JP4915072B2 - Microreactor - Google Patents

Description

  The present invention relates to a microreactor used for, for example, biological substance inspection / analysis by gene amplification reaction, antigen-antibody reaction, etc., inspection / analysis of other chemical substances, chemical synthesis of target compounds by organic synthesis, etc. In particular, the present invention relates to an improvement in technology for sending a mixed reagent having a stable mixing ratio to a subsequent process when a mixed reagent obtained by mixing a plurality of reagents and a sample are mixed and reacted.

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. A system integrated on a chip has been developed (Patent Document 1). This is a micro-TAS (Micro total Analysis System), bioreactor, love
It is also called “Lab-on-chips” or biochip, and its application is expected in the medical examination / diagnosis field, environmental measurement field, and agricultural production field. In reality, as seen in genetic testing, automated, faster, and simplified microanalysis systems are costly and necessary when complex processes, skilled techniques, and equipment operations are required. It can be said that not only the amount of sample and the time required, but also the benefits of enabling analysis at any time and place are great.

  In various types of analysis and inspection, importance is attached to the quantitativeness of analysis, the accuracy of analysis, and the economic efficiency of these analysis chips. For that purpose, it is a problem to establish a highly reliable liquid feeding system with a simple configuration. There is a need for a microfluidic control element with high accuracy and excellent reliability. The present inventors have already proposed a micropump system and a control method thereof suitable for this (Patent Documents 2 to 4).

In the analysis chip as described above, in which the reaction of each component is performed in the fine flow path, a process of combining and mixing these components in the fine flow path is required (Patent Document 5). For example, when there are a plurality of reagents to be reacted with the sample to be analyzed, it is necessary to obtain a mixed reagent by previously joining a plurality of reagents in the fine channel.
JP 2004-28589 A JP 2001-322099 A JP 2004-108285 A JP 2004-270537 A JP 2005-66400 A

  However, when mixing and mixing multiple reagents, even if each reagent is sent to the junction by the micro pump at the same timing, as long as the timing is not accurate, It is difficult to stabilize the ratio. When the initial mixed reagent at the head part is combined with the sample downstream to carry out the reaction, problems often occur because the mixing ratio is not stable at the head part.

  The present invention uses a micropump to send a plurality of reagents downstream from each reagent storage unit, join these reagents together, mix them, and join the obtained mixed reagents with the sample further downstream to react with the reaction unit It is an object of the present invention to provide a microreactor capable of delivering only a mixed reagent having a stable mixing ratio to a subsequent step when performing a reaction.

The microreactor of the present invention comprises a plate-shaped chip,
A plurality of reagent storage sections each including a storage chamber in which a plurality of reagents are individually stored;
A reagent mixing unit that mixes a plurality of reagents delivered from the plurality of reagent storage units to generate a mixed reagent; and
A sample receiver having an inlet for injecting a sample from the outside;
A mixed reagent sent from the reagent mixing part and a reaction part for mixing and reacting the sample sent from the sample receiving part,
The plurality of reagent storage units, the reagent mixing unit, the sample receiving unit, and the reaction unit are incorporated in the chip and communicated with each other through a flow path,
It comprises a delivery prevention mechanism for preventing the initial mixed reagent mixed in the initial stage from among the mixed reagents generated in the reagent mixing section.

In a preferred aspect of the above invention, the reagent mixing section is a flow path for mixing the plurality of reagents, and is a mixing flow path provided with a downstream end where the flow of the mixed reagent is stopped,
In the middle part of the mixing channel, a delivery channel for sending the mixed reagent to the reaction unit is branched,
The delivery prevention mechanism is composed of the mixing channel and the delivery channel,
The initial mixed reagent is accommodated between the intermediate portion and the downstream end of the mixing channel, and is prevented from being sent from the sending channel to the reaction unit.

  In this case, the delivery preventing mechanism sends a mixed reagent to the delivery channel when the pressure in the mixing channel becomes a predetermined pressure or higher at the connection portion between the mixing channel and the delivery channel. It is preferable to provide a part.

  Such a liquid feeding control unit may be formed of a narrow channel having a cross-sectional area smaller than the cross-sectional area of the delivery channel, provided on the delivery channel side in the connection portion between the mixing channel and the delivery channel. it can.

  In the above invention, since the initial mixed reagent at the beginning of mixing is trapped and discarded to the downstream end side of the mixing flow path, even if the timing of merging of the reagents is slightly shifted, only the mixed reagent with a stable mixing ratio is used. Can be sent to a subsequent process.

  According to the present invention, even if the timing of merging of the reagents is slightly shifted, only the mixed reagent with a stable mixing ratio can be sent to the subsequent process.

  Hereinafter, the present invention will be described in detail with reference to the drawings. The microreactor according to the present invention performs a reaction between a sample and a reagent for various inspections, chemical analysis, chemical synthesis, sample processing / separation, etc., in a fine channel or structure provided in a plate-shaped chip. Is.

  Applications of the microreactor of the present invention include, for example, biological substance inspection / analysis by gene amplification reaction, antigen-antibody reaction, etc., inspection / analysis of other chemical substances, chemical synthesis of target compounds by organic synthesis, drug efficacy screening, chemicals, etc. Extraction, formation and separation of metal complexes are included.

The microreactor of the present invention is in a plate-shaped chip,
(I) A plurality of reagent storage units each including a storage chamber in which a plurality of reagents are individually stored (ii) A reagent mixing unit that generates a mixed reagent by mixing a plurality of reagents delivered from the plurality of reagent storage units ( iii) Sample receiving part having an inlet for injecting a sample from outside (iv) A reaction part for mixing and reacting the mixed reagent sent from the reagent mixing part and the sample sent from the reagent receiving part It has.

The plurality of reagent storage units, the reagent mixing unit, the sample receiving unit, and the reaction unit are incorporated in the chip and communicated with each other through a flow path.
In addition to the above-described parts, structural parts having various functions are provided in the chip as needed. Specific examples of such a structural part include a part for controlling liquid feeding, a storage part for storing a processing liquid other than the sample and the reagent, a reagent for removing unnecessary components contained in a biological sample, etc. A pre-processing unit for pre-processing prior to the reaction, a detection unit for detecting a target substance contained in the liquid after the reaction, a waste liquid storage unit for storing the waste liquid, a micro for sending the liquid Examples include pumps.

Specific examples of the part for controlling the liquid feeding include a check valve, a valve part such as an active valve, and the like.
Specific examples of the storage section for storing each processing liquid include a cleaning liquid storage section for cleaning with a necessary substance adsorbed on a channel wall, a carrier such as a bead, and a reaction between the reagent and the sample. For stopping reaction, for storing reaction product for denaturing reaction products to be suitable for detection, and for labeling reaction products with fluorescent substances for optical detection Examples include a reagent storage part, and other parts such as an extraction liquid, an eluent, a lysis reagent, and a hemolysis reagent.

In the case of optically detecting the reaction product in the microreactor, the detection unit is composed of, for example, a flow path part or a liquid reservoir part formed using a light transmissive member.
The pretreatment unit is a part that concentrates, separates, lyses, and the like of the analysis target contained in the sample. For example, protein, ionic substances, and the like contained in the biological sample are removed. Such a process is performed by, for example, arranging a carrier such as a filter, a bead, a gel, or a membrane in a flow path, adsorbing a biological substance or the like on the carrier, and flowing a lysis reagent, a hemolysis reagent, or the like in the flow path, Subsequently, it can carry out by flowing a washing | cleaning liquid.

  The waste liquid storage part is configured by a space that communicates with the flow path, which is provided, for example, by bonding a base material in which a recess having a required size is formed to the lower side of the base material for the flow path. . If necessary, the space contains a porous body for absorbing a waste liquid such as a sponge.

  The micropump sends out each liquid, such as each reagent stored in the reagent storage section and a liquid stored in another storage section, to the downstream side of the flow path. In general, a plurality of micropumps are installed corresponding to the liquid to be delivered, and each micropump delivers an aqueous or oily driving liquid to the downstream side, and liquids such as samples and reagents are downstream with the driving liquid. Extrude and feed.

  Although the micropump may be formed directly in the microreactor, it can be installed separately from the microreactor, that is, independently of the plate-like chip. For example, a sample inspection device is composed of a micropump and its control device, an optical detection device for reaction detection, a temperature control device, a device main body including a driving liquid tank containing a driving liquid, and a microreactor in which a reagent is sealed in advance. The case where it comprises is mentioned.

In this case, after injecting the sample into the sample receiving portion of the microreactor, the microreactor is attached to the apparatus main body, and the plurality of micropumps on the apparatus main body side and the flow paths of the microreactors corresponding to these micropumps are communicated. Let In this state, the driving liquid from the driving liquid tank is sent out to the flow path of the microreactor by the micropump, thereby pushing out the liquid in the flow path, for example, the reagent in the reagent storage unit and the sample in the sample receiving unit to the downstream side. Mixing each other, mixing a reagent and a sample, and the like.

  The micropump is manufactured by a photolithography technique or the like, and is inserted into an inlet / outlet hole of a valve chamber provided with an actuator and a micropump driven by a piezo element described in Japanese Patent Application Laid-Open Nos. 2001-322099 and 2004-108285. Various types such as a check valve type micro pump provided with a check valve can be used. Note that the micropump driven by the piezo element has a first flow path in which the flow path resistance changes according to the differential pressure, and a change rate of the flow path resistance with respect to the change in the differential pressure is smaller than that of the first flow path. Two flow paths, a pressure chamber connected to the first flow path and the second flow path, and a piezo actuator that changes the internal pressure of the pressure chamber. The liquid can be fed in the forward and reverse directions by driving with.

  The microreactor is manufactured by applying a fine processing technique such as a photolithography technique using a plate-like substrate. Usually, a microreactor is manufactured by forming a channel and a concave portion to be a structure portion on one or more base materials and then bonding a plurality of base materials together.

  Various materials are used for the base material constituting the microreactor depending on the purpose. Specific examples thereof include plastic resins such as polystyrene, rubber-based materials such as polydimethylsiloxane, various inorganic glasses, silicon, ceramics, and metals. Moreover, you may perform various surface treatments, such as a hydrophobization process and a hydrophilic treatment, with respect to the flow-path wall surface according to the objective.

  The size of the channel width in the microreactor is appropriately designed in consideration of the advantages of the microscale space, the channel resistance, etc. For example, the width is several tens to several hundreds μm, preferably 50 to 200 μm, and the depth Is 25 to 300 μm, preferably 50 to 100 μm. The vertical and horizontal sizes of the entire chip of the microreactor are typically several tens of mm and the height is typically several millimeters although it depends on the application.

  The sample receiving portion in the microreactor of the present invention includes a storage chamber for temporarily storing a sample and an injection port for injecting the sample from the outside into the storage chamber. The storage chamber may have various shapes such as a flow channel shape and a liquid reservoir shape.

  The injection port of the sample receiving unit is configured so that a small amount of sample can be injected from the upper surface of the chip, for example. In order to prevent external leakage, contamination, infection when using a biological sample, etc., and to ensure sealing performance, a plug made of an elastic material such as a rubber-like material is formed at the injection port, or polydimethylsiloxane It is desirable that the injection port is covered with a rubber material such as reinforced film. For example, a sample in a syringe is injected with a needle pierced with a rubber stopper or a needle passed through a pore with a lid. In the former case, it is preferable that the needle hole is immediately closed when the needle is pulled out. Further, a sample injection mechanism other than these may be installed.

  The upstream side of the sample receiving part communicates with a flow path communicating with the micropump, and the downstream side communicates with a mixing part with the reagent. The sample injected into the sample receiving part is pushed downstream by the driving liquid supplied by the micropump, pretreated as necessary, and then mixed with the reagent.

Various samples can be injected into the sample receiving portion depending on the purpose. For example, in the case of examining / analyzing biological substances, analyte-containing samples derived from living organisms such as whole blood, plasma, serum, buffy coat, urine, feces, saliva, sputum and the like can be mentioned. In the case of genetic testing, a gene, DNA, or RNA is an analyte as a nucleic acid that serves as a template for an amplification reaction. A sample prepared or isolated from a sample possibly containing such a nucleic acid may be used. Other analytes include various metabolites, hormones, proteins (including enzymes and antigens), and the like.

  In addition, for the purpose of chemical synthesis, the raw material compound to be reacted with the reagent becomes a sample, and in the case of drug screening, drug extraction, metal complex formation / separation, etc. The object to be reacted is a sample.

  The reagent storage unit in the microreactor of the present invention is composed of a storage chamber for storing the reagent. The storage chamber may have various shapes such as a flow channel shape and a liquid reservoir shape. The upstream side of the reagent storage unit communicates with a flow path that communicates with the micropump, and the downstream side communicates with a mixing unit with other reagents. The reagent stored in the reagent storage unit is pushed downstream by the driving liquid supplied by the micropump, and merges and mixes with other reagents.

Various types of reagents are targeted depending on the purpose. For example, reagents for amplifying a gene contained in a specimen by PCR include at least 2′-deoxynucleoside 5′-triphosphate, Taq DNA polymerase, Vent DNA polymerase, or Pfu DN.
A polymerase is included.

In addition, a reagent for amplifying a gene contained in a specimen by the ICAN (Isothermal chimera primer initiated nucleic acid amplification) method described in Japanese Patent No. 3433929 includes at least 2′-deoxynucleoside 5′-triphosphate, A chimeric primer capable of specifically hybridizing to a gene to be detected, a DNA polymerase having strand displacement activity, and an RNase of endonuclease are included.

  In addition, when chemical synthesis is intended, the raw material compound that reacts with the sample serves as a reagent, and when the purpose is drug screening, drug extraction, metal complex formation / separation, etc. Substances to be reacted with are reagents.

  In applications such as genetic testing, it is desirable that the reagent is previously stored in the reagent storage unit so that the test can be performed quickly regardless of location or time. In this case, it is preferable to seal the upstream side and the downstream side of the reagent storage unit in order to prevent evaporation, leakage, bubble mixing, contamination, and denaturation of the built-in reagent.

  The reagent container can be sealed with a sealant. This sealant is solidified or gelled under refrigerated conditions in which the microreactor is stored before use, and melts and becomes fluidized at room temperature during use. For example, oils and fats, aqueous solutions of gelatin, and the like can be used.

  Moreover, you may provide the water-repellent valve | bulb of FIG. 4 mentioned later in the upstream edge part of the storage chamber of a reagent storage part, a downstream edge part, or both of these. Accordingly, the reagent can be effectively prevented from flowing out during storage of the microreactor, and at the time of use, the reagent can be easily pushed out by the micropump.

  In the reaction part, after the liquid mixture of the sample and the mixed reagent is introduced, the reaction is started by, for example, raising the temperature. The reaction section may have various shapes such as a liquid reservoir shape and a flow channel shape. For example, an embodiment in which a sample and a mixed reagent are stored in the liquid reservoir to perform a reaction, and the sample and the mixed reagent are combined in a fine flow channel. The reaction direction of the combined liquid is changed by a micropump, and the reaction form is changed appropriately according to the type of the sample and the reagent, such as an aspect in which the combined liquid is repeatedly moved back and forth in the fine channel. Can do.

  In the microreactor described above, a plurality of reagents are sent from each reagent container to the downstream side by a micropump, and these reagents are merged and mixed, and the obtained mixed reagent is merged with the sample further downstream. The reaction is performed in the reaction section, and when mixing and mixing multiple reagents, even if each reagent is sent to the merge section at the same timing, the top of the liquid will be used unless the timing is accurate. In the part, it is difficult to stabilize the mixing ratio of each reagent. When the initial mixed reagent at the head part is combined with the sample downstream to carry out the reaction, problems often occur because the mixing ratio is not stable at the head part. Therefore, it is desirable to send the mixed reagent to the next step after the mixing ratio is stabilized.

  Therefore, in the present invention, among the mixed reagents generated in the reagent mixing section that generates a mixed reagent by mixing a plurality of reagents delivered from a plurality of reagent storage sections, an initial mixed reagent that is initially mixed is the reaction section. A transmission prevention mechanism is provided for preventing transmission to the camera. Hereinafter, the mechanism for preventing delivery of the initial mixed reagent will be described with reference to FIGS. 1 to 3 and FIGS. 5 to 7.

  FIG. 1 is a diagram showing a configuration of an initial mixed reagent delivery preventing mechanism included in a microreactor according to an embodiment of the present invention. In the present embodiment, a case where the mixed reagent 7c is obtained by using two types of reagents, the reagent 7a and the reagent 7b, is shown.

In the figure, reference numeral 5a denotes a flow path through which the reagent 7a is sent, and its upstream side communicates with a reagent storage section (not shown) in which the reagent 7a is stored.
Reference numeral 5b denotes a flow path through which the reagent 7b is delivered, and its upstream side communicates with a reagent storage unit (not shown) in which the reagent 7b is stored.

  5c is a mixing channel (reagent mixing unit) that mixes the reagent 7a and the reagent 7b to generate the mixed reagent 7c, and the flow of the mixed reagent 7c flows from the junction 6 between the channel 5a and the channel 5b. It consists of a flow path up to the downstream end 8 to be stopped.

  In the middle part of the mixing channel 5c, a delivery channel 5d for sending the mixed reagent 7c to a downstream reaction unit (not shown) is branched. The delivery flow path 5d is composed of a flow path from the branch point to the mixing flow path 5c.

  A water repellent valve 51a is provided on the side of the delivery channel 5d at the connecting portion between the mixing channel 5c and the delivery channel 5d. This water repellent valve (liquid feeding control unit) has the structure shown in FIG. The water repellent valve 51 shown in the figure includes a liquid feeding control passage 52 having a small diameter. The liquid feeding control passage 52 is a narrow flow passage whose cross-sectional area (cross-sectional area of a cross section perpendicular to the flow passage) is smaller than the cross-sectional areas of the upstream flow passage 53a and the downstream flow passage 53b.

  When the flow path wall is formed of a hydrophobic material such as plastic resin, the liquid 54 in contact with the liquid feed control path 52 is transferred to the downstream flow path 53b due to a difference in surface tension with the flow path wall. Passing is restricted.

  When the liquid 54 flows out to the downstream flow path 53b, a liquid supply pressure of a predetermined pressure or more is applied by a micropump, thereby causing the liquid 54 to flow downstream from the liquid supply control passage 52 against the surface tension. Push out to the road 53b. After the liquid 54 flows out to the flow path 53b, the liquid flows to the downstream flow path 53b without maintaining the liquid supply pressure required to push the tip of the liquid 54 to the downstream flow path 53b. That is, until the liquid supply pressure in the positive direction from the upstream side to the downstream side reaches a predetermined pressure, the passage of the liquid from the liquid supply control passage 52 is blocked, and the liquid supply pressure exceeding the predetermined pressure is applied. 54 passes through the liquid feed control passage 52.

When the flow path wall is formed of a hydrophilic material such as glass, it is necessary to apply a water-repellent coating, for example, a fluorine-based coating, to at least the inner surface of the liquid feeding control passage 52.

  In FIG. 1, the initial mixed reagent 7d is selectively sent to the downstream end 8 side at the branch point of the mixing flow channel 5c with the delivery flow channel 5d by the action described below, and is downstream of the mixing flow channel 5c. Trapped in This prevents the initial mixed reagent 7d from flowing out toward the delivery flow path 5d.

  As shown in FIG. 1, the reagent 7a sent from the reagent container to the channel 5a by the micropump and the reagent 7b sent from the other reagent container to the channel 5b by the separate micropump are merged. It merges in the part 6 and is mixed in the mixing channel 5c.

  At this time, the feeding pressure of the mixed reagent 7c by the micropump is set to a pressure lower than the pressure at which the initial mixed reagent 7d and the mixed reagent 7c can pass through the water repellent valve 51a, and the mixed reagent 7c in the mixing channel 5c is fed. To do. As a result, as shown in FIG. 2, the initial mixed reagent 7d does not flow out to the delivery channel 5d side at the connection portion between the mixing channel 5c and the delivery channel 5d, and the downstream end 8 of the mixing channel 5c. Sent to the side.

  Then, after the initial mixed reagent 7d reaches the downstream end 8 of the mixing channel 5c, the liquid feeding pressure of the mixed reagent 7c by the micropump is set to a pressure higher than the pressure at which the mixed reagent 7c can pass through the water repellent valve 51a. . Thereby, as shown in FIG. 3, the mixed reagent 7c in the mixing channel 5c passes through the water repellent valve 51a, and the mixed reagent 7c flows out to the delivery channel 5d.

  By controlling the liquid feeding pressure by the micropump as described above, the initial mixed reagent 7d is accommodated between the intermediate portion of the mixing flow path 5c and the downstream end 8, and the initial mixed reagent 7d is contained in the delivery flow path 5d. Only the mixed reagent 7c that has been truncated is sent out. That is, only a mixed reagent with a stable mixing ratio can be supplied to the subsequent process.

  In this embodiment, a water repellent valve 51b is also provided at the downstream end 8 of the mixing channel 5c. In the water repellent valve 51b, the liquid feeding pressure at which the initial mixed reagent 7d can pass to the downstream flow path is larger than the liquid feeding pressure at which the mixed reagent 7c can pass to the delivery flow path 5d in the water repellent valve 51a. It is a water repellent valve.

  Therefore, after the initial mixed reagent 7d reaches the downstream end 8 of the mixing channel 5c, the liquid feeding pressure of the mixed reagent 7c by the micropump can be passed through the water repellent valve 51a, and the initial mixing can be performed. By setting the pressure so that the reagent 7d cannot pass through the water repellent valve 51b, the mixed reagent 7c is allowed to pass beyond the water repellent valve 51a as shown in FIG. 3, and initial mixing is performed at the downstream end 8 of the mixing channel 5c. The reagent 7d can be prevented from passing beyond the water repellent valve 51b.

  For example, the liquid feed control passages (reference numeral 52 in FIG. 4) in the water repellent valve 51a are made larger in cross-sectional area than the liquid feed control passages in the water repellent valve 51b, thereby making these liquid feed control passages liquid. It is possible to make a difference in the liquid feeding pressure that can pass through. In addition, in some cases, the liquid feeding pressure that allows the liquid to pass by making the surface tension difference between the liquid and the flow path wall of the liquid feeding control passage different between the water repellent valve 51a and the water repellent valve 51b. It is also possible to make a difference in

For example, the flow path ahead of the water repellent valve 51b communicates with the waste liquid storage unit. In this case, the initial mixed reagent 7d and the mixed reagent 7c accommodated between the intermediate portion of the mixing flow path 5c and the downstream end 8 are transferred to the micropump at an appropriate time such as after all the reaction and detection steps are completed. Is pushed out into the flow path ahead of the water-repellent valve 51b and stored in the waste liquid storage part.

  In this embodiment, the water repellent valve 51b is provided at the downstream end 8 of the mixing channel 5c. However, the downstream end 8 is completely sealed without providing the water repellent valve 51b at the downstream end 8. Also good.

  FIG. 5 is a diagram showing a configuration of an initial mixed reagent delivery preventing mechanism provided in a microreactor according to another embodiment of the present invention. Components corresponding to those in FIGS. 1 to 3 are denoted by the same reference numerals, and detailed description thereof is omitted.

  In this embodiment, the basic configuration is the same as that of the above-described embodiment, but an active valve 9 is provided on the delivery flow path 5d side in the connection portion between the mixing flow path 5c and the delivery flow path 5d. The initial mixed reagent 7d is selectively sent to the downstream end 8 side and trapped on the downstream side of the mixing channel 5c at the branch point of the mixing channel 5c with the delivery channel 5d by the action described below. . This prevents the initial mixed reagent 7d from flowing out toward the delivery flow path 5d.

  As shown in FIG. 5, the reagent 7a sent from the reagent container to the channel 5a by the micropump and the reagent 7b sent from the other reagent container to the channel 5b by the separate micropump are merged. It merges in the part 6 and is mixed in the mixing channel 5c.

  At this time, the active valve 9 is in a closed state, and as shown in FIG. 6, the initial mixed reagent 7d is placed on the delivery channel 5d side at the connection portion between the mixing channel 5c and the delivery channel 5d. It is sent out to the downstream end 8 side of the mixing channel 5c without flowing out.

  Then, after the initial mixed reagent 7d passes through the branch point between the mixing channel 5c and the delivery channel 5d, the active valve 9 is opened. Thereby, as shown in FIG. 7, the mixed reagent 7c in the mixing channel 5c passes through the active valve 9, and the mixed reagent 7c flows out to the delivery channel 5d.

  By controlling the active valve 9 as described above, the initial mixed reagent 7d is accommodated between the intermediate portion of the mixing flow path 5c and the downstream end 8, and the initial mixed reagent 7d is cut off in the delivery flow path 5d. Only the mixed reagent 7c is delivered. That is, only a mixed reagent with a stable mixing ratio can be supplied to the subsequent process.

  As the active valve 9, various known structures can be formed in the microreactor. For example, a base material made of a flexible material is pressed from above by a pneumatic piston, a hydraulic piston, a hydraulic piston, a piezoelectric actuator, a shape memory alloy actuator, etc., and an opening disposed below the base material is covered. An active valve with a structure that closes the valve, bimetal is used as the valve body, and the valve body is deformed by energization heating and the flow path is blocked by using a shape memory alloy as the valve body, and the valve body is deformed by energization heating. An active valve having a structure for closing the flow path can be used. Valve opening / closing control is performed by voltage drive or the like by an external control device.

  The initial mixed reagent delivery prevention mechanism provided in the microreactor of the present invention described above is composed of a mixing flow path and a delivery flow path, and the initial mixed reagent is composed of an intermediate portion and a downstream end of the mixing flow path. It is accommodated in between, and the delivery from the delivery channel to the reaction part is prevented.

  Two embodiments having such an initial mixed reagent delivery prevention mechanism have been described. In addition, for example, by making the cross-sectional area of the mixing channel different from the cross-sectional area of the delivery channel, the capillary force By selectively introducing the initial mixed reagent to the channel end side of the mixing channel, it is possible to prevent the initial mixed reagent from being sent to the sending channel side.

  Alternatively, the surface tension of the channel wall of the mixing channel and the surface tension of the channel wall of the delivery channel are made different depending on the channel material or surface treatment of the channel wall, and the initial mixed reagent is selected based on the surface tension. In particular, by guiding to the channel end side of the mixing channel, it is possible to prevent the initial mixed reagent from being sent to the sending channel side.

FIG. 8 is a diagram showing the channel configuration of a microreactor according to another embodiment of the present invention, and shows the channel configuration on the upstream side of the mixing portion with the sample.
Each of the three types of reagents is stored in each of the reagent storage units 33h, 33i, and 33j. Micro pumps 12h, 12i, and 12j built in a device separate from the microreactor are pumped upstream of each reagent storage unit. The reagents are fed by these micropumps from the reagent storage units to the downstream mixing channel 5c, which are connected by the connection unit 31.

  The mixing channel 5c, the delivery channel 5d branched from the mixing channel 5c, and the water-repellent valves 51a and 51b constitute an initial mixed reagent delivery prevention mechanism similar to that shown in FIG. The tip of the mixed solution of each reagent sent from 33i and 33j and merged in the mixing channel 5c is discarded, and the mixed reagent is sent to the next step after the mixed state is stabilized.

  FIG. 9 is a plan view showing another embodiment of the microreactor of the present invention. In the microreactor 1 of this embodiment, three types of reagents are accommodated in each of the flow path-shaped reagent accommodating portions 33a, 33b, and 33c, which are reagent accommodating chambers. A water repellent valve having the structure shown in FIG. 4 is provided at both ends of the reagent storage portion (upstream end portion 34a and downstream end portion 34b in the reagent storage portion 33a). A reagent is sealed in the channel between the two.

  Although not described in detail, the water repellent valve in FIG. 4 is provided in positions other than both ends of the reagent storage portions 33a to 33c in the fine flow path of the microreactor 1 in FIG. The above-described water repellent valve is also provided at the mixed reagent inlet and the sample inlet at the mixed reagent / sample confluence section 38, and the timing of the start of liquid feeding to the flow path beyond that is provided by the water repellent valve. Be controlled.

  Openings 32c to 32e opened from one surface of the microreactor 1 to the outside are provided on the upstream side of the reagent containers 33a to 33c in FIG. These openings 32c to 32e are aligned with flow path openings provided on the connection surface of the micropump unit when the microreactor 1 is connected to a micropump unit to be described later and connected to the micropump.

  Similarly, the openings 32a, 32b, and 32f to 32k communicate with the micropump by connecting the microreactor 1 and the micropump unit. A pump connection portion is constituted by a chip surface including these openings 32a to 32k, and the microreactor 1 and the micropump unit are connected by bringing the pump connection portion into close contact with the connection surface of the micropump unit.

  In order to ensure the necessary sealing performance and prevent leakage of the driving fluid, this pump connection part is formed with an adhesive surface made of a flexible (elastic, shape following) resin such as polytetrafluoroethylene or silicone resin. It is preferred that The close contact surface having such flexibility may be, for example, due to the constituent substrate itself of the microreactor, or by a separate member having flexibility attached around the flow path opening in the pump connection portion. It may be a thing.

Reagents stored in the reagent storage units 33a to 33c are water repellent valves (not shown) provided at the downstream ends of the reagent storage units 33a to 33c by separate micro pumps communicating with the openings 32c to 32e, respectively. The three types of reagents are mixed in the mixing flow path 5c (reagent mixing section 36), which is a flow path following the flow path.

  The flow path system including the mixing flow path 5c and the delivery flow path 5d constitutes an initial mixed reagent delivery prevention mechanism as shown in the embodiment of FIG. 1 or FIG. Is trapped at the channel end 8 of the mixing channel 5c.

  The mixed reagent mixed in the mixing channel 5 c and sent out to the delivery channel 5 d merges with the sample stored in the sample receiving unit 37 at the merging unit 38. The mixed reagent is pushed downstream by the driving liquid by the micro pump communicated with the opening 32b, and the sample is pushed downstream by the driving liquid by the micro pump communicated with the opening 32a. The mixed solution of the mixed reagent and the sample is accommodated in the reaction unit 39 and the reaction is started by heating or the like.

  The liquid after the reaction is sent to the detection unit 40, and the target substance is detected by, for example, an optical detection method. Each of the reagents (for example, a solution for stopping the reaction between the mixed reagent and the sample, a substance to be detected) previously stored in the flow path ahead of these openings by separate micro pumps communicating with the openings 32f to 32j. On the other hand, a liquid for performing necessary processing such as a label, a cleaning liquid, and the like) are extruded downstream at a predetermined timing and fed.

  10 is a perspective view of a micropump unit used in the microreactor of FIG. 9, and FIG. 11 is a sectional view thereof. The micropump unit 11 includes three substrates: a silicon substrate 17, a glass substrate 18 thereon, and a glass substrate 19 thereon. The substrate 17 and the substrate 18 and the substrate 18 and the substrate 19 are joined by anodic bonding, respectively.

A micro pump 12 (piezo pump) is constituted by an internal space between the silicon substrate 17 and the glass substrate 18 bonded thereto by anodic bonding.
The substrate 17 is obtained by processing a silicon wafer into a predetermined shape by a photolithography technique. For example, by fine processing including formation of an oxide film on the silicon substrate surface, resist application, resist exposure and development, oxide film etching, silicon etching by ICP (Inductively Coupled Plasma), etc. A pressurizing chamber 22, a first channel 23, a first liquid chamber 25, a second channel 24, and a second liquid chamber 26 are formed.

  At the position of the pressurizing chamber 22, the silicon substrate is processed into a diaphragm, and a piezoelectric element 21 made of lead zirconate titanate (PZT) ceramic or the like is attached to the outer surface thereof.

  The micropump 12 is driven by the control voltage to the piezoelectric element 21 as follows. The piezoelectric element 21 is vibrated by the applied voltage having a predetermined waveform, and the silicon diaphragm at the position of the pressurizing chamber 22 is vibrated, thereby increasing or decreasing the volume of the pressurizing chamber 22. The first flow path 23 and the second flow path 24 have the same width and depth, and the length of the second flow path 24 is longer than that of the first flow path 23. In the first flow path 23, when the differential pressure increases, turbulent flow is generated in the flow path, and the flow path resistance increases. On the other hand, in the second flow path 24, the flow path width is long, so that even if the differential pressure increases, it tends to be laminar flow, and the change rate of the flow resistance with respect to the change in differential pressure is smaller than that of the first flow path 23. .

  For example, by adjusting the control voltage for the piezoelectric element 21, the volume of the pressurizing chamber 22 is decreased while applying a large differential pressure by quickly displacing the silicon diaphragm in the direction toward the inside of the pressurizing chamber 22, and then pressurizing. When the volume of the pressurizing chamber 22 is increased while slowly displacing the silicon diaphragm from the chamber 22 toward the outside to give a small differential pressure, the driving liquid is sent in the forward direction from left to right in FIG. To be liquidated.

  On the contrary, the volume of the pressurizing chamber 22 is increased while giving a large differential pressure by quickly displacing the silicon diaphragm in the direction from the pressurizing chamber 22 toward the outside, and then the direction toward the inside of the pressurizing chamber 22. When the volume of the pressurizing chamber 22 is reduced while slowly displacing the silicon diaphragm to give a small differential pressure, the driving liquid is fed in the reverse direction from right to left in FIG.

  Note that the difference in flow rate resistance change ratio with respect to the change in differential pressure between the first flow channel 23 and the second flow channel 24 is not necessarily due to the difference in the length of the flow channel. It may be based.

Control of the flow rate by the micropump 12 can be performed by adjusting the voltage applied to the piezoelectric element 21.
A flow path 20 is patterned on the substrate 19. As an example, the size and shape of the channel 20 are rectangular in cross section with a width of about 150 μm and a depth of about 300 μm. On the downstream side of the flow path 20, an opening 15 for allowing the micro pump 12 to communicate with the fine flow path of the inspection chip by being aligned with the openings 32 a to 32 k of the microreactor in FIG. 9 is provided.

  The upstream side of the flow path 20 communicates with the micro pump 12 through the flow path provided in the substrate 17 via the through hole 16 b of the substrate 18. The upstream side of the micropump 12 communicates with the opening 14 provided in the glass substrate 19 from the flow path provided in the substrate 17 through the through hole 16 a of the substrate 18. The opening 14 is connected to a driving liquid tank (not shown). The opening 14 is connected to the driving liquid tank through, for example, PDMS (polydimethylsiloxane) packing.

  Each of the openings 15a, 15b, and 15c communicates with the openings 32c, 32d, and 32e of the microreactor shown in FIG. 9 (note that only a part of the entire micropump unit is shown in FIG. 10). The micropump 12 sends the driving liquid through the flow path 20, the opening 15a, and the opening 32c, pushes the reagent stored in the reagent storage section 33a downstream, and sends the driving liquid through the flow path 20, the opening 15b, and the opening 32d. The reagent stored in the reagent storage unit 33b is pushed downstream, the driving liquid is fed through the flow path 20, the opening 15c, and the opening 32e to push the reagent stored in the reagent storage unit 33c downstream.

  For example, the microreactor is subjected to reaction and analysis by being mounted on a separate system body. The system main body and the microreactor constitute a micro total analysis system. An example of this micro total analysis system will be described below. FIG. 12 is a perspective view showing an example of a micro total analysis system, and FIG. 13 is a diagram showing an internal configuration of a system main body in the micro total analysis system.

  The system main body 3 of the micro comprehensive analysis system 2 includes a casing-like storage body 62 that stores each device for analysis. Inside the storage body 62 is disposed a micropump unit 11 provided with a chip connection portion 13 having a channel opening for communicating with the microreactor 1 and a plurality of micropumps (not shown). .

Further, inside the container 62, a detection processing device for detecting a reaction in the microreactor 1 (LED, photomultiplier, light source 68 such as a CCD camera, and optical detection by visible spectroscopy, fluorescence photometry, etc. And a control device (not shown) for controlling the detection processing device and the micropump unit 11 are provided. In addition to the control of liquid feeding by the micropump and the control of the detection processing device for detecting the reaction in the microreactor 1 by optical means, etc., the temperature control of the microreactor 1 by the heating / cooling unit described later, the reaction in the microreactor 1 Control, data collection (measurement) and processing. Control of the micropump is performed by applying a driving voltage corresponding to the program according to a program in which various conditions relating to the liquid feeding sequence, flow rate, timing, and the like are set in advance.

  In the micro integrated analysis system 2, a pump connection portion 31 including a flow channel opening provided on the upstream side of a micro flow channel of the microreactor 1 (for example, upstream of a reagent storage unit, a sample receiving unit, etc.) and a chip surface around the channel opening. Then, the microreactor 1 is mounted in the housing 62 in a state where the chip connection portion 13 of the micropump unit 11 is in liquid tight contact, and then the target substance in the specimen is analyzed in the microreactor 1. The microreactor 1 is placed on the transport tray 65 and introduced into the storage body 62 from the insertion port 63. However, if the microreactor can be fixed inside the storage body 62 in a state where the microreactor is pressurized with respect to the micropump unit, the transport tray is not necessarily used.

  Inside the container 62, a heating / cooling unit (Peltier element 66, heater 67) for locally heating or cooling the microreactor 1 mounted at a predetermined position is provided. For example, the Peltier element 66 is pressed against the region of the reagent storage unit in the microreactor 1 to selectively cool the reagent storage unit, thereby preventing deterioration of the reagent and the like, and in the flow channel region constituting the reaction unit. The reaction part is selectively heated by press-contacting the heater 67, thereby bringing the reaction part to a temperature suitable for the reaction.

  The micro pump unit 11 is connected to one driving liquid tank 61, and the upstream side of the micro pump communicates with the driving liquid tank 61. On the other hand, the downstream side of the micropump is communicated with a channel opening provided on one side of the micropump unit 11, and is connected to each channel opening communicated with each micropump and the pump connection portion 31 of the microreactor 1. The microreactor 1 is connected to the micropump unit 11 so that each provided flow path opening is connected.

  An oil-based or water-based driving liquid such as mineral oil stored in the driving liquid tank 61 is sent out by the micropump to the liquid storage section in the microreactor 1 via the pump connection section 31, and each storage section is driven by the driving liquid. The liquid is extruded to the downstream side of the microreactor 1 and fed.

  A series of analysis steps of pretreatment, reaction, and detection of a sample as a measurement sample is performed in a state where the microreactor 1 is mounted on a system body 2 in which a micropump, a detection processing device, and a control device are integrated. Preferably, predetermined reactions and optical measurements based on sample and reagent delivery, pretreatment, and mixing are automatically performed as a series of continuous steps, and the measurement data is stored in a file along with the necessary conditions and recorded items. Stored in In FIG. 12, the analysis result is displayed on the display unit 64 of the container 62.

Specific examples of the reaction between the sample (specimen) and the reagent using the microreactor of the present invention and the detection thereof will be shown below. In a preferred embodiment of the microreactor, in one chip,
A sample receiving part into which a sample or an analyte extracted from the sample (for example, DNA, RNA, gene) is injected;
A sample pretreatment unit for preprocessing the sample;
A reagent container for storing reagents used for probe binding reaction, detection reaction (including gene amplification reaction or antigen-antibody reaction);
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 that can be connected to each of the accommodating portions and a separate micropump for feeding the liquid in the flow path is provided.

  A micropump is connected to the microreactor via a pump connection unit, and the sample is injected into the sample receiving unit or a biological material extracted from the sample (for example, DNA or other biological material) and stored in the reagent storage unit. The reagent is fed to the downstream flow path, and mixed and reacted in a reaction section of the fine flow path, for example, a reaction section that performs a gene amplification reaction (such as an antigen-antibody reaction in the case of protein). Next, the processing solution obtained by treating this reaction solution and the probe accommodated in the probe accommodating portion are sent to the detection portion in the downstream flow channel, mixed in the flow channel, and combined with the probe (or high). A biological substance is detected based on the reaction product.

In addition, the above reaction and detection are performed 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 .

  The sample injected into the sample receiving unit is pretreated, for example, by mixing the sample and the treatment liquid in a sample pretreatment unit provided in advance in the flow path before mixing with the reagent, if necessary. The The specimen pretreatment unit may include a separation filter, an adsorption resin, beads, and the like. Preferable sample pretreatment includes separation or concentration of analyte, deproteinization, and the like. For example, lysis treatment and DNA extraction treatment are performed using a lysis agent such as a 1% SDS mixed solution. In this process, DNA is released from the inside of the cell and adsorbed on the membrane surface of the bead or filter.

  A predetermined amount of a necessary reagent is sealed in advance in the reagent storage section of the microreactor. Therefore, it is not necessary to fill a necessary amount of the reagent each time it is used, and it is ready for use. When analyzing a biological substance in a specimen, reagents necessary for the measurement are generally known. For example, when analyzing an antigen present in a specimen, a reagent containing an antibody against it, preferably a monoclonal antibody, is used. The antibody is preferably labeled with biotin and FITC.

  In addition to various reagents used for gene amplification, reagents stored in the microreactor for genetic testing include probes used for detection, coloring reagents, pretreatment reagents used for the sample pretreatment, and the like. .

  By supplying the driving liquid from the micropump, the reagent is pushed out from each reagent container and joined together to generate a mixed reagent. At this time, the initial mixed reagent is discarded by the initial mixed reagent delivery mechanism shown in FIGS. 1 to 3 and the like, and only the mixed reagent having a stable mixing ratio is sent downstream.

  After that, by supplying the driving liquid from the micropump, the specimen is pushed out from the sample receiving part and merged with the mixed reagent having a stable mixing ratio, so that in the reaction part, gene amplification reaction, analyte trap or antigen antibody Reactions required for analysis such as reactions are started.

As a DNA amplification method, a PCR amplification method described in various documents including improvements and actively used in various fields can be used. In the PCR amplification method, 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. 2005-318867). 2004-108285). 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.

Recently developed ICAN (Isothermal chimera primer initiated nucleic acid
The amplification method is a suitable amplification technique even in the system of the present invention because DNA amplification can be performed in a short time at an arbitrary constant temperature of 50 to 65 ° C. (Japanese Patent No. 3343929). By hand, the method, which takes one hour, ends in 10-20 minutes, preferably 15 minutes, in the system of the present invention.

  A detection unit for detecting an analyte, for example, an amplified gene, is provided downstream of the reaction unit in the microchannel of the microreactor. At least the detection part is made of a transparent material, preferably a transparent plastic, in order to enable optical measurement.

  The biotin-affinity protein (avidin, streptavidin) adsorbed on the detection section 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 gene is trapped at the detection site.

A method for detecting the separated analyte or the amplified DNA of the target gene is not particularly limited, but as a preferred embodiment, it is basically performed in the following steps.
(1a) Fine flow channel downstream from the containing portion of a sample or DNA extracted from the sample or cDNA synthesized by reverse transcription reaction from RNA extracted from the sample or sample and a biotin-modified primer at the 5 ′ position To liquid.

After performing the gene amplification reaction in the microchannel of the reaction section, the amplification reaction solution containing the gene amplified in the microchannel and the denaturing solution are mixed, and the amplified gene is single-stranded by denaturation treatment. This is hybridized with a probe DNA fluorescently labeled with FITC (fluorescein isothiocyanate) at the end.

Next, the solution is sent to the detection site in the microchannel to which the biotin-affinity protein is adsorbed, and the amplified gene is trapped in the detection site in the microchannel (the probe labeled with the fluorescence after trapping the amplified gene at the detection site) It may be hybridized with DNA.)
(1b) A reagent containing an antibody, preferably a monoclonal antibody, specific to an analyte such as an antigen, metabolite or hormone present in the sample is mixed with the sample. In that case, the antibody is labeled with biotin and FITC. Therefore, the product obtained by the antigen-antibody reaction has biotin and FITC. This is sent to a detection site in a microchannel to which a biotin affinity protein (preferably streptavidin) is adsorbed, and is immobilized on the detection site through the binding of biotin affinity protein and biotin.
(2) A colloidal gold solution whose surface has been modified with an anti-FITC antibody that specifically binds to FITC flows through the fine channel, and this hybridizes to the immobilized FITC of the analyte / antibody reaction product or to the gene. The gold colloid is adsorbed to the FITC-modified probe.
(3) Optically measure the concentration of colloidal gold in the fine channel.

As mentioned above, although embodiment of this invention was described, this invention is not limited to these embodiment, In the range which does not deviate from the summary, various deformation | transformation and change are possible.

FIG. 1 is a diagram showing a configuration of an initial mixed reagent delivery prevention mechanism provided in a microreactor according to an embodiment of the present invention, and shows a state in which two reagents are joined and mixed in a mixing channel 5c. Yes. FIG. 2 is a view showing a state in which the front end of the mixed reagent has passed to the downstream end side of the mixing channel in the delivery preventing mechanism of FIG. FIG. 3 is a view showing a state in which the mixed reagent flows out into the delivery channel after the leading end of the mixed reagent reaches the downstream end of the mixing channel in the delivery preventing mechanism of FIG. FIG. 4 is a view showing a water repellent valve. FIG. 5 is a diagram showing a configuration of an initial mixed reagent delivery prevention mechanism provided in a microreactor according to another embodiment of the present invention, and shows a state in which two reagents are joined and mixed in the mixing channel 5c. ing. FIG. 6 is a view showing a state in which the leading end portion of the mixed reagent has passed to the downstream end side of the mixing channel in the delivery preventing mechanism of FIG. FIG. 7 is a view showing a state in which the mixed reagent flows out into the delivery channel after the leading end of the mixed reagent reaches the downstream end of the mixing channel in the delivery prevention mechanism of FIG. FIG. 8 is a diagram showing the channel configuration of a microreactor according to another embodiment of the present invention, and shows the channel configuration on the upstream side of the mixing portion with the sample. FIG. 9 is a plan view showing another embodiment of the microreactor of the present invention. FIG. 10 is a perspective view of a micropump unit used in the microreactor of FIG. FIG. 11 is a cross-sectional view of the micropump unit of FIG. FIG. 12 is a perspective view showing an example of a micro total analysis system. FIG. 13 is a diagram showing an internal configuration of a system main body in this micro comprehensive analysis system.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microreactor 2 Micro comprehensive analysis system 3 System main body 5a Flow path 5b Flow path 5c Mixing flow path 5d Sending flow path 6 Junction part 7a Reagent 7b Reagent 7c Mixed reagent 7d Initial mixed reagent 8 Downstream end 9 Active valve 11 Micro pump unit 12, 12h to 12j Micropump 13 Chip connection portion 14 Opening 15, 15a to 15c Opening 16a Through hole 16b Through hole 17 Substrate 18 Substrate 19 Substrate 20 Channel 21 Piezoelectric element 22 Pressurizing chamber 23 First channel 24 Second channel 25 1st liquid chamber 26 2nd liquid chamber 31 Pump connection part 32a-32k Opening 33a-33c, 33h-33j Reagent storage part 34a, 34b End part 35 Merge part 36 Reagent mixing part 37 Sample receiving part 38 Merge part 39 Reaction part 40 Detectors 51, 51a to 51e Water repellent valve 52 Liquid feed control passages 53a, 53b Flow path 54 Liquid 61 Dynamic liquid tank 62 accommodating body 63 insertion port 64 display unit 65 the carrier tray 66 Peltier element 67 heater 68 light source 69 detector

Claims (4)

A plate-shaped chip;
A plurality of reagent storage sections each including a storage chamber in which a plurality of reagents are individually stored;
A reagent mixing unit that mixes a plurality of reagents delivered from the plurality of reagent storage units to generate a mixed reagent; and
A sample receiver having an inlet for injecting a sample from the outside;
A mixed reagent sent from the reagent mixing part and a reaction part for mixing and reacting the sample sent from the sample receiving part,
The plurality of reagent storage units, the reagent mixing unit, the sample receiving unit, and the reaction unit are incorporated in the chip and communicated with each other through a flow path,
A microreactor comprising a delivery preventing mechanism for preventing an initial mixed reagent mixed in the initial stage from among the mixed reagents generated in the reagent mixing unit from being sent to the reaction unit. The reagent mixing part is a flow path for mixing the plurality of reagents, and includes a mixing flow path provided with a downstream end where the flow of the mixed reagent is stopped,
In the middle part of the mixing channel, a delivery channel for sending the mixed reagent to the reaction unit is branched,
The delivery prevention mechanism is composed of the mixing channel and the delivery channel,
2. The microreactor according to claim 1, wherein the initial mixed reagent is accommodated between an intermediate portion and a downstream end of the mixing channel, and is prevented from being sent from the sending channel to the reaction unit. .   The delivery preventing mechanism includes a liquid delivery control unit that delivers a mixed reagent to the delivery channel when the pressure in the mixing channel exceeds a predetermined pressure at a connection portion between the mixing channel and the delivery channel. The microreactor according to claim 2.   The liquid feeding control unit is a narrow channel having a cross-sectional area smaller than the cross-sectional area of the delivery channel, provided on the delivery channel side in the connection portion between the mixing channel and the delivery channel. The microreactor according to claim 3.

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