reservoir 13, R14: Reservoir 14, R15: Free adsorption reaction tank, W11: Discharge port 11, W12: Recovery port 12, S11: Sample introduction port, H
11: Heating device

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 4B024 AA19 AA20 CA01 CA11 CA20                       HA20                 4B029 AA21 AA23 BB20 CC01 CC02                       CC11 CC13                 4D017 AA11 BA07 CA05 CB01 CB05                       DB02                 4G075 AA02 AA39 AA62 AA63 BA10                       BB05 BB10 CA02 CA03 CA51                       CA54 DA02 EC30 ED20 EE04                       EE12 EE31 FA01 FA12 FC17                       FC20

Claims (18)

[Claims] 1. A liquid or gaseous sample, a reagent, or a mixed fluid of the sample and the reagent, which comprises at least one reservoir and / or reaction tank and at least one flow path, A cartridge for flowing a fluid from a reservoir and / or a reaction tank into the at least one channel to apply a chemical reaction to a predetermined component in the sample, the reagent, or the mixed fluid, comprising: (1) elasticity At least one flat plate member for airtightness made of a material,
(3) a laminated structure of three or more layers formed by alternately sandwiching and crimping at least two flat plate-like members for substrates made of a material having elasticity and hardness higher than that of the elastic material; Is a groove and / or hole provided in the flat plate member for substrate, a groove and / or hole provided in the flat plate member for airtightness, or for airtightness by the pressure of the sample, the reagent or the mixed fluid. (3) The reservoir and / or the reaction tank are formed of at least one member selected from the group consisting of voids formed by deforming portions of the flat plate member, and (3) the flat plate member for airtightness and / or A chemical reaction cartridge comprising a groove and / or a hole provided in the flat plate member for a substrate. 2. The chemical reaction cartridge according to claim 1, further comprising at least one valve that controls opening and closing of the flow path. 3. At least one of said valves has a rod-shaped member, said rod-shaped member being movable with respect to a flow path on said cartridge, said rod-shaped member having an open section and a closed section. ,
The open section has a structure in which the projected area on a plane perpendicular to the moving direction is smaller than that of the closed section, and the flow path on the cartridge and the open section of the rod-shaped member communicate with each other due to the movement of the rod-shaped member. Then, the valve is opened, and the movement of the rod member closes the flow passage on the cartridge by the closed section of the rod member, thereby closing the valve and controlling the opening / closing of the flow passage on the cartridge. The cartridge for chemical reaction according to claim 2, wherein the cartridge is a valve. 4. At least one of the valves controls the formation of a void formed by deforming a portion of the airtight flat plate member by the pressure of the sample, the reagent and / or the mixed fluid. The chemical reaction cartridge according to claim 2, which is a valve that controls opening and closing of the flow path. 5. The chemical reaction cartridge according to claim 2, further comprising a controller that controls the opening and closing of at least one of the valves by an actuator. 6. A step of releasing a nucleic acid from a material containing a nucleic acid by a chemical reaction, a step of adsorbing the released nucleic acid on a nucleic acid-binding carrier, a step of washing the nucleic acid-binding carrier on which the nucleic acid is adsorbed, and a nucleic acid The chemical reaction cartridge according to claim 1, comprising a nucleic acid isolation reaction comprising a step of eluting the nucleic acid from the adsorbed nucleic acid-binding carrier. 7. A release liquid for releasing nucleic acid from a material containing nucleic acid, a nucleic acid-binding carrier, an adsorption liquid for adsorbing the released nucleic acid on the nucleic acid-binding carrier, a washing liquid for washing the nucleic acid-adsorbing carrier on which nucleic acid is adsorbed, The chemical reaction cartridge according to claim 6, further comprising at least one of an eluent for eluting the nucleic acid from the nucleic acid-binding carrier to which the nucleic acid is adsorbed. 8. A nucleic acid-binding carrier, a free adsorbing liquid for releasing nucleic acid from a nucleic acid-containing material and adsorbing the released nucleic acid on the nucleic acid-binding carrier, a washing solution for washing the nucleic acid-adsorbing carrier. 7. The chemical reaction cartridge according to claim 6, further comprising at least one eluent for eluting the nucleic acid from the nucleic acid-binding carrier on which the nucleic acid is adsorbed. 9. The chemistry according to claim 6, wherein the nucleic acid-binding carrier comprises silica or a silica derivative, and the step of adsorbing the released nucleic acid to the nucleic acid-binding carrier uses a solution containing chaotropic ions. Reaction cartridge. 10. The cartridge for chemical reaction according to claim 7, wherein the nucleic acid-binding carrier is made of silica or a silica derivative, and the adsorbing liquid contains chaotropic ions. 11. The chemical reaction cartridge according to claim 8, wherein the nucleic acid-binding carrier is made of silica or a silica derivative, and the free adsorption liquid contains chaotropic ions. 12. The cartridge for chemical reaction according to claim 6, wherein the nucleic acid-binding carrier is a nucleic acid-binding magnetic substance-containing particle. 13. The nucleic acid-binding carrier is a film made of silica or a silica derivative, which is characterized in that
11. The chemical reaction cartridge according to item 11. 14. The chemical reaction cartridge according to claim 1, wherein the chemical reaction includes a nucleic acid amplification reaction. 15. The chemical reaction cartridge according to claim 6, wherein the chemical reaction further includes a nucleic acid amplification reaction for amplifying the nucleic acid isolated by the nucleic acid isolation reaction. 16. The nucleic acid amplification reaction is a polymerase chain reaction (PCR).
Or the cartridge for chemical reaction according to item 15. 17. The chemical reaction cartridge according to claim 1, wherein the temperature is controlled by heating or cooling at least a part of the cartridge. 18. The chemical reaction cartridge according to claim 1, wherein at least two locations of the cartridge are heated and / or cooled to control different temperatures. .