JP2007135504A - Microreactor used for nucleic acid inspection and holding beads at amplification site - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microreactor used for nucleic acid inspection and capable of quickly, highly reliably and highly sensitively analyze, and to provide an inspection device. <P>SOLUTION: This nucleic acid inspection device is a system structure which separately has a chip component used for each specimen and loading elements for reagents/liquid-sending systems, and a control/detection component as an inspection device main body. Thereby, cross-contamination, carry-over contamination, and insufficient amplification or error amplification are hardly caused on a fine amount analysis and an amplification reaction. In this microreactor structure, nucleic acid non-adsorbing beads are held in a passage at a nucleic acid amplification site in which the nucleic acid amplification reaction is carried out. When the nucleic acid is amplified, the beads are moved in the passage to enhance the amplification efficiency of the amplification, thereby enabling the highly reliable and highly sensitive analysis in a short time. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a nucleic acid test microreactor that holds non-nucleic acid-adsorbing beads in a flow path of an amplification site, and a nucleic acid test apparatus including the microreactor.

In recent years, by making full use of micromachine technology and ultrafine processing technology, devices and means (for example, pumps, valves, flow paths, sensors, etc.) for performing conventional sample preparation, chemical analysis, chemical synthesis, etc. have been miniaturized. Systems integrated on a chip have been developed. this is,
It is also called μ-TAS (Micro total Analysis System), bioreactor, Lab-on-chips, biochip, and its application is expected in medical examination / diagnosis field, environmental measurement field, agricultural production field Has been. When complicated processes, skilled techniques, and instrumentation are required, automated, faster, and simplified microanalysis systems are not only cost, required sample volume, time required, but also time and Enables analysis anywhere.

  In the field where various tests such as clinical tests are performed, quantitativeness, accuracy of analysis, and the like are regarded as important in the measurement using these analysis chips that produce results quickly regardless of location. Since the analysis chip has severe restrictions in terms of its size and form, it is necessary to establish a highly reliable liquid delivery system with a simple configuration. Therefore, there is a need for a microfluidic control element that has high accuracy and excellent reliability. The present inventors have already proposed a micropump system suitable for this (Patent Documents 1 and 2).

The microreactor that provides a simple and quick inspection means has raised specific problems and requests that should be solved as actual problems, and the solution is desired. For example, in order to perform an inspection or the like on a chip, minimizing the amount of sample and reducing the amount of necessary reagent is the greatest challenge required for a microreactor. However, depending on the sample, the concentration of the gene or nucleic acid to be detected may be extremely dilute. Since the amount of sample that can be introduced into the chip is limited, such a sample amount does not fall within the measurement range unless it is concentrated. As a physical method for extracting and separating nucleic acid from a biological sample on a microchip, the present inventors have proposed a method (Patent Document 3) of adsorbing to a bead filled in a flow channel, followed by elution, separation and concentration (Patent Document 3) Disclosed. In detection of genes and nucleic acids, it is usual to use an amplification reaction by PCR (polymerase chain reaction). In that case, it is desirable to appropriately control the nucleic acid amplification reaction so that amplification desired from the viewpoint of amplification efficiency proceeds sufficiently in a short time. Therefore, a method for performing a PCR reaction on beads has been reported (Patent Document 4).
JP 2001-322099 JP 2004-108285 A Japanese Patent Application No. 2004-312314 JP 2004-65199 A

  The present invention made in view of the above situation is that a nucleic acid non-adsorbing bead is held in a nucleic acid amplification site where a nucleic acid amplification reaction is performed, and the nucleic acid is amplified when the nucleic acid is amplified. We propose a microreactor for nucleic acid testing with improved nucleic acid amplification process by reciprocating inside to increase amplification efficiency.

The microreactor for nucleic acid testing of the present invention has a nucleic acid amplification site that holds a non-nucleic acid-adsorbing bead inside the channel, and when the nucleic acid is amplified, the bead is reciprocated in the channel of the nucleic acid amplification site. It is characterized by letting.

  The non-nucleic acid bead is preferably a hydrophobic bead. Furthermore, the beads are preferably formed from zirconia, silicon nitride, polystyrene or ceramics.

It is desirable that the liquid repeatedly move back and forth in the flow path to reciprocate the beads by changing the liquid feeding direction of the micropump that feeds the nucleic acid amplification site.
The beads may be further magnetic and may be reciprocated in the flow path by a magnetic field applied from the outside.

The microreactor of the present invention includes a merging section for sending and merging at least two kinds of liquids including a specimen and a reaction reagent for nucleic acid amplification by the micropump,
A wide channel for holding the beads in the nucleic acid amplification site, which is provided in advance from the merging portion and where the reaction with the reaction reagent is performed;
Control means for controlling the micropump so as to switch the flow direction of the merged liquid fed from the merge section into the flow path in the reverse direction and to repeatedly move the merged liquid back and forth in the flow path; It is characterized by providing.

The microreactor has at least a sample receiving part, a reagent storage part, a waste liquid storage part, a micropump connection part, and a fine channel, and each part is communicated with the fine channel,
Nucleic acid amplification site provided downstream through the microchannel by the action of a separate micropump that connects the sample in the sample receiving unit and the reagent in the reagent storage unit to the micropump connection unit, and then the detection site To measure the nucleic acid,
As a result of the nucleic acid measurement, the waste liquid produced is transferred to the waste liquid reservoir and confined.

The micropump is
A first channel in which the channel resistance changes according to the differential pressure;
A second flow path in which the ratio of change in flow path resistance to change in differential pressure is smaller than the first flow path;
A pressurizing chamber connected to the first flow path and the second flow path;
A piezo pump provided with an actuator for changing the pressure inside the pressurizing chamber is desirable.

As a liquid feed dividing means,
Branched microchannels,
The pump pressure of the micropump that allows the liquid to pass by blocking the passage of the liquid until the liquid feeding pressure in the positive direction reaches a preset pressure and applying a liquid feeding pressure equal to or higher than the preset pressure. A liquid feed control unit capable of controlling the passage of liquid by
A backflow prevention unit for preventing backflow of liquid in the flow path;
And is characterized by controlling the liquid feeding and the amount of the liquid feeding in the branched flow path.

  A device body in which a micropump and a temperature control device are integrated together with a detection device for optically detecting nucleic acid, and a microreactor for nucleic acid testing that can be attached to the device body, and the microreactor is attached to the device body Thus, a nucleic acid test apparatus characterized by automatically measuring a nucleic acid is also included in the present invention.

Furthermore, nucleic acid non-adsorbing beads are held inside the wide flow channel of the nucleic acid amplification site, and when the nucleic acid is amplified, the inside of the flow channel is changed by changing the liquid feed direction of the micropump that feeds the nucleic acid amplification site. A nucleic acid amplification method is also included in the present invention, in which amplification efficiency is increased by reciprocating or moving the bead within the flow path by repeatedly moving the liquid back and forth.

  The nucleic acid test apparatus of the present invention has a system configuration in which a chip component, which is a microreactor for each specimen, and a control / detection component, which is a main body of the apparatus, are equipped with elements necessary for the feeding and amplification reaction of specimens and reagents. is there. Thus, in the amplification process, problems of erroneous amplification or insufficient amplification, cross contamination, carry over contamination are avoided.

  According to the configuration of the microreactor of the present invention, the nucleic acid non-adsorbing beads are held in the flow path inside the nucleic acid amplification site where the nucleic acid amplification reaction is performed. By reciprocating in the flow path to increase amplification efficiency, nucleic acid amplification can be performed in a short time.

Detailed Description of the Invention
Hereinafter, a nucleic acid test microreactor of the present invention and a nucleic acid test apparatus including the microreactor and a micropump, various control devices, and a detection device will be described. In the present specification, the “specimen” is a fluid containing the nucleic acid to be measured. “Gene” refers to DNA or RNA carrying genetic information that expresses some function, but may also be simply referred to as DNA or RNA that is a chemical entity. These and the polynucleotide are collectively referred to as “nucleic acid”. “Element” refers to a functional component installed in the microreactor. The “fine channel” is a channel formed in the microreactor of the present invention.

Overview of Microreactor and Nucleic Acid Testing Device FIG. 1 is a system schematic diagram of a nucleic acid testing device comprising a nucleic acid testing microreactor detachable from the device body and the device body, and FIG. 2 is a schematic diagram of one embodiment of the testing device. is there. In the various embodiments, the present invention can be arbitrarily modified and changed in accordance with the spirit of the present invention, and these are included in the present invention. That is, as for the whole or a part of the microreactor (inspection chip) and the inspection apparatus of the present invention, various structures, configurations, arrangements, shapes, dimensions, materials, methods, methods, etc. are applicable as long as they match the purpose of the present invention. Can be.

  The test chip 2 shown in FIGS. 1 and 2, that is, the nucleic acid test microreactor, is a single chip that is manufactured by appropriately combining one or more members such as plastic resin, glass, silicon, and ceramics. The vertical and horizontal sizes are usually about several tens mm and the height is about several mm. Preferably, the fine flow path and the casing of the microreactor are formed of a plastic resin that is easy to process and inexpensive, and easy to dispose of by incineration. Among these, polyolefin and polystyrene resins such as polypropylene are preferable because they are excellent in moldability, have a strong tendency to adsorb streptavidin and the like as described later, and can easily form a detection site on a fine channel. The fine channel is formed to have a width and height of about 10 μm to several hundreds of μm, for example, a width of about 100 μm and a depth of about 100 μm by a fine processing technique. In addition, in such a microreactor, when a fluorescent substance or a coloring substance is generated and optically detected, the detection part that covers at least the detection part of the microchannel on the surface of the microreactor is transparent. A member, preferably a transparent plastic, is preferred. Moreover, it is preferable that additives, such as antioxidant, are not contained in resin used for shaping | molding as much as possible from a viewpoint of reducing the fluorescence background. FIG. 3 shows an example of a typical flow path configuration of the microreactor for nucleic acid test of the present invention.

  The nucleic acid test apparatus of the present invention includes a device body 1 in which a micropump, a control device that controls the micropump, a temperature control device that controls temperature, a detection device, and the like are integrated, and a microreactor 2 that can be attached to the device body. Consists of. When the sample liquid is injected into the sample receiving part of the microreactor 2 in which the reagent is sealed in advance and the microreactor is attached to the main body 1 of the nucleic acid test apparatus, the mechanical connection for operating the liquid feed pump is controlled, if necessary. Electrical connections are also made. When the main body and the microreactor are joined, the flow path of the microreactor is also activated. Accordingly, in an example of a preferred embodiment, when analysis is started, a series of procedures such as feeding of specimens and reagents, amplification of nucleic acid based on mixing, reaction such as binding of nucleic acid and probe, detection of reactants and optical measurement are performed. It is automatically performed as a continuous process, and measurement data is stored in a file together with necessary conditions and recorded items, and nucleic acid measurement is automatically performed.

  The detection device is a device that irradiates a detection portion on a flow path with measurement light from, for example, an LED, and detects transmitted light or reflected light by means of optical detection such as a photodiode or a photomultiplier tube. is there. As optical detection means, there are various optical devices having different principles, but an ultraviolet / visible spectrophotometer is desirable. A device incorporated in the inspection device may be used, or a separate device may be connected at the time of use. Preferably, in the nucleic acid test device of the present invention, the detection device for optically detecting the nucleic acid contained in the specimen is incorporated and integrated with the liquid feeding means including the micropump and the temperature control device. It has become.

  A control system relating to each control of liquid feeding, temperature, and reaction, optical detection, data collection and processing constitute a main body 1 of the nucleic acid test apparatus of the present invention together with a micropump and an optical apparatus. The apparatus main body 1 is commonly used for samples by mounting the chip 2 on the apparatus main body 1. The above-described reactions such as amplification and detection are software installed in the nucleic acid test apparatus as a program together with micropump and temperature control, optical detection data processing, as preset conditions for the order of feeding, volume, timing, etc. It is built in. In the conventional analysis chip, when performing different analysis or synthesis, it is necessary to configure a microfluidic device corresponding to the contents to be changed each time. In contrast, in the present invention, only the above-described detachable chip needs to be replaced. If it is necessary to change the control of each element, the control program stored in the apparatus main body may be appropriately modified.

  In the nucleic acid test apparatus of the present invention, since all components are miniaturized and are in a form that is convenient to carry, the workability and operability are good regardless of the place and time of use. Since a large number of micropump units used for liquid feeding are incorporated in the apparatus main body, the chip can be used as a disposable type.

  The microreactor and nucleic acid test apparatus of the present invention can be suitably used particularly for testing genes or nucleic acids (DNA). In that case, a mechanism for nucleic acid amplification is mounted on the microreactor. However, it can be said that the basic configuration of nucleic acids other than genes is almost the same. For example, a reverse transcriptase containing part may be installed for RNA. Alternatively, as another aspect of the microreactor and the nucleic acid test apparatus of the present invention, a microreactor having a simple configuration with fewer steps performed on the microreactor may be used. For example, a microreactor specialized in DNA amplification is a chip that can collect an amplification product by injecting a specimen and a reagent into a predetermined site and flowing them into a flow path to perform an amplification reaction. In any case, normally, the reagent storage unit, the sample pretreatment unit, the reagents, the probes, and the like may be appropriately changed or omitted, and the arrangement and number of liquid feeding elements will change.

For nucleic acid testing microreactor of nucleic acid testing microreactor present invention, the following an example of the preferred embodiment are described. The microreactor of this aspect has at least a sample receiving part, a reagent storage part, a waste liquid storage part, a micropump connection part, and a fine flow path, and the respective parts are communicated with each other through the fine flow path.
The sample liquid is flowed to the flow path constituting the nucleic acid amplification site provided downstream of the sample receiving part and then to the flow path constituting the detection site to measure the nucleic acid, and the waste liquid generated as a result of the nucleic acid measurement is stored in the waste liquid. It is a microreactor characterized by being moved to a part and confined.

  Furthermore, in addition to each storage unit, flow path, and pump connection unit, each element such as a liquid feed control unit, backflow prevention unit, reagent quantification unit, and mixing unit is installed at a functionally appropriate position by microfabrication technology. Yes.

  Note that some of the components of the microreactor and nucleic acid test apparatus of the present invention are included as options, and such components can be omitted as appropriate when the system is simplified. For example, the reagent storage unit and the waste liquid storage unit may not be installed depending on circumstances.

Sample Receiving Unit The sample receiving unit 20 of the microreactor of the present invention has the following configuration (FIG. 3). The sample receiving unit 20 communicates with the sample injection unit to temporarily store a sample (containing nucleic acid) and supply the sample to the mixing unit. The sample injected into the sample receiving unit 20 is connected to the micropump 11 and the pump connection unit 12, and is sent to the mixing unit immediately before the downstream amplification site by these actions.

  In addition, the part that injects the specimen on the upper surface of the specimen receiving part (specimen injecting part) is formed with a stopper made of an elastic material such as rubber to prevent leakage, infection and contamination to the outside and to ensure sealing performance. It is desirable that it is covered with a resin such as polydimethylsiloxane (PDMS) or a reinforced film.

-Specimen The specimen to be measured in the present invention is not particularly limited to a biological sample itself, but most biological specimens such as whole blood, plasma, serum, buffy coat, urine, feces, saliva, sputum, etc. Is applicable. In the case of a genetic test, a gene, DNA, or RNA is an analysis target as a nucleic acid that serves as a template for an amplification reaction. The specimen may be prepared or isolated from a sample that may contain such nucleic acid. Therefore, in addition to the above-mentioned samples, cell cultures; nucleic acid-containing samples such as viruses, bacteria, molds, yeasts, plants, animals; samples that may contain or contain microorganisms, and other nucleic acids All possible samples are targeted.

The microreactor of the present invention requires a very small amount of specimen as compared with the case of manual work using a conventional apparatus. For example, a blood sample of about 2 to 3 μL is simply injected into a chip having a length and width of several centimeters. For example, in the case of a gene, the DNA is 0.001 to 100 ng.

Reagent storage section In the microreactor for nucleic acid testing of the present invention, a reagent storage section may be provided as necessary. In a preferred embodiment, necessary reagents (including a cleaning solution) are sealed in a predetermined amount in the reagent storage unit 18 in the microreactor (FIG. 3). It is further desirable that a plurality of reagent storage units are provided corresponding to a plurality of reagents. With such a reagent container, the microreactor of the present invention does not need to be filled with the necessary amount of reagent each time it is used, and is ready to use, so it can be inspected quickly regardless of location or time. . When analyzing a nucleic acid in a specimen, reagents necessary for the measurement are generally known. Examples of the reagents for genetic testing include various reagents used for nucleic acid amplification, probes used for detection, and coloring reagents.

  In order to prevent evaporation, leakage, mixing of bubbles, contamination, denaturation, etc., the reagent parts built in the chip are sealed and sealed with a sealing material (sealing liquid). Preferably it is. These sealing materials (contained in the sealing liquid container 25 in FIG. 5) are solidified or gelled under refrigerated conditions in which the microreactor chip is stored before use, and when used at room temperature. It melts into a fluid state. As such a sealing material, a water-insoluble plastic material can be used. Preferably, the water solubility is 1% or less and the melting point is 8 ° C. to room temperature (25 ° C.). Examples include oils and fats and aqueous solutions of gelatin.

Each part of the reagent storage part and the mixing part for mixing the reagents (and the sample receiving part if necessary) is cooled by a Peltier element. The Peltier element 3 may be provided on the upper surface of the chip of each of these parts or on both upper and lower surfaces. By cooling the reagent container or the like, it is possible to prevent the reagent from being altered by heating. In particular, PCR amplification requires temperature management that raises and lowers between three temperatures. Even when the ICAN (Isothermal chimera primer initiated nucleic acid amplification) method is applied for gene amplification, the amplification reaction section is 50
Since it is necessary to set the temperature to ~ 65 ° C, amplification can be started as soon as the temperature of the reaction part is raised in advance, and the inspection time can be shortened. To be easily altered. In order to prevent this, it is desirable to cool by providing a Peltier element as described above. Usually, if the temperature is kept at about 4 ° C. or less, the reagent can be sufficiently prevented from being altered.

Micropump and pump connection part In this embodiment, a micropump 11 is provided for each of the specimen receiving part 20, the reagent storage part 18 and, if necessary, the control storage part, to feed the contents of these storage parts. ing. The micropump 11 is connected to the upstream side of the reagent storage unit 18, and supplies the driving liquid to the reagent storage unit side by the micropump 11, thereby pushing out the reagent to the flow path and feeding the reagent. The micropump unit is incorporated in an apparatus main body (nucleic acid test apparatus) separate from the microreactor, and is connected to the microreactor from the pump connection portion 12 by mounting the microreactor on the apparatus main body (see FIG. 5).

In this embodiment, a piezo pump is used as the micro pump. That is,
A first channel in which the channel resistance changes according to the differential pressure;
A second flow path in which the ratio of change in flow path resistance to change in differential pressure is smaller than the first flow path;
A pressurizing chamber connected to the first flow path and the second flow path;
A piezo pump provided with an actuator for changing the pressure inside the pressurizing chamber (FIG. 6). The details are described in Patent Documents 1 and 2 above.

A specimen receiving part in which the specimen is accommodated in each flow path upstream of a joining part that joins a liquid of a specimen containing a nucleic acid to be measured with a nucleic acid amplification site and a reagent (or a mixture of reagents); A reagent container for storing the reagent liquid, and a pump connection part provided upstream of each of the container parts. The micropump is connected to the pump connection part, and the driving liquid is supplied from each micropump. The reaction solution necessary for the nucleic acid amplification analysis is started by extruding the sample solution and the reagent in each of the storage portions by supplying them and joining them together (FIGS. 4, 7, and 8). . The mixing of the reagent and the reagent and the mixing of the sample and the reagent may be performed at a desired ratio in a single mixing part, or a plurality of merging parts are provided by dividing one or both, and finally They may be mixed so as to have a desired mixing ratio. The mode of such a reactive site is not particularly limited, and various forms and modes are conceivable. The reaction part of the nucleic acid amplification site is basically desirably a wide reservoir-like channel (FIG. 4), but may be a fine channel sufficiently long for the reaction. Nucleic acid non-adsorbing beads are filled and held in the wide channel in the nucleic acid amplification site. Therefore, a preferred embodiment of the microreactor chip of the present invention is:
A merging section for sending and merging at least two kinds of liquids including a reagent and a specimen from a fine channel by a micropump;
A flow channel wider than the fine flow channel in the nucleic acid amplification site provided in advance from the junction,
And amplifying the reaction by diffusing and mixing the combined solution by reciprocating or moving the nucleic acid non-adsorbing beads held in the wide channel when the nucleic acid is amplified. (FIG. 7).

The beads may be prepared from any insoluble or solid material as long as the surface does not substantially adsorb nucleic acids. Specifically, nylon, magnetic beads, Dynabeads,
Materials such as metal surfaces (eg, steel, gold, silver, aluminum, silicon and copper), plastic materials (eg, polyethylene, polypropylene, substituted polyalkenes, polyamides, polyesters, polyvinylidene difluoride (PVDF)), celite, ceramics, etc. Is mentioned. Even if the beads made of a material other than the above are beads having a property of adsorbing nucleic acid, the beads may be further surface-treated with fluorine or the like to be non-adsorbing nucleic acid. Preferably, the beads are formed from zirconia, silicon nitride (SiN), polystyrene or ceramics. “Nucleic acid non-adsorbing” means that nucleic acid does not substantially adhere, and is defined, for example, as a bead whose nucleic acid concentration change is 1% or less when placed in a 10 μM nucleic acid solution.

In the case where the beads are in the form of particles, the size of the particles is usually in the range of 10 μm to 2 mm. However, in order to be held in the wide flow channel of the nucleic acid amplification site and always stop in the wide flow channel, the size must be larger than the flow channel width of the micro channel immediately before the site and the downstream micro channel. It is better to avoid the same size because it may be clogged by such a fine flow path and clogged, preventing the smooth flow of liquid.

Such particles, referred to herein as “beads,” are often, but not necessarily, spherical. However, such beads do not constrain the shape of the matrix and may take any shape, including random shapes, needles, fibers and elongated ones. The number of beads filled in the channel of the amplification site is arbitrary and is not particularly limited, but is preferably set in consideration of the width and length of the channel, the speed of the liquid, and the like.
As an example, zirconia beads having a particle diameter of 100 to 1000 μm are filled in a fine channel at a density of 0.05 to 1.3 g / ml.

・ Liquid movement in the reaction site of the nucleic acid amplification site The beads flow actively reciprocating or moving in the wide channel of the amplification site, and the flow of the liquid in the channel changes, and the stirring action causes the reagent solution and the sample solution Is promoted (FIG. 7). Therefore, the number of opportunities for the amplification template polynucleotide and the amplification reagent including the primer and the enzyme to be increased increases the probability that the reaction will occur and the amplification process proceeds more smoothly, resulting in the efficiency of amplification. Will be increased. If the beads are not accommodated, the mixed solution of the specimen and the reagent flows in a laminar flow through the fine channel. Therefore, in order to sufficiently perform the mixing by diffusion and sufficiently advance the amplification reaction, it is necessary to configure a longer flow path. On the other hand, even in the case of a wide liquid reservoir-like flow path, the flow path in the amplification region can be short because the mixed liquid is effectively stirred by the reciprocation of the beads.

Reciprocating or moving the beads in the wide channel can be easily realized by changing the liquid feeding direction of the micropump. In particular,
A confluence unit for sending and merging at least two kinds of liquids including a sample liquid and a reaction reagent for nucleic acid amplification by a micropump;
A wide channel for holding the beads in the nucleic acid amplification site, which is provided in advance from the junction and where the reaction with the reaction reagent is performed;
A control means for controlling a micropump for switching back and forth in the flow path by switching the flow direction of the merged liquid fed from the merge section into the flow path;
(FIG. 8).

  Preferably, the liquid mixture of the specimen and the reaction reagent sent to the amplification site is switched back and forth as indicated by the arrows in FIGS. 7 and 8, so that the liquid is in the wide channel of the amplification site. The micropump 11 is driven so as to repeatedly move back and forth in the flow path direction. In order to switch the liquid feeding direction back and forth, the driving direction of the micro pump may be periodically reversed. Thereby, the probability that the nucleic acid in the sample and the reaction reagent meet can be increased, and the nucleic acid can be amplified in a short time to improve the efficiency. Depending on the case, for example, the reciprocation may be performed with an amplitude of 5 mm and a period of about 5 seconds.

  FIG. 8 is a diagram showing a control system of the liquid-feeding micropump 11 for moving the liquid containing the beads back and forth in the wide flow path in the amplification reaction site. As shown in the figure, the liquid-feeding micropump 11 is connected to a microcomputer 34 via an amplifier 32 and a D / A converter 33. The 32 to 35 systems for controlling the micropump 11 may be in the chip. However, by incorporating the chip 2 in the main body of the microreactor and mounting the chip 2 on the main body of the apparatus, the pump connecting portion of the chip 2 is connected to the main body of the apparatus. It is also possible to control the operation by connecting to the micropump.

  FIG. 8 shows a control system for the micropump 11 for feeding liquid and the valve 57, and an air cylinder 36 for opening and closing the valve 57 is connected to the microcomputer 34 via the D / A converter 33. Yes. The microcomputer 34 is equipped with a timer 35 and controls the liquid feeding of the liquid feeding micropump 11 and the opening / closing of the valve 57 at a preprogrammed timing. The micropump 11 may be connected to the sample receiving unit 20 so that the sample feeding from the sample receiving unit 20 and the feeding of the amplification reaction reagent from the reagent storage unit 18 may be controlled separately. In this case, for example, when a valve 57 such as a check valve is provided in the fine channel on the specimen receiving unit 20 side, and when the mixed solution containing the nucleic acid and the reagent introduced into the reaction unit of the nucleic acid amplification site is moved back and forth, The valve 57 is closed and the liquid feed is switched by the micropump 11 connected to the reaction site.

  If the beads are further magnetic, they may be reciprocated by a magnetic field applied from outside rather than by the action of the micropump. That is, the movement of the beads is realized by reciprocating an external magnet separate from the chip along the flow path directly above the amplification site where the beads are present.

Nucleic acid amplification method In the genetic test using the microreactor of the present invention, the nucleic acid amplification method is not limited. For example, the DNA amplification technique can use a PCR amplification method that is actively used in various fields. Various conditions for carrying out the amplification technique have been examined in detail and described in various documents including improvements. In PCR amplification, it is necessary to manage the temperature by raising and lowering between three temperatures. However, a channel device that enables temperature control suitable for a microchip has already been proposed by the present inventors (Japanese Patent Application Laid-Open No. 2004-2000). -108285). This device system may be applied to the amplification channel of the chip of the present invention. As a result, the heat cycle can be switched at high speed, and the micro flow path is a micro reaction cell with a small heat capacity. Therefore, DNA amplification can be performed in a much shorter time than the conventional method that is performed manually.

The recently developed ICAN (registered trademark) method as an improvement of PCR has the feature that DNA amplification can be carried out in a short time at an arbitrary constant temperature of 50 to 65 ° C. (Patent No. 3433929). Therefore, the ICAN method is a suitable amplification technique because the microreactor of the present invention requires simple temperature control. By hand, the method, which takes 1 hour, ends in 10-20 minutes, preferably 15 minutes, in the bioreactor of the present invention.

  The DNA amplification reaction may be another PCR modification method, and the microreactor of the present invention is flexible enough to cope with any change in the design of the flow path. When using any DNA amplification reaction, details of the technique are disclosed, and those skilled in the art can easily introduce them.

  In the amplification method of the present invention, non-nucleic acid non-adsorbing beads are held inside the wide flow channel of the nucleic acid amplification site, and the liquid is flown in the flow channel by changing the liquid feeding direction of the micropump that feeds the nucleic acid amplification site. The method is characterized in that amplification efficiency is improved by moving back and forth repeatedly and reciprocating or moving the beads within the flow path at the site.

  The nucleic acid amplification method of the present invention will be specifically described. FIG. 7 is a diagram showing an example of a flow path configuration for mixing and reacting a specimen and a reagent. As shown in the figure, the specimen liquid fed from the specimen feeding side channel 54 by the micropump (not shown) and the reagent fed from the reagent reservoir 7 by the micropump 11 are in the Y-shaped channel. They are merged at the merging portion 58 and fed to the wide flow channel 9 a that holds the beads in the subsequent nucleic acid amplification portion 9.

  An amplification reaction, for example, an ICAN reaction is performed in the wide channel 9a. After filling the sample liquid and the reagent in the wide channel 9a, the micropump 11 for feeding the reagent is driven so as to repeat the liquid feeding direction and alternately switch between the forward direction and the reverse direction. The combined liquid in the inside is moved back and forth in the flow path 9a to perform the ICAN reaction. For example, when the width of the wide channel 9a is 0.2 mm, the depth is 0.2 mm, and the amount of liquid is 25 μl, the reciprocation may be performed with an amplitude of 25 mm and a period of about 5 seconds. By this back-and-forth movement, the beads move and the reagent and the sample in the wide channel 9a are actively diffused and mixed, and the nucleic acid and the reagent are separated by the flow velocity gradient of the liquid between the central part of the channel and the vicinity of the wall surface. Increases the chance of encounter and improves reaction speed. Therefore, amplification proceeds in a short time, and amplification efficiency can be increased.

  In addition, a valve 57 such as a check valve or an active valve is provided in the flow path 54 on the specimen liquid supply side, and is closed during mixing, and the combined liquid in the flow path 9a is removed by the micropump 11 that supplies the reagent. When moved back and forth, there is no need to arrange a separate micro pump for mixing in the flow path. The micro pump 11 is preferably a piezo pump as shown in FIG. In FIG. 7, the combined liquid is reciprocated by only one micropump 11. However, without providing the valve 57, the micropump (not shown) on the specimen feeding side, the micropump 11 on the reagent storage unit 7 side, May be made to reciprocate the combined liquid in the wide flow path 9a.

-Production of reaction part The nucleic acid amplification site 9 of the microreactor is formed by cutting a groove in a polystyrene substrate as a flow path in the reaction part, for example, with a width of 0.2 mm and a depth of 0.2 mm. In consideration of the volume of the wide liquid reservoir, the beads are filled with an appropriate number, for example, about 10 to 50, and the substrate to be the upper surface of the chip is bonded together.

・ Quantitative liquid feeding mechanism A check valve is provided at an appropriate position in the flow path between the reagent storage unit, the nucleic acid amplification site, and the detection unit. In addition, it is preferable to provide a check valve at an appropriate position for preventing contamination such as cross contamination as described above.

  As a check valve used in the flow path of this microreactor, a check valve that allows and blocks the passage of liquid by opening and closing the opening formed in the substrate by moving the microsphere using a microsphere as a valve body Is preferred. Alternatively, even if the flexible substrate that is laminated on the substrate and whose end portion extends to the upper side of the opening is a check valve that opens and closes the opening by moving the upper side of the opening up and down by hydraulic pressure Good.

  Although it is desirable to provide a check valve in order to prevent the back flow of the liquid and accurately carry out the predetermined liquid feeding, as a suitable mechanism using the check valve, the quantitative liquid feeding mechanism shown in FIG. Can be mentioned. In this mechanism, a predetermined amount of reagent is filled in the flow path (reagent filling flow path 15A) between the check valve 16 and the hydrophobic valve 13a. A branch channel 15B that branches from the reagent-filled channel 15A and communicates with the micropump 11 that feeds the driving liquid is provided.

  The reagent is quantitatively fed as follows. In FIG. 9, the reagent solution 60 is first filled from the check valve 16 side by supplying the reagent solution 60 to the reagent filling channel 15 </ b> A at a solution feeding pressure at which the reagent solution 60 does not pass from the hydrophobic valve 13 a forward. Next, the driving liquid 70 is fed by the micropump 11 in the direction from the branch flow path 15B toward the reagent filling flow path 15A at a liquid feed pressure that allows the reagent liquid 60 to pass through the hydrophobic valve 13a. Thus, the reagent solution 60 filled in the reagent filling channel 15A is pushed forward from the solution feeding control unit 15A, and thereby the reagent solution 60 is quantitatively fed. In addition, by providing the large-volume storage unit 7 in the reagent filling channel 15A, the variation in the quantity is reduced. This quantitative liquid feeding mechanism is also used for quantitative mixing of reagents and quantitative liquid feeding of specimens.

Detection Site In the microreactor of the present invention, a detection site for detecting the amplified nucleic acid is provided downstream from the nucleic acid amplification site. The biotin-affinity protein (avidin, streptavidin) adsorbed at the detection site on the microchannel is specific to biotin labeled on the probe substance or biotin labeled on the 5 'end of the primer used in the gene amplification reaction. Join. Thereby, the probe labeled with biotin or the amplified nucleic acid is trapped at the detection site.

A method for detecting the separated amplified nucleic acid is not particularly limited, but a preferred embodiment is basically performed in the following steps. That is, using the microreactor,
(1) DNA synthesized from a sample or DNA extracted from a sample, or cDNA synthesized by reverse transcription reaction from RNA extracted from the sample or sample, and a primer modified with biotin at the 5 ′ position Liquid is sent to the fine channel. A step of amplifying a nucleic acid in a channel in a nucleic acid amplification site, a step of mixing an amplification reaction solution containing an amplified nucleic acid and a denaturing solution in a fine channel, and denaturing the amplified DNA into a single strand, amplification The treated solution obtained by denaturing the resulting DNA into a single strand is sent to the detection site in the microchannel where biotin-affinity protein is adsorbed, and the amplified nucleic acid is trapped through the step of trapping the amplified gene. A probe DNA whose end is fluorescently labeled with FITC (fluorescein isothiocyanate) is flowed to the detection site in the microchannel, and this is hybridized with the immobilized gene. (A previously hybridized gene amplified with fluorescently labeled probe DNA may be trapped at the detection site.)
(2) A gold colloid solution whose surface is modified with an anti-FITC antibody that specifically binds to FITC is allowed to flow through the fine channel, and thereby the gold colloid is adsorbed to the FITC-modified probe hybridized with DNA.
(3) The concentration of the gold colloid in the fine channel is optically measured.

When immobilizing streptavidin in the microchannel formed in the polystyrene substrate, no special chemical treatment is required. It is only necessary to apply the biotin affinity protein onto a fine channel downstream of the amplification reaction site and adsorb the biotin affinity protein on the channel. As a probe DNA for genetic testing, fluorescently labeled oligodeoxynucleotides are preferably used. As the DNA base sequence, a sequence that is complementary to a part of the base sequence of the gene to be detected is selected. By appropriately selecting the base sequence of the probe DNA, it is possible to detect with high sensitivity without being affected by coexisting DNA and background, which specifically binds to the target gene.

  Known fluorescent substances such as FITC, RITC, NBD, Cy3, and Cy5 can be used as the fluorescent dye for labeling the probe. In particular, FITC is desirable due to the availability of anti-FITC antibodies such as gold colloid anti-FITC anti-mouse IgG.

  Although it is possible to measure the fluorescence of the fluorescent dye FITC, it is necessary to consider light fading of the fluorescent dye, background noise, and the like. A system that can finally measure with high sensitivity by visible light is preferable. This is because the equipment is more versatile than fluorescence photometry, and there are few interference factors and data processing is easy. Instead of using optical detection of colloidal gold using anti-FITC anti-mouse IgG, the probe may be labeled with horseradish peroxidase (HRP) instead of the fluorescent dye. For the detection, a coloring reaction catalyzed by the enzyme can also be used.

  An example of the best mode of the microreactor for nucleic acid test of the present invention will be described below. The present invention is not limited to this example, and modifications, omissions, changes, additions, and the like can be made without departing from the spirit of the invention.

A preferred embodiment of the microreactor for nucleic acid testing of the present invention is:
In one chip, a sample receiving part into which a sample containing at least nucleic acid is injected,
A reagent container for storing reagents used for probe binding reaction, detection reaction (including gene amplification reaction), and the like;
A positive control accommodating part for accommodating a positive control;
A negative control accommodating portion for accommodating a negative control;
A probe accommodating portion that accommodates a probe (for example, a probe that hybridizes to a gene to be detected amplified by a gene amplification reaction);
A fine flow path communicating with each of these accommodating portions,
A pump connection portion connectable to a separate micropump for feeding the liquid in each storage portion and the flow path;
And a waste liquid storage part,
A micropump is connected to the chip via a pump connection unit, the sample contained in the sample receiving unit and the reagent contained in the reagent containing unit are sent to the microchannel, and the confluence just before the nucleic acid amplification site The reaction solution generated at the nucleic acid amplification site and the probe accommodated in the probe accommodating portion are sent to the detection portion in the downstream flow path, mixed in the flow path, and combined with the probe (or Hybridization) and detecting nucleic acid based on the reaction product,
While performing the above reaction and detection in the same manner for the positive control housed in the positive control housing section and the negative control housed in the negative control housing section,
A waste liquid storage part that communicates with the specimen pretreatment part and the detection site through a through-hole, is a hollow chamber provided at the bottom of the microreactor, and is a sealed waste liquid reservoir that contains waste liquid and the like generated as a result of specimen measurement It is characterized by having it.

Simultaneously performing positive control and internal control is particularly important in gene amplification by PCR. This is because it is particularly necessary to check that the PCR reaction is occurring correctly. For example, when any problem occurs, it is optimal for verifying whether it originates from set conditions, reagents, operation, analysis system, or sample. Since the PCR method can amplify a very small amount of gene present in a sample several hundred thousand to several million times or more, the influence of contamination such as cross contamination is extremely serious. These control settings, useful for determining false positives and false negatives, follow conventional analytical technique practice. According to the flow channel configuration of the microreactor of the present invention, the analysis can be performed simultaneously in the analysis flow channel separate from the specimen under the same reagent and the same conditions. As described above, in the flow path of the nucleic acid amplification site, sufficient mixing is realized by moving the mixed solution back and forth and reciprocating or moving the beads. Therefore, differences such as amplification failure due to insufficient diffusion of nucleic acids, primers, etc. are unlikely to occur between the specimen and the control.

The waste liquid storage part is completely sealed in the microreactor and is a disposable chip, so that it is discarded in the hazard box as it is after use. For this reason, after a clinical sample is injected into the microreactor, the person involved in the test does not need to come into contact with the sample or the like at all during or after the test, and safety from infection is ensured.
In the present invention, when the positive control and the negative control are simultaneously analyzed for one sample, it is necessary to divide the reagents and the sample into two or more and send them to the respective analysis channels. The liquid feed dividing means is provided for that purpose. The liquid feed dividing means is composed of a branched fine channel, a liquid feed control unit 13 and a backflow prevention unit.

  The liquid feeding control unit 13 blocks the passage of the liquid until the liquid feeding pressure in the forward direction (usually, the direction in which the liquid is pushed out by the pump, that is, the downstream direction) reaches a predetermined pressure. Addition allows the passage of liquid. Further, the backflow prevention part for preventing the backflow of the liquid in the flow path is a check valve in which the valve body closes the flow path opening due to the backflow pressure, or the valve body is pressed against the flow path opening by the valve body deforming means. And an active valve for closing the opening.

  In the microchannel of the microreactor of the present invention, a flow branched by the micropump, a liquid feed control unit capable of controlling the passage of liquid by the pump pressure, and a backflow prevention unit for preventing the backflow of the liquid in the channel. The division of each liquid in the channel is controlled and a fixed amount of liquid is sent. By the action of the liquid feeding and dividing means and the micropump 11, the reagents and the specimen are divided at an appropriate ratio.

  The genetic test has been described above with reference to the drawings shown as an example of the best mode for carrying out the present invention. However, the present invention is not limited to such embodiments and examples.

FIG. 1 is a system schematic diagram of a nucleic acid test apparatus comprising a microreactor and an apparatus main body. FIG. 2 is a schematic view of a nucleic acid test apparatus according to an embodiment of the present invention. FIG. 3 is a schematic view of a microreactor (chip) for nucleic acid testing. The micropump belongs to a device separate from the microreactor. FIG. 4 shows an embodiment of the microreactor for nucleic acid testing of the present invention. The amplification site is filled with nucleic acid non-adsorbing beads and held. FIG. 5 shows a configuration around the pump connection portion of the microreactor when the micropump 11 is separated from the microreactor. (A) is the figure which showed the structure of the pump part which sends a drive liquid, (b) is the figure which showed the structure of the pump part which sends a reagent. FIG. 6 shows a piezo pump, (a) is a sectional view showing an example of the pump, and (b) is a top view thereof. (C) is sectional drawing which showed the other example of the piezo pump. FIG. 7 is a diagram showing an example of a flow path configuration of an amplification part that performs mixing of a specimen and a nucleic acid amplification reagent and performing a nucleic acid amplification reaction. FIG. 8 is a diagram for explaining an amplification part that performs an amplification reaction and a flow path configuration around the amplification site, and a control system that repeatedly switches in the front-rear direction the liquid mixture of the sample and the reagent introduced into the flow path. is there. FIG. 9 is a diagram for explaining a quantitative liquid feeding mechanism using a check valve.

Explanation of symbols

1 Device body 2 Microreactor (inspection chip)
3 Peltier 4 Heater 5 Photodiode 6 LED
7 Reagent storage part 9 Nucleic acid amplification site (reaction part)
9a Flow path in nucleic acid amplification site 10 Drive liquid tank 11 Micro pump (piezo pump)
12 pump connection unit 13 liquid supply control unit 15 flow channel 15A reagent filling flow channel 15B branch flow channel 16 check valve 18 reagent storage unit 19 sample 20 sample receiving unit 24 driving liquid storage unit 25 sealing liquid storage unit 26 air vent flow Path 31 reagent 32 amplifier 33 D / A converter 34 microcomputer 35 timer 36 air cylinder 41 upper substrate 42 substrate 43 diaphragm 44 piezoelectric element 45 pressurization chamber 46 first channel 47 second channel 48 first liquid chamber 49 Second liquid chamber 54 Sample liquid supply side flow path 55 Bead 56 Nucleic acid 57 Valve 58 Junction section 60 Reagent liquid 70 Driving liquid 71 Silicon substrate 72 Port 73 Port

Claims (11)

  For nucleic acid testing, comprising a nucleic acid amplification site for holding a non-nucleic acid-adsorbing bead inside the flow channel, and reciprocating the bead in the flow channel of the nucleic acid amplification site when amplifying the nucleic acid Microreactor.   The microreactor for nucleic acid testing according to claim 1, wherein the non-nucleic acid bead is a hydrophobic bead.   The microreactor for nucleic acid test according to claim 1 or 2, wherein the beads are formed of zirconia, silicon nitride, polystyrene, or ceramics.   2. The nucleic acid test according to claim 1, wherein liquid is repeatedly moved back and forth in the flow path to reciprocate the beads by changing a liquid feeding direction of a micropump for feeding liquid to the nucleic acid amplification site. Microreactor.   The microreactor for nucleic acid test according to claim 1 or 2, wherein the beads are further magnetic and reciprocate in the flow path by a magnetic field applied from the outside. A merging section for feeding and merging at least two kinds of liquids including a specimen and a reaction reagent for nucleic acid amplification by the micropump;
A wide channel for holding the beads in the nucleic acid amplification site, which is provided in advance from the merging portion and where the reaction with the reaction reagent is performed;
Control means for controlling the micropump so as to switch the flow direction of the merged liquid fed from the merge section into the flow path in the reverse direction and to repeatedly move the merged liquid back and forth in the flow path; The microreactor according to claim 4, comprising: It has at least a sample receiving part, a reagent storage part, a waste liquid storage part, a micropump connection part, and a fine flow path, and each part is communicated with the fine flow path,
Nucleic acid amplification site provided downstream through the microchannel by the action of a separate micropump that connects the sample in the sample receiving unit and the reagent in the reagent storage unit to the micropump connection unit, and then the detection site To measure the nucleic acid,
The microreactor for nucleic acid test according to any one of claims 1 to 6, wherein a waste liquid generated as a result of the nucleic acid measurement is transferred to and confined in the waste liquid reservoir. The micropump is
A first channel in which the channel resistance changes according to the differential pressure;
A second flow path in which the ratio of change in flow path resistance to change in differential pressure is smaller than the first flow path;
A pressurizing chamber connected to the first flow path and the second flow path;
The microreactor for nucleic acid test according to any one of claims 1 to 7, wherein the microreactor is a piezo pump provided with an actuator for changing the pressure inside the pressurizing chamber. As a liquid feed dividing means,
Branched microchannels,
The pump pressure of the micropump that allows the liquid to pass by blocking the passage of the liquid until the liquid feeding pressure in the positive direction reaches a preset pressure and applying a liquid feeding pressure equal to or higher than the preset pressure. A liquid feed control unit capable of controlling the passage of liquid by
A backflow prevention unit for preventing backflow of liquid in the flow path;
The nucleic acid testing microreactor according to claim 1, wherein the liquid feeding and the amount of the liquid feeding in the branched flow path are controlled.   A device body in which a micropump and a temperature control device are integrated together with a detection device for optically detecting nucleic acid, and a microreactor for nucleic acid testing that can be attached to the device body, and the microreactor is attached to the device body Thus, a nucleic acid test apparatus for automatically measuring a nucleic acid.   Nucleic acid non-adsorbing beads are held inside the wide flow channel of the nucleic acid amplification site, and when amplifying nucleic acid, the liquid in the flow channel is changed by changing the liquid feed direction of the micropump that feeds the nucleic acid amplification site. A method of nucleic acid amplification characterized in that amplification efficiency is improved by repeatedly moving back and forth repeatedly to move the beads back and forth within the flow path.

Images