JP2007120399A - Micro fluid chip and micro comprehensive analysis system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a micro fluid chip for micro comprehensive analysis system capable of making clear up temperature distribution in the chip distinct by shutting off heat conduction in areas of different temperature environments as much as possible. <P>SOLUTION: A heat insulating means is applied to a boundary in the micro fluid chip in which different kinds of temperature areas exist in parallel. An air gap penetrating through the micro fluid chip and a groove formed on a surface of the micro fluid chip substrate but not communicating with a minute flow passage of the chip are given as the heat insulating means. The boundary of the different kind of temperature areas is, in an embodiment, a boundary of a heating area and a cooling area. The heating area includes, for instance, a reaction part performing a gene amplifying reaction and the cooling area includes, for instance, a specimen storage part and/or a regent storage part. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

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. Therefore, it is a problem to establish a highly reliable liquid feeding system with a simple configuration. A microfluidic control element having high accuracy and excellent reliability is required, and the present inventors have already proposed a suitable micropump system and a control method thereof (Patent Documents 2 to 4).

The above chip is formed with a series of fine flow paths including a specimen and reagent storage section, a reagent mixing section, a reaction section, a detection section, and a flow path that communicates these parts. There are areas to be selectively heated or cooled. For example, a fine channel constituting a reaction site for performing an amplification reaction by a PCR (polymerase chain reaction) method needs to be heated to a predetermined temperature during analysis. On the other hand, for example, a specimen that easily changes in quality when heated, a part that contains a reagent, or a part where such reagents are mixed with each other is not desirable to be heated, and the part can selectively release heat or Must be cooled.

However, in a chip in which a series of fine channels are integrated in a limited space, it is difficult to arrange portions that require heating or portions that are not desired to be heated at sufficiently wide intervals. If these parts are close to other parts, various problems may be caused by heat conduction. As an example, when a cooling site is present in the vicinity of a heating site for performing a gene amplification reaction by the PCR method, the temperature distribution of the heating site becomes halfway, causing a problem in the reaction.
JP 2004-28589 A JP 2001-322099 JP 2004-108285 A JP 2004-270537 A

  The present invention has been made in view of the above-described problems, and provides a microfluidic chip capable of blocking heat conduction between regions having different temperature distributions as much as possible and making the temperature distribution in the chip sharp. Let it be an issue. Another object of the present invention is to provide a micro total analysis system in which adverse effects due to heat on the reaction are eliminated by using such a chip.

The micro total analysis system of the present invention is
Two or more types of temperatures selected from the group consisting of a “heating region” in contact with a separate heating device, a “cooling region” in contact with a separate cooling device, and a “room temperature region” in non-contact with the heating device and the cooling device A microfluidic chip in which regions coexist and thermal insulation is applied at the boundary of different types of temperature regions;
A system unit body;
The system apparatus main body includes at least a base main body,
A micropump unit including a chip connection portion disposed in the base body and having a channel opening for communicating with the chip; and the micropump;
A heating device and / or a cooling device;
A control device for controlling at least the function of the micropump unit and the heating device and / or cooling device;
With
The micropump
A first flow path in which the flow path resistance changes according to the differential pressure;
A second flow path whose rate of change in flow path resistance with respect to a change in differential pressure is smaller than the first flow path;
A pressurization chamber connected to the first flow path and the second flow path;
An actuator for changing the internal pressure of the pressurizing chamber;
It is a micro pump provided with the drive device which drives this actuator.

  The heat insulating means is preferably a gap penetrating the microfluidic chip, or may be a groove formed on the surface of the microfluidic chip substrate that is not in communication with the microchannel of the chip. .

  The boundary between the different types of temperature regions is, for example, a boundary between a heating region and a cooling region. This heating region includes, for example, a reaction unit that performs a gene amplification reaction, and the cooling region includes, for example, a specimen storage unit and / or a reagent storage unit.

  According to the present invention, there is provided a microfluidic chip that sufficiently blocks heat conduction between different temperature regions and has a clear temperature distribution. With the heat insulating means used in the present invention, different temperature regions can be arranged adjacent to each other in a narrow range of the microfluidic chip, so that it is possible to provide a microfluidic chip with a smaller size. Also provided is a micro total analysis system that can efficiently inspect biological substances such as genes by suppressing the occurrence of side reactions due to the formation of an intermediate temperature range and the loss of reagents from the flow path. Is done.

  In this specification, “microfluidic chip” refers to a chip in a micro total analysis system used for various applications such as synthesis and inspection. Sometimes called a “chip”. “Flow path element” refers to a functional component installed in a microfluidic chip. “Fine channel” refers to a minute groove-like channel formed in the microfluidic chip of the present invention. A container, a reaction unit, or a detection unit for reagents and the like communicating with the channel. However, even when it is formed in a wide liquid reservoir shape with a large capacity, it may be referred to as a “fine channel” including these parts. In many cases, the fluid flowing in the fine channel is actually a liquid, and specifically, various reagents, sample liquids, denaturant liquids, cleaning liquids, driving liquids, and the like are applicable. “Gene” refers to DNA or RNA carrying genetic information that expresses some function, but may also be simply referred to as DNA or RNA that is a chemical entity. The target substance to be analyzed is sometimes referred to as “analyte”.

In the various embodiments, the present invention can be arbitrarily modified and changed in accordance with the spirit of the present invention, and these are included in the present invention. That is, the structure, configuration, arrangement, shape, size, material, method, method, etc. of the whole or a part of the micro total analysis system of the present invention can be varied as long as they match the purpose of the present invention. it can.

Micro Comprehensive Analysis System FIG. 1 is a conceptual diagram showing a configuration in one embodiment of a micro comprehensive analysis system of the present invention. As shown in the drawing, in this embodiment, there is a system device body 1 as a device that accommodates the microfluidic chip 2 and the chip. This device includes a Peltier 3 (cooling device), a heater 4 (heating device) used for the reaction, a micropump 11 for liquid feeding, a driving liquid tank 10 and a micropump unit having a chip connecting portion, and A control device (not shown) for controlling the liquid supply, temperature and reaction of the liquid, and the optical detection system (photodiode 5)
And a detection processing device (not shown) that also collects and processes measurement data. The micro total analysis system is desirably configured so that components other than the microfluidic chip 2 are integrated into the system apparatus main body 1, and the microfluidic chip 2 is attached to and detached from the system apparatus main body 1.

The microfluidic chip 2 is equivalent to what is generally called a test chip, an analysis chip, a microreactor chip, or the like. Usually, the vertical and horizontal sizes of this chip are several tens of mm and the height is about several mm. A fine flow path is formed there by a fine processing technique. The various liquids in the reagent storage unit and the sample storage unit communicating with the fine flow path are communicated with each storage unit via the pump connection unit 12 having a flow channel opening for communicating with the micropump. Liquid is fed by the pump 11.

Although the micropump can be provided in the microfluidic chip 2, a plurality of micropumps are usually incorporated in the system apparatus body 1. With these multiple micropumps,
A micropump unit including a chip connection portion having a channel opening for communicating with the microfluidic chip 2 is disposed in the base body of the system apparatus body 1. As shown in the figure, the microfluidic chip 2 is mounted on the system apparatus main body 1 and is superposed on each other so that the pump connecting part of the chip is connected to the chip connecting part in the micropump unit.

The device of the electric control system that controls the micropump 11 sets a target value of the flow rate and supplies a drive voltage corresponding to the target value to the micropump. As will be described later, a control device responsible for such control is also incorporated into the main body of the system of the present invention, and the microfluidic chip 2
When the pump connection part is connected to the chip connection part of the micro pump unit, the operation may be controlled.

  A detection processing device (not shown), which is a unit responsible for optical detection, data collection and processing, is particularly limited as a means of optical measurement when a technique such as visible spectroscopy or fluorescence photometry is applied. However, it is desirable that an LED, a photomultiplier, a photodiode, a CCD camera, and the like be appropriately installed in the system apparatus body as its constituent elements.

The Peltier 3 and the heater 4 are disposed so as to abut on specific parts of the microfluidic chip, and cool or heat these parts.
A control device (not shown) that controls at least the function of the micropump unit is incorporated in the main body of the system of the present invention. This control device further integrates and controls the temperature control by the heating device and / or the cooling device and the measurement data recording and processing function in the detection processing device so that the system can be controlled and controlled in an integrated manner. May be. Various conditions such as the order of liquid feeding by the micropump 11, the control of the volume and timing, or the temperature control by the heating device and / or the cooling device are set in advance as the contents of the program and installed in the micro total analysis system. These controls can be performed according to software such as a microcomputer.

  A series of analysis steps of pretreatment, reaction, and detection of a specimen as a measurement sample is performed in a state where a chip is mounted on the system apparatus body 1 in which the micropump 11, the detection processing apparatus, and the control apparatus are integrated. . The analysis may be started after the sample is injected into the mounted chip, or after the chip into which the sample has been injected is mounted on the apparatus main body. Predetermined reactions and optical measurements based on sample and reagent delivery, pretreatment, and mixing are automatically performed as a series of continuous processes, and the measurement data is stored in a file along with the necessary conditions and recorded items. Desirable form is desirable.

  In the conventional analysis chip, when performing different analysis or synthesis, it is necessary to configure a microfluidic device corresponding to the contents to be changed each time. In contrast to this, in the micro total analysis system of the present invention, only the above-described detachable chip need be replaced. If it is necessary to change the control of each flow path element, the control program stored in the apparatus main body may be changed as appropriate.

Microfluidic chip The microfluidic chip of the present invention is designed such that each flow path element or structure is finely located at a functionally appropriate position so as to be used for chemical analysis, various inspections, sample processing / separation, chemical synthesis, etc. It is arranged by processing technology. The chip is provided with a plurality of reagent storage units for storing each reagent in addition to a sample storage unit for storing a sample liquid as a fluid storage unit, and the reagent storage unit includes reagents used for a predetermined reaction, A cleaning solution, a denaturing treatment solution and the like are accommodated. This is because it is desirable that the reagent is stored in advance so that the examination can be performed quickly regardless of the place or time. Reagents built in the chip are sealed on the surface of the storage portion in order to prevent evaporation, leakage, mixing of bubbles, contamination, denaturation, and the like.

  The preferable aspect in said chip | tip has as a structure the basic substrate which consists of a groove | channel formation board | substrate and a coating substrate. At least on the groove forming substrate, the pump connection part, the valve base part and the liquid reservoir part (reagent storage part, each storage part such as the specimen storage part, waste liquid storage part), liquid feed control part, backflow prevention part, reagent quantification part, mixing A fine channel including a structural part such as a part is formed. The coated substrate needs to cover at least the structure portion, the flow path, and the detection portion of the groove forming substrate, and may cover the entire surface of the groove forming substrate.

・ Material The microfluidic chip is required to have excellent processability, non-water absorption, chemical resistance, heat resistance, and low cost. Considering the chip structure, application, detection method, etc. Appropriate selection of the chip material is required. Various known materials can be used as the material, and usually the substrate and the flow path element are formed by appropriately combining one or more materials in accordance with individual material characteristics.

  For example, it is desirable that a chip intended for a large number of measurement specimens, particularly clinical specimens at risk of contamination and infection, be of a disposable type. Therefore, a plastic resin that can be mass-produced, is light in weight, is strong against impact, and can be easily disposed of by incineration. Among them, polystyrene that is excellent in transparency, mechanical properties, and moldability and is easy to be finely processed is preferably used.

  When it is necessary to heat the chip to near 100 ° C. in analysis, it is preferable to use a resin having excellent heat resistance (for example, polycarbonate). Resin and glass have low thermal conductivity, and by using these materials in the locally heated region of the microfluidic chip, heat conduction in the surface direction is suppressed, and only the heated region is selectively heated. Can do.

  Further, since the detection part of the fine channel optically detects the fluorescent substance or the product of the color reaction, it is necessary to use a light transmissive material for the substrate at this part. As such a material, alkali glass, quartz glass, and transparent plastics can be used.

Microchannel The microchannel of the microfluidic chip is formed on the substrate according to a channel arrangement designed in advance according to the purpose. The flow path through which the fluid flows is, for example, a micro flow path with a micrometer order width formed to have a width of several tens to several hundreds μm, preferably 50 to 100 μm, and a depth of about 25 to 200 μm, preferably 50 to 100 μm. If the channel width is less than 50 μm, the channel resistance increases, which is inconvenient for fluid delivery and detection. The advantage of the microscale space is diminished in the flow path exceeding 500 μm in width.

  A conventional microfabrication technique can be used as a method for forming the microchannel, but a photolithography technique is typically preferable. With this technique, the fine structure is transferred to the photosensitive resin, unnecessary portions are removed, and the fine flow path is formed. As the photosensitive resin used as the material of the groove-formed substrate at this time, a plastic having a good mechanical property and capable of accurately transferring a submicron structure is preferably used. Polystyrene, polydimethylsiloxane, etc. are excellent in shape transferability. If necessary, processing by injection molding, extrusion molding or the like may be used.

-Temperature region The microfluidic chip of the present invention has a "heating region" that contacts a separate heating device, a "cooling region" that contacts a separate cooling device, and a "normal temperature region" that does not contact the heating device and the cooling device. Two or more temperature regions selected from the group consisting of coexist.

  The form in which the heating area and the cooling area are provided on the same chip is not a rare aspect in a biological material analysis chip. This is because specimens and reagents are often easily denatured when heated, and in general, it is desirable to cool them and keep them at a low temperature, while high temperatures are required for detection and reaction.

  As an example of the fine channel included in the heating region that is selectively heated by a site, for example, a channel that constitutes a reaction site for performing a gene amplification reaction between DNA and a reagent by a PCR method can be mentioned. In addition, as an example of a micro flow path included in a cooling region that is selectively cooled by a site, a specimen storage unit, a reagent (for example, a gene amplification reagent) storage unit, and / or a plurality of reagents are joined and mixed. A flow path can be mentioned.

Heating / cooling device In the present invention, in a microfluidic chip formed with a series of fine flow paths, a heating element or a heating element is connected to a chip surface of a heating region including a flow path part to be selectively heated. A heating device such as a heat conducting member is brought into contact to heat the heating region. Further, a cooling device such as a cooling body or a heat conduction member connected to the cooling body is brought into contact with the chip surface of the cooling area including the flow path portion to be selectively cooled, thereby cooling the cooling area. Such a heating region and a cooling region are placed under temperature control of the present system as a temperature control region of the microfluidic chip. Note that the heating device or the cooling device may be arranged not only on one side of the chip but also on both sides if necessary for uniform temperature distribution in the region and rapid temperature rise or fall.

  As the above heating device, for example, a sheet heating element (heater) that heats a resistor by energization and transfers heat directly or via a dielectric or the like, a member with high thermal conductivity ( For example, a metal member (such as aluminum) is connected and the surface of the member is brought into contact with the chip surface, and a Peltier element.

  Further, the heating device is provided with a temperature sensor, and this temperature sensor is preferably connected to a controller having a memory in which a control program relating to the heating operation is stored. The controller controls energization and the like to the heating device according to the program based on the temperature measured by the temperature sensor. The configuration of such an embodiment requires a PCR method that requires a rapid temperature increase, specifically, a rapid increase in temperature from about 40 ° C. to about 90 ° C., and the need to frequently increase and decrease the temperature. It is preferably used when performing.

  On the other hand, a Peltier element is preferable as the cooling device. A heat sink may be disposed in contact with the Peltier element in order to dissipate the heat. Moreover, you may use together the block which consists of members (for example, metal members, such as aluminum) with high heat conductivity. As for the cooling device, similarly to the heating device, it is desirable to connect a control device for temperature management.

  By using the apparatus as described above, uniform heating or cooling can be performed selectively in each of a plurality of functional parts in a series of fine channels formed in the microfluidic chip.

・ Necessity of temperature distribution management When areas where different temperature distributions are required are adjacent to each other and heat conduction occurs between these areas, several problems may occur.

  In bioassays and synthesis reactions including the PCR method, unless the temperature conditions are strict, the success or failure of the reaction is often affected, or the control of the reaction is hindered. For example, in PCR, it is required to strictly manage not only the temperature conditions but also the heating time. If an intermediate temperature region is present in the reaction part flow path, non-specific amplification occurs as a side reaction, which inhibits amplification of the target sequence. For this reason, the target amplification reaction may not occur sufficiently. Therefore, it is desirable that the temperature difference between a region (for example, a heating region) including a reaction portion flow path where gene amplification or the like is performed and a region adjacent to the region are distinct.

  This also has an advantage from the viewpoint of preventing the reagent, the specimen, and the mixed liquid thereof from being lost from the flow path. The reason why the liquid in the fine channel is lost due to evaporation, etc. is that the air trapped in the contact surface of the chip with the system device body expands and becomes bubbles, increasing the channel pressure in the channel and thereby the liquid It is assumed that the reagent or the like enters the gap formed on the contact surface when the air is further removed and the reagent or the like leaks from there. In addition, the formation of the bubbles in the heating region or in the vicinity of the liquid is also related to the loss of the liquid in the flow path and the occurrence of trouble due to the bubbles.

Insulating means From the above, it is necessary to sufficiently block heat conduction between different temperature regions in the microfluidic chip, especially between the heating region and the cooling region. In the microfluidic chip of the present invention, it is possible to insulate sufficiently so that a temperature gradient and heat conduction do not occur between them by applying a heat insulating means to the boundary between these regions. The problem is effectively suppressed. When the microfluidic chip is made of, for example, a polypropylene resin having a thickness of 2 mm, the heating area and the cooling area have conventionally been required to be separated from each other by about 5 to 10 mm. In this case, the microfluidic chip can be miniaturized by greatly reducing the distance between these regions.

In the present invention, the above “boundary” refers to a portion located in the middle of different temperature regions. For example, when the cooling region and the heating region are adjacent to each other, the vicinity of the outer peripheral line surrounding each region can be considered as the boundary. In addition, when a normal temperature region exists between the cooling region and the heating region, the normal temperature region can be considered as a boundary between the cooling region and the heating region, and heat insulation means can be provided at an appropriate place in the normal temperature region. is there.

In the microfluidic chip of the present invention, the specific mode of the heat insulating means for separating the regions is not particularly limited, but for example, the following means can be preferably used.
-Air gap As one aspect of the heat insulating means provided by the present invention, it is possible to provide a space that penetrates the microfluidic chip at the boundary between different temperature regions. “Void” refers to a space in which air is interposed, such as a slit or slit. The size and shape of the gap can be appropriately adjusted in consideration of the arrangement of the fine flow path, the thermal conductivity of the material of the chip substrate, and the like. The same applies to the location and number of gaps.

  Such gaps are produced, for example, by manufacturing a groove-formed substrate and a coated substrate designed so that a gap is formed in each corresponding part by injection molding, and stacking the two substrates into a chip. Can be produced. Moreover, after manufacturing the chip | tip which does not provide the space | gap, you may make it create the space | gap which penetrates it by a conventional method, The production method is not ask | required.

-Groove | channel As another aspect of the heat insulation means provided by this invention, providing a groove | channel in the boundary of a different temperature range is mentioned. This groove does not communicate with the fine flow path formed in the chip, and is distinguished from the fine flow path. The width, depth, shape, installation location, number, and the like of the grooves can also be adjusted as appropriate in consideration of the arrangement of the fine channels, the thermal conductivity of the chip substrate material, and the like.

Such a groove can be formed by, for example, scraping a part of the surface of a chip on which two substrates are overlapped by a conventional method, and the method for producing the groove is not limited.
A preferred embodiment of the heat insulating means in the heating region, the cooling region, and the boundary of the microfluidic chip in the micro total analysis system of the present invention as described above will be described with reference to FIGS.

FIG. 2 is a top view showing an embodiment of the microfluidic chip 2 which has a heating region 32 and a cooling region 35 and is provided with a heat insulating means 37 at the boundary. In the heating region 32, there is a reaction portion for performing a gene amplification reaction by, for example, the PCR method. In the cooling area 35, for example, there are a specimen storage section and a reagent storage section for use in the gene amplification reaction. Further, the heat insulating means at the boundary between the heating region and the cooling region is a gap penetrating the chip or a groove formed in the chip surface as described above. Although a heating device and a cooling device provided in the apparatus main body are not shown, they contact the microfluidic chip from the upper surfaces of the heating region and the cooling region, respectively.

  FIG. 3 and FIG. 4 are schematic views of cross sections including a reaction portion, a sample storage portion and a reagent storage portion of the microfluidic chip, a heating member and a cooling member in contact with the chip surface, and the above-described heat insulating means.

  In FIG. 3, the heating device 33 as described above is brought into contact with the chip surface in the heating region 32 including the reaction portion flow path 31 (for example, the site where the gene amplification reaction is performed by the PCR method) to perform local heating. Yes. Further, the cooling device 36 as described above is pressed against the cooling region 35 including the reagent mixing channel 34 in which the reagents are mixed to cool to a predetermined temperature. A gap that penetrates the chip is provided at the boundary between the heating region 32 and the cooling region 35, whereby the heat conduction between the heating region 32 and the cooling region 35 is effectively blocked.

Further, in FIG. 4, a groove formed on the chip surface exists at the boundary between the heating region 32 and the cooling region 35, and such an aspect is also effective in suppressing heat conduction.
Micro-pump unit A system apparatus main body of the present invention includes a base body, a micro-pump including a chip connecting portion disposed in the base main body and having a channel opening for communicating with the microfluidic chip, and a plurality of micro-pumps. It has a unit as a structural unit. The micropump may be incorporated in the system apparatus main body as a chip-like pump unit in which a plurality of micropumps are formed by photolithography technology or the like, for example.

  As the micro pump, various types such as a check valve type pump in which a check valve is provided in an inflow / outflow hole of a valve chamber provided with an actuator can be used, but a piezo pump is preferably used. In general, the piezo pump includes a first flow path in which the flow path resistance changes in accordance with the differential pressure, a second flow path in which the change rate of the flow path resistance with respect to the change in the differential pressure is smaller than the first flow path, A pressurizing chamber connected to the first flow path and the second flow path, an actuator for changing the internal pressure of the pressurizing chamber, and a driving device for driving the actuator, By changing the voltage value and the frequency, the liquid feeding direction and the liquid feeding speed can be controlled. The present inventors have already proposed such a micropump and a system using the micropump, and details thereof are disclosed in JP 2001-322099 A, JP 2004-108285 A, JP 2004-270537 A, and the like. Can be referred to.

  FIG. 6A is a cross-sectional view showing an example of a piezo pump, and FIG. 6B is a top view thereof. In this micropump, a substrate 42 on which a first liquid chamber 48, a first flow path 46, a pressurizing chamber 45, a second flow path 47, and a second liquid chamber 49 are formed and laminated on the substrate 42. Upper substrate 41, diaphragm 43 laminated on upper substrate 41, piezoelectric element 44 laminated on the side of diaphragm 43 facing pressure chamber 45, and drive unit for driving piezoelectric element 44 ( (Not shown). The drive unit and two electrodes on the surface of the piezoelectric element 44 are connected by wiring such as a flexible cable, and a voltage having a specific waveform is applied to the piezoelectric element 44 by the drive circuit of the drive unit through the connection. It has become. Although not shown in FIGS. 6A and 6B, the first liquid chamber 48 is provided with a port 72 connected to the driving liquid tank 10, and the first liquid chamber is provided in the “reservoir”. It plays the role and receives the driving fluid from the driving fluid tank 10 at the port 72. The second liquid chamber 49 forms a flow path of the micropump unit, and a port 73 of the chip connection portion is provided at the end of the flow path, and is connected to the “pump connection portion” 12 of the microfluidic chip.

  FIG. 6C shows another example of this pump. In this example, the pump is composed of a silicon substrate 71, a piezoelectric element 44, and a flexible wiring (not shown). A pressurizing chamber 45, a diaphragm 43, a first channel 46, a first liquid chamber 48, a second channel 47, and a second liquid chamber 49 are formed. The first liquid chamber 48 is provided with a port 72, and the second liquid chamber 49 is provided with a chip connection port 73. For example, when this piezo pump is separated from the chip 2 in FIG. The port 73 communicates with the pump connection portion 12 of the microfluidic chip 2. For example, the pump can be connected to the microfluidic chip 2 by superimposing the substrate 74 in which the ports 72 and 73 are perforated and the vicinity of the pump connecting part of the microfluidic chip. Further, as described above, a plurality of pumps can be formed on one silicon substrate. In this case, it is desirable that the driving liquid tank 10 is connected to a port opposite to the port connected to the chip 2. When there are a plurality of pumps, their ports may be connected to a common drive fluid tank.

The relationship between the micro pump and the micro total analysis system of the present invention shown in FIG. 1 will be described. In the example of FIG. 1, the micropump 11 belongs to the system apparatus main body 1 as an apparatus different from the microfluidic chip 2 and communicates with the driving liquid tank 10. The micropump 11 is connected to the microfluidic chip 2 and the port 73 of the chip connection part in the micropump unit when the two are joined to each other in a predetermined form, and to communicate with the micropump in the microfluidic chip. The pump connection part 12 having the channel opening is connected to communicate with the channel of the microfluidic chip.

  As another aspect, the micropump itself can also be incorporated on the microfluidic chip. In particular, this mode can be adopted in the case where the flow path on the chip is relatively simple and the chip is used for a purpose or application that assumes repetitive use, for example, a chip for chemical synthesis reaction.

Analysis Embodiment The micro-analysis system of the present invention can be suitably used particularly for examination of genes or nucleic acids (DNA, RNA). In that case, the microchannel of the microfluidic chip is configured to be suitable for gene amplification by the PCR method or ICAN (Isothermal chimera primer initiated nucleic acid amplification) method. It can be said that the road configuration is almost the same. Usually, the specimen pretreatment unit, reagents, and probes may be changed. In this case, the arrangement and number of liquid feeding elements will change. A person skilled in the art can easily change the type of analysis by mounting the reagents necessary for immunoassay, for example, on the microfluidic chip and making modifications including slight changes to the flow path elements and specifications. Can be changed. Biological substances other than genes mentioned here refer to various metabolites, hormones, proteins (including enzymes and antigens), and the like.

In a preferred embodiment of the microfluidic chip of the present invention, in one chip,
A specimen storage part into which a specimen or an analyte extracted from the specimen (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.

FIG. 5 shows one embodiment of the configuration of the fine channel in such a microfluidic chip. The micropump 11 connected via the pump connection unit 12 of the microfluidic chip includes a sample housed in the sample storage unit 20 or a biological material extracted from the sample (for example, DNA or other biological material), and a reagent storage unit The reagent contained in 18 is sent to the flow path 15. These are mixed and reacted at the reaction site of the fine channel, for example, the site of gene amplification reaction (in the case of protein, antigen-antibody reaction, etc.), and then this reaction solution is transferred to the detection unit 22a in the downstream channel. The treated processing solution and the probe accommodated in the probe accommodating part 21d are fed, mixed in the flow path and combined (or hybridized) with the probe, and detection of biological material is performed based on the reaction product. Do. In addition, reaction and detection are performed in the same manner as described above for the positive control housed in the positive control housing section 21h and the negative control housed in the negative control housing section 21i.

-Sample The sample storage unit of the microfluidic chip communicates with the sample injection unit, and temporarily stores the sample and supplies the sample to the mixing unit. The sample is injected from a sample injection unit provided on the upper surface of the sample storage unit, for example. The specimen injection part is preferably formed with a stopper made of an elastic material such as a rubber-like material, or covered with a resin such as polydimethylsiloxane (PDMS) or a reinforced film. For example, a specimen in a syringe is injected with a needle that has been pierced with a stopper made of the rubber material or a needle that has passed through a pore with a lid.

  A specimen to be a target for genetic testing is a sample containing DNA or RNA. For example, whole blood, plasma, serum, buffy coat, urine, stool, saliva, sputum, biological samples, viruses, bacteria, mold, etc. , Yeast, animal and plant cells, and the like. Alternatively, DNA or RNA isolated from these samples by conventional techniques may be used.

  The amount of specimen required is very small compared to the manual work performed using conventional devices. For example, for a chip having a length and width of several centimeters, it is only necessary to inject a blood sample of about 2 to 3 μL. As DNA, it is 0.001-100 ng.

  In addition, the sample injected into the sample storage unit may be pre-processed in a sample pre-processing unit provided in advance in the channel before mixing with the reagent, if necessary. Preferred sample pretreatments include analyte separation or concentration, deproteinization, and the like. For example, there may be mentioned DNA extraction treatment in which lysis is performed using a lysis agent such as a 1% SDS mixed solution, and the released DNA is adsorbed on the membrane surface of beads, an adsorption resin or a filter.

-Reagents Reagents contained in the microfluidic chip are filled with a predetermined amount of necessary reagents according to the intended reaction method and detection method 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. Reagents necessary for analyzing a biological material in a specimen are the same as in the past. For example, when analyzing an antigen present in a specimen, a reagent containing an antibody (preferably a monoclonal antibody) against the antigen is used. The antibody is preferably labeled with biotin and FITC. Examples of reagents for genetic testing include various reagents used for gene amplification, probes used for detection, coloring reagents and the like, and if necessary, pretreatment reagents used for the sample pretreatment are also included. It is.

・ Mixing By supplying the driving liquid from the micropump, the specimen liquid and the reagent liquid are pushed out from each container and joined together, so that reactions such as gene amplification reaction, analyte trap or antigen-antibody reaction can be performed. Be started.

  The mixing of the reagent and the reagent and the mixing of the sample and the reagent may be performed at a desired ratio in a single mixing part, or a plurality of merging parts are provided by dividing one or both, and finally You may mix so that it may become a desired mixing ratio.

  The mode of such a reactive site is not particularly limited, and various forms and modes are conceivable. As an example, a micro flow channel in which each liquid is diffused and mixed is provided in advance of a joining portion (flow channel branching point) that joins two or more liquids containing reagents, and from the downstream end of the micro flow channel. The reaction is carried out in a liquid reservoir provided in advance and having a space wider than the fine channel.

-Reaction As a DNA amplification method, the PCR method described in various literatures including an improvement point and utilized actively in many fields can be used. In the PCR method, it is necessary to manage the temperature by raising and lowering between three temperatures. However, the temperature of the microfluidic chip may be controlled using an appropriate device. In the microfluidic chip, the heat cycle can be switched at high speed in the fine flow path, and DNA amplification can be performed in a much shorter time than manually. In addition, the recently developed ICAN method (Takara Bio Inc., registered trademark) allows DNA amplification to be carried out in a short time at an arbitrary constant temperature of 50 to 65 ° C. (Japanese Patent No. 3343929). It is also a suitable amplification technique in the system.

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

  Furthermore, the biotin affinity protein (avidin, streptavidin) adsorbed and immobilized on the detection site on the fine channel is labeled on the 5 ′ end of the biotin labeled on the probe substance or the primer used in the gene amplification reaction. Bind specifically to biotin. Thereby, the probe labeled with biotin or the amplified gene is trapped at the detection site.

The method for analyzing the DNA of the analyte or the target gene is not particularly limited, but as a preferred embodiment, it is basically carried out 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.

  The step of amplifying the gene in the microchannel of the reaction site, the amplification reaction solution containing the gene amplified in the microchannel and the denaturing solution are mixed, and the amplified gene is made into a single strand 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, A various deformation | transformation and change are possible within the range which does not deviate from the summary.

FIG. 1 is a diagram showing a schematic configuration in an embodiment of a micro comprehensive analysis system. FIG. 2 is a top view showing an embodiment of a microfluidic chip having a heating region and a cooling region, and having a heat insulating means at the boundary. FIG. 3 is a schematic diagram of a cross section including a reaction part, a specimen storage part and a reagent storage part of the microfluidic chip, a heating member and a cooling member in contact with the chip surface, and a gap penetrating the chip as the above-described heat insulating means. FIG. 4 is a schematic diagram of a cross section including a reaction portion, a specimen storage portion and a reagent storage portion of a microfluidic chip, a heating member and a cooling member that are in contact with the chip surface, and a groove formed on the chip surface as the above-described heat insulating means. is there. FIG. 5 is a diagram showing one embodiment of the configuration of the fine channel in the microfluidic chip. FIG. 6A is a cross-sectional view showing an example of a piezo pump, and FIG. 6B is a top view thereof. FIG. 6C is a cross-sectional view showing another example of the piezo pump.

Explanation of symbols

1 Body 2 Microfluidic chip 3 Peltier (cooling device)
4 Heater (heating device)
5 Photodiode 6 LED
DESCRIPTION OF SYMBOLS 10 Drive liquid tank 11 Micro pump 12 Pump connection part 13 Liquid feeding control part 15 Fine flow path 15a-15d Fine flow path 16 Check valve 17a Reagent storage part 17b Sample storage part 18 Reagent storage part 18a-18c Reagent storage part 20 Sample Storage unit 21a Reaction stop solution storage unit 21b Denaturation solution storage unit 21c Hybridization buffer storage unit 21d Wash liquid storage unit 21e Colloidal gold storage unit 21f Probe DNA storage unit 21g Internal control probe DNA storage unit 21h Positive control storage unit 21i Negative control Storage unit 21j Buffer storage unit 22 Detection unit 23 Waste liquid storage unit 31 Reaction unit flow path 32 Heating region 33 Heating device 34 Reagent storage unit 35 Cooling region 36 Cooling device 37 Thermal insulation means 37a Gap 37b Groove 38 Groove formation substrate 39 Covered substrate 41 Upper substrate 42 Substrate 43 Diaphragm 44 Piezoelectric element 45 Pressurization chamber 46 First flow path 47 Second flow path 48 First liquid chamber 49 Second liquid chamber 71 Silicon substrate 72 Port 73 Port 74 Substrate

Claims (7)

  Two or more types of temperatures selected from the group consisting of a “heating region” in contact with a separate heating device, a “cooling region” in contact with a separate cooling device, and a “room temperature region” in non-contact with the heating device and the cooling device A microfluidic chip in which regions coexist, wherein a heat insulating means is provided at a boundary between different types of temperature regions.   2. The microfluidic chip according to claim 1, wherein the heat insulating means is a gap penetrating the microfluidic chip.   2. The microfluidic chip according to claim 1, wherein the heat insulating means is a groove formed on the surface of the microfluidic chip substrate, and the groove is not in communication with a fine channel of the chip.   The microfluidic chip according to any one of claims 1 to 3, wherein a boundary between the different types of temperature regions is a boundary between a heating region and a cooling region.   The microfluidic chip according to claim 1, wherein the heating region includes a reaction unit that performs a gene amplification reaction.   The microfluidic chip according to any one of claims 1 to 4, wherein the cooling region includes a specimen container and / or a reagent container. The microfluidic chip according to any one of claims 1 to 6,
A system unit body;
The system apparatus main body includes at least a base main body,
A micropump unit including a chip connection portion disposed in the base body and having a channel opening for communicating with the chip; and the micropump;
A heating device and / or a cooling device;
A control device for controlling at least the function of the micropump unit and the heating device and / or cooling device;
With
The micropump
A first flow path in which the flow path resistance changes according to the differential pressure;
A second flow path whose rate of change in flow path resistance with respect to a change in differential pressure is smaller than the first flow path;
A pressurization chamber connected to the first flow path and the second flow path;
An actuator for changing the internal pressure of the pressurizing chamber;
A micro total analysis system, characterized in that it is a micro pump provided with a drive device for driving the actuator.

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