JP4682874B2 - Microreactor - Google Patents

Description

  The present invention relates to a microreactor used for biological substance inspection / analysis by, for example, gene amplification reaction, antigen-antibody reaction, etc., inspection / analysis of other chemical substances, chemical synthesis of a target compound by organic synthesis or the like.

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, and such a technique has already been disclosed in Patent Document 1 and the like.

  Such a technology is also called micro-TAS (Micro Total Analysis System), bioreactor, Lab-on-chip, biochip, medical examination / diagnosis field, environmental measurement field, agricultural production Its application is expected in the 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 economy of these analysis chips. For this purpose, there is a problem to establish a highly reliable liquid feeding system with a simple configuration, and there is a demand for a microfluidic control element that is highly accurate and excellent in reliability. The present inventors have already proposed a control method (see Patent Documents 2 to 4).

  By the way, in the flow path of such an analysis chip (hereinafter referred to as a microreactor), the liquid is placed in a desired position by utilizing the flow resistance or surface tension of the liquid in the flow path as a part of the flow path. A water-repellent valve is provided that can be stopped at and restarted from the stopped position.

  Specifically, as shown in FIGS. 12A and 12B, such a water-repellent valve 100 has a flow path 102 between the upstream flow path 102 and the downstream flow path 104. A liquid feed control flow path 106 having a smaller cross-sectional area than 104 is provided.

  For example, when the flow path walls of these flow paths 102 and 104 are formed of a hydrophobic material such as plastic resin, the liquid 112 in contact with the liquid feed control flow path 106 is downstream of the control flow path. The passage to the downstream channel 104 is restricted by the difference in the surface tension of the channel wall between the end 110 and the downstream channel 104.

  When the liquid 112 flows out to the downstream flow path 104, a liquid feed pressure of a predetermined pressure or higher is applied by a micropump (not shown), thereby causing the liquid 112 to flow against the surface tension. It pushes out to the downstream flow path 104 from 106.

  Note that after the liquid 112 has flowed into the downstream channel 104, the liquid 112 does not have to be maintained on the downstream side without maintaining the liquid feeding pressure required to push the tip of the liquid 112 to the downstream channel 104. It flows to the flow path 104.

  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 112 from the liquid supply control flow path 106 is blocked at the control flow path downstream end 110, The liquid 112 passes through the end portion 110 on the downstream side of the control flow path by applying a liquid feeding pressure equal to or higher than a predetermined pressure.

As shown in FIG. 13, such a water repellent valve 100 is provided at both ends of the reagent storage unit 114 in the microreactor, for example.
In such a reagent container 114, the reagent 116 is sealed in advance, and the reagent 116 does not flow out of the reagent container 114 by the water repellent valves 100 and 100 provided at both ends of the reagent container 114. It is like that.

  As shown in FIG. 14, when the driving liquid 122 is poured into the flow path 120 from the opening 118 by a micropump (not shown) from the state of FIG. 13, the driving liquid 122 enters the branch flow path 126 branched from the branch point 124. Will be filled.

At this time, the air existing in the channel 120 and the branch channel 126 passes through the air vent channel 128 and is released to the outside.
Further, when the driving liquid 122 is filled in the branch flow path 126 while expelling air from the air vent flow path 128, the air vent flow path 128 blocks the passage of the drive liquid 122 due to water repellency or is a high flow path. A small amount of driving fluid 122 is allowed to flow in a state where the resistance is loaded.

  Then, the liquid feeding pressure by the micropump (not shown) strongly acts on the reagent 116 via the driving liquid 122, and the water repellent valve 100 on the downstream side of the reagent storage unit 114 has a liquid holding force higher than that. The liquid feeding pressure is given.

As a result, the reagent 116 is pushed out of the reagent container 114 by the driving liquid 122, passes through the water repellent valve 100, and is sent out to the downstream flow path.
JP 2004-28589 A JP 2001-322099 A JP 2004-108285 A JP 2004-270537 A

  A microreactor provided with such a water repellent valve 100 is molded by injection molding using a mold 200 as shown in FIG. Since the liquid control flow path equivalent portion 202 has a very fine shape, the resin 300 is not sufficiently filled up to the tip end portion 204 at this portion, and a liquid feed control flow path having a desired shape may not be obtained.

  In the water-repellent valve 100 in which a liquid-feeding control flow path having a desired shape is not obtained, the water-repellent valve 100 has a larger dimension than the originally required water-repellent valve or the corner of the water-repellent valve is rounded. The holding force cannot be obtained, and the liquid cannot be stopped accurately.

  Further, in the conventional water-repellent valve, as shown in FIG. 16, when the reagent 116 is originally stopped at the downstream end portion 110 of the control channel, it is stopped at the upstream end portion 108 of the control channel. Will occur.

When the reagent 116 stops at the upstream end 108 of the control channel in this way, the driving liquid 122 is stopped.
A small bubble 132 may be generated in the space 130 between the reagent 116 and the reagent 116.
The liquid feed timing control is based on the premise that the reagent 116 is stopped at the downstream end portion 110 of the control flow path. When the reagent 116 is stopped at a location different from the original in this way, bubbles 132 are generated. As a result, the timing of liquid feeding is delayed, and there is a problem that accurate liquid feeding cannot be performed.

  In view of such a current situation, the present invention can reliably stop a liquid such as a reagent and a specimen at a desired position, and can be surely restarted from the stopped position, and further has a water-repellent property with stable liquid feeding properties. An object is to provide a microreactor having a valve.

  Another object of the present invention is to provide a microreactor having a desired flow path shape in which resin is reliably filled up to the tip of the flow path using injection molding.

The present invention was invented in order to achieve the problems and objects in the prior art as described above, and the microreactor of the present invention comprises:
A flow path is provided on at least one surface of the plate-shaped chip, and a liquid feed control unit constituting a water repellent valve is provided in a part of the flow path, whereby the liquid feed speed and liquid feed of the liquid in the flow path are provided. A microreactor with timing control,
The liquid feeding control unit is
An upstream connection port having a cross-sectional area smaller than the cross-sectional area of the channel on the upstream side;
A downstream connection port having a cross-sectional area smaller than the cross-sectional area of the flow path on the downstream side;
A connection port communicating portion for communicating the upstream connection port and the downstream connection port,
The upstream connection port is configured to have a cross-sectional area larger than the cross-sectional area of the downstream connection port,
The connection port communication portion is configured so that a cross-sectional area continuously changes.

By configuring in this way, it is possible to stop the liquid such as the reagent and the specimen at a desired position and restart from the stopped position.
Furthermore, since the cross-sectional area of the connection port communication portion changes continuously, the liquid can be smoothly fed, and it is ensured that the liquid such as the reagent or the sample stops in the connection port communication portion. Can be prevented.
In addition, since the cross-sectional area of the upstream connection port is larger than the cross-sectional area of the downstream connection port, the liquid is reliably stopped at the downstream connection port without stopping the liquid at the upstream connection port. Can be stopped.
Furthermore, if the connection port communication portion is configured so as to expand from the downstream connection port toward the upstream connection port, the injection port communication portion is connected to a corresponding portion of the mold connection port during injection molding of the microreactor. Thus, the resin filling is reliably performed, and the yield at the time of molding the microreactor can be improved.

The microreactor of the present invention is
The microreactor is
Comprising a reservoir for storing reagents or specimens;
The liquid feeding control unit is provided on each of one side and the other side of the storage unit.

Thus, if the liquid feeding control unit is provided on one side and the other side of the storage unit, the driving liquid is fed by the micropump, and the reagent in the storage unit is pushed to the downstream side of the flow path by this driving liquid. Even in this case, since the reagent is surely stopped at a desired position, the reagent can be reliably pushed downstream without generating bubbles.
The microreactor of the present invention is
A width from the upstream connection port to the downstream connection port of the connection port communication portion is 40 μm to 300 μm.

  According to the present invention, a microreactor having a water-repellent valve that can reliably stop a liquid such as a reagent or a specimen at a desired position and can be reliably restarted from the stopped position and has a stable liquid feeding property. Can be provided.

  In addition, the present invention can provide a microreactor having a desired flow path shape in which resin is reliably filled up to the tip of the flow path using injection molding.

Hereinafter, embodiments (examples) of the present invention will be described in more detail with reference to the drawings.
<Microreactor 10>
FIG. 1 is a top view of a microreactor in an embodiment of the present invention, and FIG. 2 is a partial sectional view perpendicular to the chip surface of the microreactor in an embodiment of the present invention.

  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.

  Microreactor applications 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, drug extraction, This includes the formation and separation of metal complexes.

The microreactor 10 schematically shown in FIG. 1 includes a storage unit 29 (hatched portion in FIG. 1) in which a reagent 30 is preliminarily sealed, and a flow path 12 extending downstream from the storage unit 29.
The reagent 30 is supplied from the reagent supply hole 11. When the reagent 30 is supplied into the microreactor 10, the reagent 30 is filled up to just before each of the water repellent valves 21 and 21.

Further, downstream of these storage units 29, a plurality of storage units 29 merge at the merge unit 13, and a fine channel 15 is provided at the end.
A sample collection unit (not shown) is provided downstream of the fine channel 15, and the sample collected in the sample collection unit (not shown) and a plurality of reagents passing through the fine channel 15. The liquid mixed with 30 is further mixed.

Further, in the storage unit 29 of the microreactor 10, valve parts such as water repellent valves 21 and 21 are arranged at appropriate positions, and control of, for example, determination of the amount of liquid fed and mixing of each liquid is performed.
A pump connection portion 94 for connecting to the chip connection portion 88 of the micropump unit 50 is provided upstream of the storage portion 29 of the microreactor 10.

  As shown in FIG. 2, such a microreactor 10 produces a base material 14 in which a groove serving as a flow path is formed by injection molding, and coats base materials 16 and 18 on both sides of the base material 14. Thus, the flow path 12 and the fine flow path 15 are formed.

Various resin materials can be used as the resin material of the base material 14 according to the purpose. Examples thereof include polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polymethyl methacrylate, and polycarbonate.

The vertical and horizontal sizes of the microreactor 10 are typically several tens of millimeters depending on the application, and the thickness is typically several millimeters.
In the microreactor 10, the flow channels 12 are provided on both surfaces, and the flow channels 12 on both surfaces are formed in the base material 14 and communicated with each other through the through holes 20.

  The channel (microchannel) has a channel width of 200 μm or less, preferably 0.1 μm to 50 μm, a channel width of 50 μm or more, preferably 70 μm to 500 μm, and a channel width. Is composed of a flow path of 200 μm or more, preferably 300 μm to 5 mm.

Here, the “flow path width” represents the horizontal width when the cross section perpendicular to the flow direction is a rectangle, and represents the average value of the horizontal width when the cross section has another shape similar to the rectangle. .
The height of the flow channel 12 is appropriately set according to the purpose regardless of the fine flow channel and the flow channel wider than that, and is, for example, 10 μm to 1000 μm.

  Typically, each reagent 30 accommodated in the plurality of storage units 29 is mixed in the downstream storage unit 29, and further, the mixed reagent in each storage unit 29 is merged in the merge unit 13, and the downstream microchannel 15 is fed.

Downstream of the fine channel 15, the sample and the mixed reagent are merged and mixed from the Y-shaped channel or the like, and the reaction is started by temperature rise or the like, and the reaction is detected at the detection site provided in the channel. It has become.
<Water repellent valve 21>
3A and 3B are views showing a water-repellent valve according to an embodiment of the present invention. FIG. 3A is a top view, FIG. 3B is a cross-sectional view taken along line XX in FIG. 4 is a view showing a water-repellent valve in another embodiment of the present invention, FIG. 4 (a) is a top view, FIG. 4 (b) is a cross-sectional view taken along line XX of FIG. 4 (a), FIG. 5 is a schematic top view showing the storage part of the microreactor and the water-repellent valves provided at both ends of the storage part in the embodiment of the present invention.

As shown in FIGS. 3A and 3B, the water repellent valve 21 provided in a part of the flow path 12 of the microreactor 10 is configured by a liquid feeding control unit 22.
The liquid supply control unit 22 includes an upstream connection port 26 having a cross-sectional area smaller than the cross-sectional area of the upstream flow path 23 and a downstream connection having a cross-sectional area smaller than the cross-sectional area of the downstream flow path 24. The port 27 includes a connection port communication portion 25 that communicates the upstream connection port 26 and the downstream connection port 27.

At this time, the connection port communication portion 25 changes from the upstream side through which the liquid 28 flows toward the downstream side so that the cross-sectional area continuously decreases.
If the connection port communication portion 25 has such a shape, the liquid 28 in the upstream flow path 23 is more likely to flow into the connection port communication portion 25 from the upstream connection port 26, so the liquid 28 is connected to the upstream side. Without stopping near the opening 26, it is possible to reliably stop near the downstream connection port 27.

  Further, since the shape of the connection port communication portion 25 changes smoothly and continuously from the upstream connection port 26 to the downstream connection port 27, the liquid 28 is in the middle of the connection port communication portion 25. Will never stop. The point is that the cross-sectional shape from the upstream side connection port 26 to the downstream side connection port 27 may be any shape as long as it continuously decreases toward the downstream side connection port 27.

  Further, as shown in FIGS. 4A and 4B, for example, in the cross-sectional shape of the connection port communication portion 25 in the XX direction, it is continuous from the upstream connection port 26 toward the downstream connection port 27. It may be made smaller.

  As shown in FIG. 5, such a water repellent valve 21 is provided, for example, at both ends of a storage unit 29 for storing reagents and samples in a microreactor, and the reagents 30 and samples in the storage unit 29 are disposed downstream. Without stopping to flow into the side flow paths 24, 24, it is surely stopped near the downstream connection port 27.

As a result, even if a driving liquid (not shown) flows into the flow path 24 from the opening 35 by a micropump (not shown), a space is left between the driving liquid (not shown) and the reagent 30. Since it does not occur, the generation of bubbles does not occur, and the reagent 30 can be reliably fed.
<Manufacturing method of microreactor 10>
6 is a partial cross-sectional view of a mold for molding a microreactor in an embodiment of the present invention, and FIG. 7 is a partial top view showing a portion corresponding to a connection port communication portion of a mold for molding a microreactor in an embodiment of the present invention. It is.

  In a microreactor, a substrate provided with a flow path is a pair of molds composed of a mold having a flow path pattern formed on one surface and a mold formed with a flow path pattern on the other surface. It is manufactured by injection molding using

As the mold, it is preferable to use a mold mainly composed of nickel or a silicon wafer capable of forming a fine flow path pattern.
Such a mold is manufactured, for example, by nickel electroforming or the like using a flow path manufactured by microfabrication using a photolithography technique as a mother mold.

  Normally, when a die is produced by nickel electroforming, each portion of the die 36 is provided with a taper (draft angle) 37 of about 1 to 3 degrees in order to improve the releasability of the base material after molding. ing.

  However, if the flow of the liquid flowing in the water repellent valve 21 is different between the upper part and the lower part of the flow path, problems are likely to occur, and the fine flow path is easily affected by the shape change due to the taper, resulting in a decrease in performance. Therefore, it is preferable that the water repellent valve equivalent portion 38 of the mold 36 is not provided with a taper (draft angle) but is a straight portion 39.

  In addition, the mold 36 preferably does not have a taper (draft) 37 other than the water repellent valve corresponding portion 38, for example, a flow channel equivalent portion having a flow channel width of 50 μm or less and a flow channel depth of 50 μm or less.

In this way, it is possible to reliably release the base material from the mold 36 without impairing the releasability at the time of molding.
Further, the flow path pattern having a relatively large flow path width can be formed by using a hard metal material, preferably a mold steel, by ordinary machining, for example, cutting by numerical control.

A pair of molds 36 in which the flow path pattern is formed in this way is set in an injection molding machine, and the mold 36 is filled with a resin, whereby a base material (not shown) is molded.
In the present invention, as shown in FIG. 7, the width t1 of the connection port communication portion corresponding portion 34 of the water repellent valve 21 in the mold is 30 μm to 1000 μm, preferably 40 μm to 300 μm. The end portion of the portion equivalent portion 34 is securely filled.
<Micro pump unit 50>
FIG. 8 is a perspective view showing an example of a micropump unit connected to the microreactor in the embodiment of the present invention, and FIG. 9 is a diagram of the micropump unit shown in FIG. 8 connected to the microreactor in the embodiment of the present invention. It is sectional drawing.

The micropump unit 50 is preferably independent from the microreactor 10.
For example, a sample is obtained from a micropump unit 50 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 10 in which a reagent is sealed in advance. The case where an inspection apparatus is comprised is mentioned.

  In this case, after injecting the sample into the sample receiving portion of the microreactor 10, the microreactor 10 is mounted on the apparatus main body, and a plurality of micropumps 58 on the apparatus main body side and each of the microreactors 10 corresponding to these micropumps 58 are installed. Communicate with the flow path.

  In this state, the driving liquid from the driving liquid tank is sent out to the flow path of the microreactor 10 by the micropump 58, whereby the liquid in the flow path, for example, the reagent 30 in the storage unit 29, the sample in the sample receiving part, etc. is downstream. Extrusion is performed to mix reagents and to mix reagents and samples.

  The micropump 58 is manufactured by a photolithography technique or the like, and is a micropump driven by a piezo element described in Japanese Patent Laid-Open Nos. 2001-322099 and 2004-108285, and an inflow / outlet hole of a valve chamber provided with an actuator Various types such as a check valve type micro pump provided with a check valve can be used.

The micro pump unit 50 shown in FIG. 8 is composed of three substrates: a silicon substrate 52, a first glass substrate 54 thereon, and a second glass substrate 56 thereon.
The silicon substrate 52 and the first glass substrate 54, and the first glass substrate 54 and the second glass substrate 56 are bonded by anodic bonding, diffusion bonding, thermal welding, adhesion, or the like, respectively.

A micro pump 58 (piezo pump) is constituted by an internal space between the silicon substrate 52 and the glass substrate 54 bonded thereto by anodic bonding or the like.
The silicon substrate 52 is obtained by processing a silicon wafer into a predetermined shape by a photolithography technique.

  For example, fine processing including formation of an oxide film on the surface of the silicon substrate 52, resist coating, resist exposure and development, etching of the oxide film, etching of silicon by ICP (High Frequency Inductively Coupled Plasma), etc. Thus, as shown in FIG. 9, a pressurizing chamber 60, a first channel 62, a first liquid chamber 66, a second channel 64, and a second liquid chamber 68 are formed.

  At the position of the pressurizing chamber 60, the silicon substrate 52 is processed into a diaphragm, and a piezoelectric element 70 made of lead zirconate titanate (PZT) ceramic or the like is attached to the outer surface thereof.

The micropump 58 is driven as follows by the control voltage to the piezoelectric element 70.
The piezoelectric element 70 is vibrated by the applied voltage having a predetermined waveform, and the silicon diaphragm at the position of the pressurizing chamber 60 is vibrated, whereby the volume of the pressurizing chamber 60 is increased or decreased.

  The first flow path 62 and the second flow path 64 have the same width and depth, and the length of the second flow path 64 is longer than that of the first flow path 62. In the first flow path 62, 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 64, the flow path width is long, so that even if the differential pressure increases, it tends to be a laminar flow, and the rate of change in flow path resistance with respect to the change in differential pressure is smaller than in the first flow path 62. .
For example, by adjusting the control voltage for the piezoelectric element 70, the volume of the pressurizing chamber 60 is decreased while the silicon diaphragm is quickly displaced in the direction toward the inside of the pressurizing chamber 60 to give a large differential pressure, and then pressurization is performed. If the volume of the pressurizing chamber 60 is increased while slowly displacing the silicon diaphragm from the chamber 60 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.

  Contrary to this, the volume of the pressurizing chamber 60 is increased while quickly displacing the silicon diaphragm in the direction from the pressurizing chamber 60 toward the outside thereof to give a large differential pressure, and then the direction toward the inside of the pressurizing chamber 60. When the volume of the pressurizing chamber 60 is decreased 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.

  The difference in flow rate resistance change ratio with respect to the change in differential pressure between the first flow channel 62 and the second flow channel 64 is not necessarily due to the difference in the length of the flow channel, but in other geometrical differences. It may be based.

The flow rate control by the micropump 58 can be performed by adjusting the voltage applied to the piezoelectric element 70.
A flow path 72 is patterned on the second glass substrate 56. As an example, the size and shape of the flow path 72 is a rectangular cross section having a width of about 150 μm and a depth of about 300 μm.

  On the downstream side of the flow path 72, an opening 74 for allowing the micropump 58 to communicate with the flow path of the microreactor 10 by being aligned with the opening of the microreactor 10 is provided.

The driving liquid is fed through the flow path 72 and the opening 74 by the micro pump 58, and each liquid such as a reagent accommodated in the flow path of the microreactor 10 is pushed downstream.
The upstream side of the flow path 72 is communicated with the micropump 58 through the flow path provided in the silicon substrate 52 through the through hole 76 of the first glass substrate 54.

  The upstream side of the micropump 58 is connected to the opening 80 provided in the second glass substrate 56 from the flow path provided in the silicon substrate 52 through the through holes 76 and 78 of the first glass substrate 54. It is communicated. The opening 80 is connected to a driving liquid tank (not shown).

The opening 80 is connected to a driving liquid tank (not shown) through, for example, PDMS (polydimethylsiloxane) packing.
<Micro total analysis system 82>
FIG. 10 is a perspective view showing an example of a micro total analysis system using a microreactor in an embodiment of the present invention, and FIG. 11 is an internal structure diagram of a system main body in the micro total analysis system shown in FIG.

For example, the microreactor 10 is attached to a separate system main body 84 to perform reaction and analysis. The system main body 84 and the microreactor 10 constitute a micro total analysis system 82.

The system main body 84 of the micro comprehensive analysis system 82 shown in FIG. 10 includes a housing 86 that houses each device for analysis.
Inside the housing 86, as shown in FIG. 11, a microchip provided with a chip connection portion 88 having a channel opening for communicating with the microreactor 10 and a plurality of micropumps (not shown). A pump unit 50 is arranged.

  Further, inside the housing 86, there are a detection processing device for detecting a reaction in the microreactor 10 (a light source 90 such as an LED and a laser, and a detector 92 that performs optical detection by visible spectroscopy, fluorescence photometry, and the like. A control device (not shown) for controlling the detector 92 and the micropump unit 50 is provided.

  With this control device (not shown), in addition to control of liquid feeding by a micropump, control of a detection processing device that detects a reaction in the microreactor 10 by optical means, etc., temperature control of the microreactor 10 by a heating / cooling unit described later. Control of reaction in the microreactor 10, data collection (measurement), processing, and the like are performed.

  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 82, a pump connection portion 94 including a flow channel opening provided on the upstream side of the micro flow channel of the microreactor 10 (for example, upstream of a storage unit, a sample receiving unit, etc.) and a chip surface around the channel opening. After the microreactor 10 is mounted inside the storage body 86 in a state where the chip connection portion 88 of the micropump unit 50 is in liquid-tight contact, the target substance in the specimen is analyzed in the microreactor 10.

The microreactor 10 is placed on the transport tray 85 and introduced into the storage body 86 from the insertion port 87.
Inside the storage body 86, a heating / cooling unit (Peltier element 96, heater 98) for locally heating or cooling the microreactor 10 mounted at a predetermined position is provided.

  For example, the Peltier element 96 is pressed against the region of the storage unit in the microreactor 10 to selectively cool the storage unit, thereby preventing alteration of the reagent and the like, and the heater 98 in the channel region constituting the reaction unit. The reaction part is selectively heated by pressure welding, thereby bringing the reaction part to a temperature suitable for the reaction.

  The micro pump unit 50 is connected to one driving liquid tank 99, and the upstream side of the micro pump 58 communicates with the driving liquid tank 99. On the other hand, the downstream side of the micropump 58 communicates with a channel opening provided on one side of the micropump unit 50, and each channel opening communicated with each micropump 58 and a pump connection part of the microreactor 10. The microreactor 10 is connected to the micropump unit 50 so that the respective channel openings provided in 94 are connected.

  The water-based driving liquid 97 stored in the driving liquid tank 99 is sent out by the micropump 58 to the liquid storage section in the microreactor 10 via the pump connection portion 94, and the liquid in each storage section is sent to the microreactor by the driving liquid 97. 10 is pushed out and sent to the downstream side.

  A series of analysis steps of pretreatment, reaction, and detection of a sample that is a measurement sample is performed in a state where the microreactor 10 is mounted on a system main body 84 in which a micropump 58, 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 Then, the analysis result is displayed on the display unit 83 of the storage body 86.

Hereinafter, specific examples of the reaction between the sample (specimen) and the reagent using the microreactor 10 of the present invention and the detection thereof will be shown.
In a preferred embodiment of the microreactor 10, in one chip,
・ Sample or specimen receiving part into which analyte (ex. DNA, RNA, gene) extracted from specimen is injected ・ Sample pretreatment part for pretreatment of specimen ・ Probe binding reaction, detection reaction (gene amplification reaction or antigen antibody) Reagent storage unit (storage unit) that stores reagents used for reactions, etc.)
・ Positive control housing for housing positive controls ・ Negative control housing for housing negative controls ・ Probe housing for storing probes (for example, probes that hybridize to a gene to be detected amplified by a gene amplification reaction) There are provided a micro-channel that communicates with each part and each accommodating part, and a pump connecting part that can be connected to each accommodating part and a separate micropump for feeding the liquid in the channel.

  The microreactor 10 is connected to the micropump unit 50 described above via a pump connection unit 94, and a specimen injected into the sample receiving part or a biological substance extracted from the specimen (for example, DNA or other biological substance), The reagent contained in the reagent container is fed to the downstream channel, and mixed and reacted in the reaction part of the fine channel, for example, the reaction part that performs gene amplification reaction (in the case of protein, antigen-antibody reaction, etc.). .

  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 as necessary, for example, by mixing the sample and the treatment liquid in the sample pretreatment unit provided in the flow path in advance before mixing with the reagent. . 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 necessary reagents are sealed in the reagent storage portion of the microreactor 10 in advance. 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.

  Reagents stored in advance in the microreactor 10 for genetic testing include various reagents used for gene amplification, probes used for detection, coloring reagents, pretreatment reagents used for the sample pretreatment, and the like. is there.

  By supplying the driving liquid 97 from the micropump 58, the reagent is pushed out from each reagent storage unit and merged to generate a mixed reagent. Thereafter, by supplying the driving liquid 97 from the micropump 58, the specimen is pushed out from the sample receiving part and joined with a mixed reagent having a stable mixing ratio, so that in the reaction part, gene amplification reaction, analyte trap or Reactions required for analysis such as antigen-antibody reaction are initiated.

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 flow channel device that enables temperature control suitable for a microreactor has already been proposed by the present inventors (Japanese Patent Laid-Open No. 2004-2004). -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.

  The recently developed ICAN (Isomeric chimera primed nucleic acid 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. .

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 10. 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 the probe DNA fluorescently labeled with FITC (fluorescein isothiocyanate).

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 amplified DNA is trapped at the detection site and then fluorescently labeled probe DNA And may be hybridized.)
(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 top view of a microreactor in an embodiment of the present invention. FIG. 2 is a partial cross-sectional view perpendicular to the chip surface of the microreactor in the embodiment of the present invention. FIG. 3 is a view showing a water repellent valve according to an embodiment of the present invention, FIG. 3 (a) is a top view, and FIG. 3 (b) is a cross-sectional view taken along line XX of FIG. 3 (a). . 4A and 4B are views showing a water-repellent valve according to another embodiment of the present invention. FIG. 4A is a top view, and FIG. 4B is a cross-sectional view taken along line XX in FIG. It is. FIG. 5 is a schematic top view showing the storage part of the microreactor and the water-repellent valves provided at both ends of the storage part in the embodiment of the present invention. FIG. 6 is a partial cross-sectional view of a mold for molding a microreactor according to an embodiment of the present invention. FIG. 7 is a partial top view showing a portion corresponding to a connection port communication portion of a mold for molding the microreactor in the embodiment of the present invention. FIG. 8 is a perspective view showing an example of a micropump unit connected to the microreactor in the embodiment of the present invention. FIG. 9 is a cross-sectional view of the micropump unit shown in FIG. 8 connected to the microreactor in the embodiment of the present invention. FIG. 10 is a perspective view showing an example of a micro total analysis system using a microreactor according to an embodiment of the present invention. FIG. 11 is an internal structure diagram of the system main body in the micro integrated analysis system shown in FIG. 12A and 12B are views showing a water-repellent valve in a conventional microreactor. FIG. 12A is a top view, and FIG. 12B is a cross-sectional view taken along line XX in FIG. FIG. 13 is a schematic top view showing a reagent container in a conventional microreactor and water-repellent valves provided at both ends of the reagent container. FIG. 14 is a diagram illustrating a process in which the driving liquid is filled into the branch channel from the state illustrated in FIG. 13 and the reagent is extruded by the driving liquid. FIG. 15 is a partial top view showing a portion corresponding to a liquid feed control flow path of a mold for forming a conventional microreactor. FIG. 16 is a state diagram showing a state when a space is formed between the driving liquid and the reagent in the conventional microreactor, and bubbles are generated in the space.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Microreactor 11 ... Reagent supply hole 12 ... Flow path 13 ... Merge part 14 ... Base material 15 ... Fine flow path 16 ... Coated base material 18 ... Coating base Material 20 ... Through hole 21 ... Water repellent valve 22 ... Liquid feeding control part 23 ... Upstream side flow path 24 ... Downstream side flow path 25 ... Connection port communication part 26.・ ・ Upstream side connection port 27 ・ ・ ・ Downstream side connection port 28 ・ ・ ・ Liquid 29 ・ ・ ・ Storage unit 30 ・ ・ ・ Reagent 34 ・ ・ ・ Equivalent part of the connection port communication unit 50 ・ ・ ・ Micro pump unit 52 ・ ・ ・-Silicon substrate 54 ... First glass substrate 56 ... Second glass substrate 58 ... Micro pump 60 ... Pressurizing chamber 62 ... First flow path 64 ... Second flow Path 66 ... First liquid chamber 68 ... Second liquid chamber 70 ... Piezoelectric element 72 ... Channel 74 ... Opening 76 ... Through hole 80 ... Opening 82 ... Micro total analysis system 83 ... Display unit 84 ... System main body 85 ... Conveying tray 86 ... Storage body 87 ... Insertion port DESCRIPTION OF SYMBOLS 88 ... Chip connection part 90 ... Light source 92 ... Detector 94 ... Pump connection part 96 ... Peltier element 97 ... Drive liquid 98 ... Heater 99 ... Drive liquid tank t1・ ・ ・ Width 100 of connection port communication portion equivalent part ・ ・ ・ Water repellent valve 102 ・ ・ ・ Upstream channel 104 ・ ・ ・ Downstream channel 106 ・ ・ ・ Liquid feed control channel 108 ・ ・ ・ Control Channel upstream end 110 ... Control channel downstream end 112 ... Liquid 114 ... Reagent storage unit 116 ... Reagent 118 ... Opening 120 ... Channel 122 ... Drive Liquid 124 ... Branch point 126 ... Branch channel 128 ... Empty Vent passage 130 ... space 132 · bubble 200 ... die 202 ... feeding control channel corresponding portions 204 ... tip 300 ... resin

Claims (3)

A flow path is provided on at least one surface of the plate-shaped chip, and a liquid feed control unit constituting a water repellent valve is provided in a part of the flow path, whereby the liquid feed speed and liquid feed of the liquid in the flow path are provided. A microreactor with timing control,
The liquid feeding control unit is
An upstream connection port having a cross-sectional area smaller than the cross-sectional area of the channel on the upstream side;
A downstream connection port having a cross-sectional area smaller than the cross-sectional area of the flow path on the downstream side;
A connection port communicating portion for communicating the upstream connection port and the downstream connection port,
The upstream connection port is configured to have a cross-sectional area larger than the cross-sectional area of the downstream connection port,
The microreactor is characterized in that the connection port communication portion is configured such that a cross-sectional area continuously changes. The microreactor is
Comprising a reservoir for storing reagents or specimens;
Microreactor according to claim 1, wherein each of the one side and the other side of the reservoir, wherein the liquid feed control section is provided. 3. The microreactor according to claim 1, wherein a width from the upstream connection port to the downstream connection port of the connection port communication portion is 40 μm to 300 μm.