JP2007292506A - Microreactor and micro-integrated analysis system using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microreactor which will not have the liquid in a storage part leak from a through-hole or the leading end of the storage part, due to the pressure being applied to the inside of the storage part inside a chip, and to provide a micro-integrated analysis system that uses it. <P>SOLUTION: The microeactor is constituted so that a flow channel is provided, at least to one side of the sheet-like chip and the through-hole communicating with the flow channel is provided, and the through-hole is sealed by a sealing material having low melting point. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microreactor and a microreactor using 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. It relates to a comprehensive analysis system.

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 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 cost effective when complex processes, skilled procedures, and complex equipment operations are required. The analysis can be performed not only on the required sample amount and the required time but also on any time and place, and the benefit of this can be said to be 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, it is a challenge to establish a highly reliable liquid feeding system with a simple configuration, and there is a need for a microfluidic control element that is highly accurate and excellent in reliability. In addition, the present inventors have already proposed a control method thereof (see Patent Documents 2 to 4).

  As shown in FIG. 8, such an analysis chip (hereinafter referred to as a microreactor) has a channel 106 formed by superimposing a coated substrate 104 on a substrate 102 having a channel groove formed on the surface thereof. Is to be formed.

The channel 106 communicates with the surface of the microreactor 100, and supplies a reagent supply hole for supplying a reagent to the reagent storage section in the chip. A through hole 108 such as an injection hole and a deaeration hole for deaerating air in the flow path is provided, and a lid member and a sealing seal 110 are formed and pasted on the through hole 108 to seal the through hole 108. It has been made.
JP 2004-28589 A JP 2001-322099 A JP 2004-108285 A JP 2004-270537 A

However, with such conventional lids and sealing seals, the chip is pressed when these are formed and adhered. For example, pressure is applied to the reagent reservoir in the chip and the reagent reservoir In some cases, the stored reagent leaks from the through hole or the tip of the reagent storage part.

Moreover, in the sealing seal, since it is necessary to stick the sealing seal at a plurality of locations, it takes time to work, and improvement in productivity is required.
In view of such a current situation, the present invention provides a microreactor and a micro total analysis system using a microreactor in which liquid in the storage portion does not leak from the through hole or the front end of the storage portion when pressure is applied to each storage portion in the chip. The purpose is to provide.

  Another object of the present invention is to provide a microreactor with excellent workability by sealing a through hole and a micro total analysis system using the microreactor.

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 is
A flow path is provided on at least one surface of the plate-shaped chip, and a microreactor having a through hole communicating with the flow path,
The through hole is
The structure is sealed with a low melting point sealing material.

  If a low melting point sealing material is used in this way, there is no need to contact the chip, so that liquid in each reservoir is not accidentally leaked from the through hole or the tip of the reservoir, and the through hole is reliably sealed. Can be stopped.

The microreactor of the present invention is
The low-melting-point sealing material is a material that is a liquid before dripping onto the chip and is solidified upon contact with the chip.

  Using a low-melting-point sealing material made of a material that solidifies at the time of contact with the chip in this way only drops the low-melting-point sealing material into the through hole, so that the sealing workability is extremely good and production Can increase the sex.

The microreactor of the present invention is
The low-melting-point sealing material is a material that is solid at room temperature and has a melting point of 20 degrees to 100 degrees.

  If a low-melting-point sealing material made of a material having a melting point of 20 to 100 degrees is used in this way, it is heated by a heater until it is dropped into the through hole and is in a liquid state. Since heat is taken away by the outside air, it is immediately solidified, so that the through hole can be sealed instantaneously.

The microreactor of the present invention is
The low-melting-point sealing material is configured to be dropped using a pipetter or a dispenser.

  If a pipetter or dispenser is used in this way, a predetermined amount of low-melting-point sealing material can be dropped into the through-hole, so that the low-melting-point sealing material may be scattered in unnecessary locations or excessively low. There is no worry of using a melting point sealing material, which is economical.

Moreover, the micro comprehensive analysis system of the present invention includes:
Any one of the above microreactors is used.
If the micro total analysis system is operated using a microreactor whose through-hole is sealed with a low-melting-point sealing material in this way, the liquid in each reservoir will accidentally leak from the through-hole or the reservoir tip. Therefore, a desired inspection can be surely performed.

  According to the present invention, it is possible to provide a microreactor and a micro total analysis system using a microreactor in which the liquid in the reservoir does not leak from the through hole or the tip of the reservoir due to pressure applied to each reservoir in the chip. it can.

  In addition, it is possible to provide a microreactor with extremely good workability by sealing the through-hole and a micro total analysis system using the microreactor.

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, and when the reagent 30 is supplied into the storage unit 29, the reagent 30 is filled up to just before each of the water repellent valves 21 and 21.

  After filling the reagent, the low-melting point sealing material 18 is dropped on the reagent supply hole 11, thereby sealing the reagent supply hole 11, and the reagent 30 is pressure-reacted by the reagent supply hole 11 and the water repellent valve 21. It is designed not to leak more.

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 container (not shown) is provided downstream of the microchannel 15, and the sample stored in the sample container (not shown) and a plurality of reagents that pass through the microchannel 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 a chip connection portion 88 of the micro pump unit 50 described later 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 flow channel groove serving as a flow channel is formed by injection molding, and a coated base material 16 is superimposed on the surface 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, polycarbonate, and cycloolefin.

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.
The channel (fine channel) 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 path is appropriately set according to the purpose regardless of the fine flow path and the flow path 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.
<Low melting point sealing material 18>
FIG. 3 is a view showing a method of dropping a low-melting-point sealing material into the through-hole in the embodiment of the present invention, and FIG. 3A is a state diagram before dropping the low-melting-point sealing material, FIG. b) is a state diagram after dropping the low-melting-point sealing material.

  A reagent supply hole provided in the microreactor 10 for supplying a reagent to a reagent reservoir in the chip, a specimen injection hole for injecting a specimen such as liquid, urine, DNA, etc. into the specimen reservoir, and air in the flow path The through hole 20 such as a deaeration hole for degassing is dripped from the upper portion of the low melting point sealing material 18 which is heated and becomes liquid, and the through hole 20 is sealed by solidifying this. ing.

  As such a low melting point sealing material 18, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, eicosane, wax, paraffin wax, etc., having a melting point of 20 to 100 degrees can be used. Among these, it is preferable to use eicosane.

  As a method for dripping the low-melting-point sealing material 18 into the through-hole 20, as shown in FIG. 3A, first, the microreactor 10 that has finished injecting the reagent from the through-hole 20 into the flow path 12 such as the reagent reservoir. Prepare.

  Further, as shown in FIG. 3B, a pipetter 22 is disposed on the upper portion of the through hole 20, and thereby a specified amount of the low melting point sealing material 18 is dropped on the upper portion of the through hole 20. In the present embodiment, the pipetter 22 is used for explanation, but any other material that can drop a predetermined amount of a low-melting-point sealing material sealing material such as a dispenser can be used.

The low melting point sealing material 18 accommodated in the pipetter 22 is kept at 100 degrees or more by a heater (not shown) provided separately.
The liquid state is maintained before dropping onto the top.

The dropped low-melting-point sealing material 18 is solidified by contacting the upper surface of the microreactor 10 and the outside air, and the through-hole 20 is sealed.
Further, as shown in FIG. 3B, in the microreactor 10 of the present invention, one pipetter 22 corresponds to one through hole 20, and the low melting point sealing material 18 is dropped. However, for example, the low-melting-point sealing material 18 is dropped using a pipetter 22 having a plurality of nozzles so that all the through-holes 20 can be sealed with respect to the plurality of through-holes 20 by one drop. It is also possible to make it.

Further, the pipetter 22 can adjust the amount of the low-melting-point sealing material 18 dripped depending on the size of the through-hole 20 or change the material depending on the location of the through-hole 20.
If the low melting point sealing material 18 is used in this manner, the through hole 20 can be reliably sealed without contacting the chip.

Moreover, since the through-hole 20 can be sealed only by dropping the low melting point sealing material 18 with the pipetter 22, workability can be improved as compared with the conventional case.
<Micro pump unit 50>
4 is a perspective view showing an example of a micropump unit connected to the microreactor in the embodiment of the present invention, and FIG. 5 is a diagram of the micropump unit shown in FIG. 4 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 micro pump unit 50 and its control device, an optical detection device for reaction detection, a temperature control device, a system main body including a driving liquid tank containing driving liquid, and a microreactor 10 in which a reagent is sealed in advance The case where a comprehensive analysis system 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 system 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, thereby pushing out the liquid in the flow path, for example, the reagent in the storage unit 29, the sample in the sample receiving part, etc. to the downstream side. Mixing reagents and mixing 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. 4 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 configured by an internal space between the silicon substrate 52 and the first 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. 5, a pressurizing chamber 60, a first flow path 62, a first liquid chamber 66, a second flow path 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. 6 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. 7 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 integrated analysis system 82 shown in FIG. 6 includes a housing 86 that houses each device for analysis.
As shown in FIG. 7, a microchip provided with a chip connecting portion 88 having a channel opening for communicating with the microreactor 10 and a plurality of micropumps (not shown) is provided in the housing 86. 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, etc. 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 region of the flow path constituting the amplification unit. Is selectively heated to bring the amplification section 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 surface 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.

  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) A fine channel downstream from these containing portions of a sample or DNA extracted from the sample, or cDNA synthesized by reverse transcription from RNA extracted from the sample or sample, and a primer modified with biotin 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 method of dropping a low-melting-point sealing material into the through-hole in the embodiment of the present invention, and FIG. 3A is a state diagram before dropping the low-melting-point sealing material, FIG. b) is a state diagram after dropping the low-melting-point sealing material. FIG. 4 is a perspective view showing an example of a micropump unit connected to the microreactor in the embodiment of the present invention. FIG. 5 is a cross-sectional view of the micropump unit shown in FIG. 4 connected to the microreactor in the embodiment of the present invention. FIG. 6 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. 7 is an internal structure diagram of the system main body in the micro integrated analysis system shown in FIG. FIG. 8 is a cross-sectional view for explaining a through hole sealing method in a conventional microreactor.

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 ... Low melting point Sealing material 20 ... through hole 21 ... water repellent valve 22 ... pipetter 29 ... storage part 30 ... reagent 50 ... micro pump unit 52 ... silicon substrate 54 ... 1st glass substrate 56 ... 2nd glass substrate 58 ... Micro pump 60 ... Pressurizing chamber 62 ... 1st flow path 64 ... 2nd flow path 66 ... 1st liquid Chamber 68 ... Second liquid chamber 70 ... Piezoelectric element 72 ... Flow path 74 ... Opening 76 ... Through hole 80 ... Opening 82 ... Micro total analysis system 83 ... Display Part 84... System main body 85... Transport tray 86. · Insertion port 88 · · · Chip connection portion 90 · · · Light source 92 · · · Detector 94 · · · Pump connection portion 96 · · · Peltier element 97 · · · drive liquid 98 · · · heater 99 · · · drive Liquid tank 100 ... Micro reactor 102 ... Base material 104 ... Coating base material 106 ... Flow path 108 ... Through hole 110 ... Sealing seal

Claims (5)

A flow path is provided on at least one surface of the plate-shaped chip, and a microreactor having a through hole communicating with the flow path,
The through hole is
A microreactor having a structure sealed with a low melting point sealing material.   2. The microreactor according to claim 1, wherein the low melting point sealing material is a material that is a liquid before dripping onto the chip and is solidified upon contact with the chip.   3. The microreactor according to claim 1, wherein the low-melting-point sealing material is a material that is solid at room temperature and has a melting point of 20 to 100 degrees.   The microreactor according to any one of claims 1 to 3, wherein the low melting point sealing material is configured to be dropped using a pipetter or a dispenser.   A micro total analysis system using the microreactor according to claim 1.

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