JP2006284451A - Micro total analysis system for analyzing target material in specimen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To further simplify and compact an analysis control system constituted by various kinds of devices for controlling liquid feed, reaction, detection and the like in a fine flow channel of a chip for analysis. <P>SOLUTION: A plurality of micro pumps 12 are disposed at respective positions in the surface direction of one chip. A compact pump mechanism is constituted by a micro pump unit 11 of which flow channel opening 15 communicated with the fine flow channel of the inspected chip is disposed on the downstream thereof, and a drive liquid tank that is connected with the upstream side of the micro pump 12 and supplies drive liquid to the plurality of micro pumps 12. The inspected chip storing a plurality of reagents in the fine flow channel and a micro pump unit are overlapped and interconnected so as to inter-couple respective flow channel openings, the drive liquid from the drive liquid tank is supplied to the upstream of each reagent by the micro pump to press each reagent to the downstream, and the reagents are merged and mixed in the flow channel. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a micro total analysis system that analyzes a target substance in a sample by using a test chip provided with a series of fine flow channels for mixing and reacting a sample and a reagent to detect the reaction.

In recent years, by making full use of micromachine technology and ultrafine processing technology, devices and means (for example, pumps, valves, flow paths, sensors, etc.) for performing conventional sample preparation, chemical analysis, chemical synthesis, etc. have been miniaturized. A system integrated on a chip has been developed (Patent Document 1). This is a micro-TAS (Micro total Analysis System), bioreactor, love
It is also called “Lab-on-chips” or biochip, and its application is expected in the medical examination / diagnosis field, environmental measurement field, and agricultural production field. In reality, as seen in genetic testing, automated, faster, and simplified microanalysis systems are costly and necessary when complex processes, skilled techniques, and equipment operations are required. It can be said that not only the amount of sample and the time required, but also the benefits of enabling analysis at any time and place are great.

In various types of analysis and inspection, importance is attached to the quantitativeness of analysis, the accuracy of analysis, and the economic efficiency of these analysis chips. For that purpose, it is a problem to establish a highly reliable liquid feeding system with a simple configuration. There is a need for a microfluidic control element with high accuracy and excellent reliability. The present inventors have already proposed a micropump system and a control method thereof suitable for this (Patent Documents 2 to 4).
JP 2004-28589 A JP 2001-322099 A JP 2004-108285 A JP 2004-270537 A

  In the analysis using the micro analysis system described above, in addition to the analysis chip, for example, a micropump, a detection device, a temperature control device, etc., control the liquid feeding, reaction, detection thereof, etc. in the micro flow channel of the chip. Various devices are necessary for this purpose. When constructing an analysis control system using these apparatuses, it is not desirable that the arrangement of each component is complicated or occupies a large space.

  It is an object of the present invention to provide a simpler and more compact structure of an analysis control system including various devices for controlling liquid feeding, reaction, detection thereof, and the like in a fine channel of an analysis chip. It is said.

In the micro total analysis system of the present invention, a plurality of reagents stored in advance in each position in the flow channel are merged and mixed on the downstream side, and then the mixed reagent and the sample are merged to react with each other. A test chip provided with a series of fine flow paths to be detected and provided with a flow path opening communicating with the micropump upstream from the position where the reagent is stored in the fine flow path;
A driving liquid tank containing a driving liquid that pushes the reagent from the upstream side and feeds it in the downstream direction of the fine flow path of the inspection chip; and
A plurality of micropumps are provided at each position in the surface direction of one chip, the upstream side of each micropump is communicated with the driving liquid tank, and the flow path opening for communicating with the fine flow path of the inspection chip is provided downstream thereof. A provided micropump unit,
After the inspection chip is connected to the micro pump unit so that these flow path openings overlap, the driving liquid is sent to the micro flow path of the inspection chip by the plurality of micro pumps, and the plurality of reagents are moved downstream. These reagents are combined and mixed by extruding,
Thereafter, the mixed reagent and the specimen are combined and reacted to detect the reaction, thereby analyzing the target substance in the specimen.

  Preferably, all the micropumps in the micropump unit are communicated with one driving liquid tank, and the driving liquid stored in the driving liquid tank is sent from each micropump to the fine flow path of the inspection chip.

  In this way, all the micropumps for feeding each reagent are arranged on one chip, and this chip and the inspection chip are overlapped at the time of analysis so as to communicate with each other. The pump mechanism that pushes the reagent in the chip downstream of the fine channel can be made compact.

  In addition, a plurality of micropumps can share the driving fluid tank, and no special piping or routing tips are required to connect the driving fluid tank and the chip-like micropump unit. The pump mechanism that pushes out downstream of the path can be made compact.

  In a preferred embodiment of the micro total analysis system of the present invention, two or more micropumps are communicated with one reagent accommodated in the fine flow path of the test chip, and the driving liquid from these micropumps is supplied to the micropump unit. The reagents are merged in the provided flow path, and the reagent is pushed by the merged driving liquid and fed in the downstream direction of the fine flow path.

  Thus, since the flow path for combining the driving liquids from the plurality of micropumps downstream is provided in the chip of the micropump unit, one reagent is fed by the plurality of parallel arranged micropumps. be able to.

  For example, the number of micropumps communicated with at least one reagent among a plurality of reagents and the number of micropumps communicated with other reagents are different from each other so that these reagents are combined and mixed. The mixing ratio of these reagents in the reagents can be varied. In particular, when the mixing ratio of a certain reagent and another reagent is high, it is necessary to greatly vary the driving voltage of each micropump when they are fed by a single micropump. The reagent can be mixed at a high mixing ratio without greatly changing the driving voltage of the micropump for feeding each reagent.

The micropump preferably used in the micro total analysis system of the present invention is:
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 driven by voltage to change the internal pressure of the pressurizing chamber;
It has.

  The micro integrated analysis system of the present invention is preferably composed of a system body in which a micropump unit and a driving liquid tank are housed and integrated in one housing body, and the inspection chip, and the inspection chip is connected to the system body. The target substance in the sample is analyzed in the state where it is mounted on the.

More preferably, the system body is
A detection processing device for detecting a reaction in the inspection chip;
A control device for controlling the micropump unit and the detection processing device;
Is provided inside the container.

  The micro integrated analysis system of the present invention has a simple and compact structure of a pump mechanism for feeding each reagent in the fine flow path of the test chip downstream.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
<Inspection chip>
The inspection chip used in the present invention is a microreactor in which each flow path element or structure is finely placed at a functionally appropriate position so that it can be used for chemical analysis, various inspections, sample processing / separation, chemical synthesis, etc. It is arranged by processing technology.

  The inspection chip is provided with a plurality of reagent storage units for storing each reagent, and the reagent storage unit stores reagents used for a predetermined reaction, a cleaning solution, a denaturing treatment solution, and the like. 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.

  The inspection chip can be manufactured using, for example, a groove-formed substrate in which grooves for configuring a flow path or the like are formed in advance on a substrate surface, and a coated substrate that is in close contact with the groove-formed substrate. The groove forming substrate is formed with each structure portion and a flow path for communicating these structure portions. Specific examples of such a structure part include liquid storage parts such as each storage part (reagent storage part, sample storage part, etc.) and a waste liquid storage part, a valve base part, and a liquid feed control part (shown in FIG. 7 described later). Examples include a water repellent valve), a backflow prevention unit (a check valve, an active valve, etc.), a reagent quantification unit, a mixing unit, and other parts for controlling liquid feeding, a reaction unit, and a detection unit. Such a structure part and a flow path may be formed also in the covering substrate. An inspection chip is formed by covering the structure portion and the flow path by bringing the covering substrate into close contact with the groove forming substrate. In addition, when detecting the reaction in a test | inspection chip | tip optically, it is necessary for at least a detection part among said structure parts to adhere | attach a light-transmitting coating substrate, and to cover it. In addition, an inspection chip may be formed by stacking three or more substrates.

  The inspection chip is usually produced by appropriately combining one or more molding materials. Examples of the molding material for the inspection chip include plastic resin, various inorganic glasses, silicon, ceramics, and metal.

  In particular, it is desirable to be disposable for a large number of samples to be measured, especially clinical samples that are at risk of contamination or infection, and it is also desirable to have versatility and mass productivity. In view of this, it is preferable to use a plastic resin as a molding material for the inspection chip.

  For a substrate for forming and processing a channel such as a groove forming substrate, a hydrophobic and water-repellent plastic is preferable so that a deformation of the channel due to water absorption is unlikely to occur and a minute amount of sample liquid is sent without loss in the middle. . Examples of such materials include resins such as polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polyethylene vinyl alcohol, polycarbonate, polymethylpentene, fluorocarbon, and saturated cyclic polyolefin. Among these, polystyrene is excellent as transparency, mechanical properties, and moldability, and can be easily finely processed. Therefore, polystyrene is preferable as a material for forming a groove forming substrate.

  When heating near 100 ° C. is necessary for the convenience of analysis, a resin having excellent heat resistance, such as polycarbonate, polyimide, polyetherimide, polybenzimidazole, polyetheretherketone, or the like is used as the substrate material.

  In order to advance the reaction for detecting the analyte, a predetermined location or reaction site of the microreactor channel is often heated to a desired temperature. The temperature for locally heating in the heating region is usually up to about 100 ° C. On the other hand, it may be necessary to cool specimens and reagents that become unstable at high temperatures. In view of such local temperature increase and decrease in the chip, it is desirable to select a material having an appropriate thermal conductivity. Examples of such a material include a resin material and a glass material. By forming these regions with a material having low thermal conductivity, heat conduction in the surface direction is suppressed, and only the heating region is selectively selected. Can be heated.

  In order to optically detect a fluorescent substance or a product of a color reaction, it is necessary to dispose a light transmissive member on at least a portion of the inspection chip surface covering the detection portion of the fine channel. Therefore, a transparent material such as alkali glass, quartz glass, or transparent plastic is used as the material for the coated substrate that covers the detection site. Note that such a light-transmitting coated substrate may cover the entire upper surface of the inspection chip.

  The flow path of the inspection chip as the microreactor is formed on the substrate according to the flow path arrangement designed in advance according to the purpose. The flow path through which the liquid flows is, for example, a micro flow path having a width of several tens to several hundreds μm, preferably 50 to 200 μm, and a depth of 25 to 300 μm, preferably 50 to 100 μm, having a micrometer order width. . When the flow path width is narrowed, the flow path resistance increases, which may cause problems in liquid delivery and the like. If the channel width is too wide, the advantage of the microscale space is diminished. The vertical and horizontal sizes of the entire inspection chip are typically several tens of mm, and the height is about several mm.

  Each structural part and flow path of the substrate can be formed by a conventional microfabrication technique. Typically, transfer of a fine structure using a photosensitive resin by a photolithography technique is preferable, and removal of an unnecessary part, addition of a necessary part, and transfer of a shape are performed using the transfer structure. For example, a pattern that models the constituent elements of the inspection chip is produced by photolithography, and this pattern is transferred and molded on a resin. As the basic substrate material for forming the microchannel of the microreactor, a plastic resin that can accurately transfer a submicron structure and has good mechanical properties is preferably used. Among them, polystyrene, polydimethylsiloxane and the like are excellent in shape transferability. If necessary, processing for forming each structural portion of the substrate and the flow path may be performed by injection molding, extrusion molding, or the like.

  A pump connection unit for connecting to a separate micropump is provided on the upstream side of the fine flow path of the test chip, for example, on the upstream side of the storage unit that stores each liquid such as a reagent and a specimen. The pump connection portion is provided with a flow passage opening communicating with the housing portion, and the driving liquid is supplied from the flow passage opening by the micro pump, and the liquid in each housing portion is pushed out to the downstream side.

  FIG. 3 is a plan view of a test chip in one embodiment of the micro total analysis system of the present invention. In the test chip 2, three types of reagents are accommodated in a total of three flow paths of reagent accommodating portions 33a, 33b, and 33c. At both end portions of these reagent storage portions (upstream end portion 34a and downstream end portion 34b in reagent storage portion 33a), a liquid feed control portion (water repellent valve) having the structure shown in FIG. 7 is provided. The reagent is sealed in the flow path between these water repellent valves.

FIG. 7 is a cross-sectional view of the liquid feeding control unit (water repellent valve). The liquid feeding control unit 51 includes a liquid feeding control passage 52 having a small diameter. The liquid feeding control passage 52 has a cross-sectional area (cross-sectional area perpendicular to the flow path) smaller than the cross-sectional areas of the upstream flow path 53a and the downstream flow path 53b.

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

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

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

  Although not described in detail, the liquid flow control unit 51 of FIG. 7 is provided in the fine flow path of the test chip 2 of FIG. 3 at positions other than both ends of the reagent storage units 33a to 33c. For example, the liquid feeding control unit is also provided at the end of the reagent mixing unit 36 and the sample storage unit 37 on the side of the merging unit 38 to control the timing of the liquid feeding start to the flow path beyond that. .

  Openings 32c to 32e opened from one surface of the test chip 2 to the outside are provided on the upstream side of the reagent containers 33a to 33c in FIG. These openings 32c to 32e are aligned with the flow path openings provided on the connection surface of the micropump unit when the inspection chip 2 is connected to the micropump unit, which will be described later, and communicated with the micropump. .

  Similarly, the openings 32a, 32b and 32f to 32j communicate with the micro pump by connecting the test chip 2 and the micro pump unit. The pump connection portion 31 is configured by a chip surface including these openings 32a to 32j, and the inspection chip 2 and the micro pump unit are connected by bringing the pump connection portion 31 into close contact with the connection surface of the micro pump unit. The pump connection portion 31 is formed with a contact surface made of a resin having flexibility (elasticity, shape followability) such as polytetrafluoroethylene or silicone resin in order to ensure necessary sealing performance and prevent leakage of driving fluid. It is preferred that Such a close contact surface having flexibility may be due to, for example, the constituent substrate itself of the inspection chip, or by a separate member having flexibility attached around the flow path opening in the pump connection portion. It may be a thing.

  Reagents stored in the flow paths 33a to 33c flow into the merge section 35 through the liquid feed control section 51 of FIG. 7 by separate micro pumps communicating with the openings 32c to 32e, and the flow paths that follow the flow path. The three types of reagents are mixed in the reagent mixing unit 36.

The mixed reagent mixed in the reagent mixing unit 36 merges with the sample stored in the sample storage unit 37 in the combining unit 38. The mixed reagent is pushed downstream with the driving liquid by the micro pump communicated with the opening 32b, and the specimen is pushed downstream with the driving liquid by the micro pump communicated with the opening 32a. The mixed solution of the mixed reagent and the specimen is accommodated in the reaction unit 39 and the reaction is started by heating or the like.

The liquid after the reaction is sent to the detection unit 40, and the target substance is detected by, for example, an optical detection method. Each of the reagents (for example, a solution for stopping the reaction between the mixed reagent and the sample, a substance to be detected) contained in the flow path ahead of these openings by separate micro pumps communicating with the openings 32f to 32j. On the other hand, a liquid for performing necessary processing such as a label, a cleaning liquid, and the like) are extruded downstream at a predetermined timing and fed.
<Micro pump unit>
FIG. 1 is a perspective view of a micropump unit in one embodiment of the micro total analysis system of the present invention, and FIG. 2 is a sectional view thereof. The micropump unit 11 includes three substrates: a silicon substrate 17, a glass substrate 18 thereon, and a glass substrate 19 thereon. The substrate 17 and the substrate 18 and the substrate 18 and the substrate 19 are joined by anodic bonding, respectively.

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

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

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

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

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

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

  Control of the flow rate by the micropump 12 can be performed by adjusting the voltage applied to the piezoelectric element 21. FIG. 8 shows an example of the waveform of the drive voltage applied to the piezoelectric element. The maximum voltage applied to the piezoelectric element is about several volts to several tens of volts, and about 100 volts at the maximum. As an example, time t1 is about 60 μs and time t2 is about 20 μs. The frequency of the drive voltage is about 11 KHz.

  Two electrodes for driving the piezoelectric element 21 are connected to a flexible wiring (not shown). An ITO film, which is a transparent electrode film, is formed on the surface of the silicon diaphragm, and one surface of the piezoelectric element 21 is adhered on the ITO film with an adhesive so that one electrode of the piezoelectric element 21 is electrically connected to the ITO film. The ITO film and the flexible wiring are connected to each other. The other surface of the piezoelectric element 21 is gold-plated, and a flexible wiring is directly connected to the gold-plated portion.

Note that a substrate other than a silicon substrate may be used as a substrate on which the micropump 12 is formed. For example, a photosensitive glass substrate may be used.
In addition, a micro pump other than the above-described piezo pump, such as a check valve type pump, may be provided, but in the present invention, a piezo pump is preferably used.

  Examples of the glass substrates 18 and 19 laminated on the silicon substrate 17 include Pyrex glass (Pyrex is a registered trademark of Corning Glass Warks), Tempax glass (Tempax is a registered trademark of Schott Glaswerk). Etc. are used.

  A flow path 20 is patterned on the substrate 19. As an example, the size and shape of the channel 20 are rectangular in cross section with a width of about 150 μm and a depth of about 300 μm. On the downstream side of the flow path 20, an opening 15 is provided for allowing the micro pump 12 to communicate with the fine flow path of the inspection chip by aligning with the openings 32 a to 32 k of the inspection chip of FIG. 3. The opening 15 may have a size larger than the width of the flow path 20 if necessary for proper alignment with the opening of the inspection chip. As shown in the figure, since the driving liquid can be discharged from the opening 15 formed at a desired position on the chip surface of the micropump unit 11, for the purpose of routing the driving liquid to the predetermined position on the inspection chip side. The flow path can be omitted or reduced.

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

  The micropump unit 11 is merely an example, and various micropump units in which a micropump, a flow path, and a connection opening for communicating with a test chip and a driving liquid tank are formed by a photolithography technique or the like. Can be produced. For example, a glass substrate is laminated on a silicon substrate, a photosensitive glass substrate, etc. formed by etching the structure of a micropump, and PDMS is laminated thereon, and further, plastic, glass, silicon, ceramics, etc. A micropump unit can be configured by bonding together a substrate on which the flow channel groove and the connection opening are formed.

In FIG. 1, the driving liquid is supplied from one opening 15 through one channel 15 by one micro pump 12. In this case, the amount of the driving liquid fed from each opening 15 can be controlled by the driving voltage applied to each micropump 12, but as shown in FIG. Control can also be achieved by providing micropumps 12a, 12b, and 12c having different sizes of pump chambers to which the piezoelectric elements are bonded.

  In this example, a piezo pump is used as the micro pump. Each of the openings 15a, 15b, and 15c communicates with the openings 32c, 32d, and 32e of the inspection chip shown in FIG. 3 (note that only a part of the entire micropump unit is shown in FIG. 16). The micropump 12a feeds the driving liquid through the flow path 20a, the opening 15a, and the opening 32c to push the reagent stored in the reagent storage section 33a downstream, and the micro pump 12b drives the flow path 20b, the opening 15b, and the opening 32d. The liquid is fed to push the reagent contained in the reagent container 33b downstream, and the driving liquid is fed through the flow path 20c, the opening 15c, and the opening 32e by the micropump 12c, and the reagent contained in the reagent container 33c. Is pushed downstream.

  The reagent housed in the reagent container 33a is pushed downstream by the micropump 12a having a small size, the reagent housed in the reagent container 33b is pushed downstream by the micropump 12b having a larger size, and the size is smaller than that. Each reagent is mixed in the reagent mixing unit 36 of FIG. 3 so that the mixing ratio increases in this order by pushing the reagent stored in the reagent storage unit 33c downstream by the large micropump 12c.

  As described above, the reagent that reacts with the sample in the fine flow path of the test chip is usually a reagent in which a plurality of reagents are mixed, and the reagents stored in separate reagent storage units are combined to form a mixed reagent. If it is necessary to combine with the sample, but it is necessary to increase the mixing ratio of the reagents, using the same type of micropump will greatly vary the voltage that drives each micropump that delivers each reagent. There must be. However, as shown in FIG. 6, the difference in driving voltage can be reduced by changing the size of the micropump for feeding each reagent.

  FIG. 4 is a perspective view showing a micropump unit in another embodiment of the micro total analysis system of the present invention. In the present embodiment, the micropumps 12 a to 12 d are communicated with one opening 15 c through the flow path 20.

  The driving liquid that pushes out the reagent is supplied from the openings 15a, 15b, and 15c. The driving liquid from the openings 15a and 15b is fed by one micropump, whereas the driving liquid from the opening 15c has four driving liquids. Liquid is fed by the micro pumps 12a to 12d. As a result, the mixing ratio of each reagent fed by the driving liquid from the openings 15a and 15b and the reagent sent by the driving liquid from the opening 15c can be changed. Thereby, even when reagents are mixed at a high mixing ratio, the difference in driving voltage for driving the micropump can be reduced.

  A specific example in which the mixing ratio of the reagents is controlled by the number of micropumps is shown in FIG. 5 (in the figure, only a part of the entire micropump unit is shown). In this example, one micropump 12a communicates with the opening 15a via the flow path 20a, three micropumps 12b to 12d communicate with the opening 15b via the flow path 20b, and five micropumps 12e ~. 12i communicates with the opening 15c via the flow path 20c. Each micropump uses a piezo pump of the same shape, and the drive voltage for each micropump is also the same.

The openings 15a, 15b, and 15c communicate with the openings 32c, 32d, and 32e of the inspection chip shown in FIG. The flow path 20a, the opening 15a, and the opening 32c are provided by the micro pump 12a.
Through which the driving liquid is fed to push the reagent contained in the reagent containing part 33a downstream, and the micropumps 12b to 12d send the driving liquid through the flow path 20b, the opening 15b, and the opening 32d to the reagent containing part 33b. The stored reagent is pushed downstream, and the driving liquid is sent through the flow path 20c, the opening 15c, and the opening 32e by the micropumps 12e to 12i to push the reagent stored in the reagent storage part 33c downstream.

The reagent housed in the reagent container 33a is pushed downstream by one micro pump 12a, the reagent housed in the reagent container 33b is pushed downstream by three micro pumps 12b to 12d, and the five micro pumps 12e to 12i are pushed. Each reagent is mixed in the reagent mixing section 36 of FIG. 3 at a mixing ratio of approximately 1: 3: 5 by pushing the reagent stored in the reagent storage section 33c downstream.
<Micro total analysis system>
The test chip is subjected to reaction and analysis, for example, by being attached to a separate system body. The system main body and the inspection chip constitute a micro total analysis system. An example of this micro total analysis system will be described below. FIG. 9 is a perspective view showing an example of a micro total analysis system, and FIG. 10 is a diagram showing an internal configuration of a system main body in the micro total analysis system.

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

  Further, inside the storage body 62 is a detection processing device (LED, photomultiplier, light source 68 such as a CCD camera, etc. for detecting a reaction in the test chip 2, optical spectroscopy such as visible spectroscopy and fluorescence photometry. A detector 69) that performs detection, and a control device (not shown) that controls the detection processing device and the micropump unit 11 are provided. With this control device, in addition to control of liquid feeding by a micro pump, control of a detection processing device that detects a reaction in the inspection chip 2 by optical means, etc., temperature control of the inspection chip 2 by a heating / cooling unit, which will be described later, Control of reaction in 2 and data collection (measurement) and processing 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 1, a pump connection portion including a flow channel opening provided on the upstream side of a fine flow channel of the test chip 2 (for example, upstream of a reagent storage unit, a sample storage unit, etc.) and a chip surface around the channel opening. 31 and the chip connection part 13 of the micropump unit 11 are in close contact with each other in a liquid-tight state, the test chip 2 is mounted inside the storage body 62, and then the target substance in the sample is analyzed in the test chip 2. The inspection chip 2 is placed on the transport tray 65 and introduced into the storage body 62 from the chip insertion port 63. However, if the inspection chip can be fixed inside the storage body 62 in a state where the inspection chip is pressed against the micropump unit, the transport tray is not necessarily used.

  Inside the storage body 62, a heating / cooling unit (Peltier element 66, heater 67) for locally heating or cooling the inspection chip 2 mounted at a predetermined position is provided. For example, the reagent containing portion is selectively cooled by pressing the Peltier element 66 against the reagent containing portion region in the test chip 2, thereby preventing the reagent from being altered and the like, and the region of the flow path constituting the reaction portion. The reaction part is selectively heated by press-contacting the heater 67, so that the reaction part is brought to a temperature suitable for the reaction.

  The micro pump unit 11 is connected to one driving liquid tank 61, and the upstream side of the micro pump communicates with the driving liquid tank 61. On the other hand, the downstream side of the micropump is communicated with a channel opening provided on one side of the micropump unit 11. Each channel opening communicated with each micropump and the pump connection portion 31 of the inspection chip 2. The inspection chip 2 is connected to the micropump unit 11 so as to be connected to the respective channel openings provided in the.

  By means of a micropump, an oil-based or water-based driving liquid such as mineral oil stored in the driving liquid tank 61 is sent to the storage part of each liquid in the inspection chip 2 via the pump connection part 13, and each storage is performed by the driving liquid. The liquid of the part is pushed out to the downstream side of the inspection chip 2 and fed.

  A series of analysis steps of pretreatment, reaction, and detection of a specimen as a measurement sample is performed in a state in which a test chip 2 is mounted on a system body 1 in which a micropump, a detection processing device, and a control device are integrated. Preferably, a predetermined reaction and optical measurement based on liquid feeding, pretreatment, and mixing of samples and reagents are automatically performed as a series of continuous steps, and the measurement data is filed with necessary conditions and recorded items. Stored in. In FIG. 9, the analysis result is displayed on the display unit 64 of the container 62.

Hereinafter, a specific example of the reaction between the specimen and the reagent using the test chip and the detection thereof will be shown. In a preferred embodiment of the inspection chip, in one chip,
A sample container into which a sample or an analyte extracted from the sample (eg, DNA, RNA, gene) is injected;
A sample pretreatment unit for preprocessing the sample;
A reagent container for storing reagents used for probe binding reaction, detection reaction (including gene amplification reaction or antigen-antibody reaction);
A positive control accommodating part for accommodating a positive control;
A negative control accommodating portion for accommodating a negative control;
A probe accommodating portion that accommodates a probe (for example, a probe that hybridizes to a gene to be detected amplified by a gene amplification reaction);
A fine flow path communicating with each of these accommodating portions,
A pump connection portion that can be connected to each of the accommodating portions and a separate micropump for feeding the liquid in the flow path is provided.

  A micropump is connected to the test chip via a pump connection unit, and the sample contained in the sample storage unit or a biological material extracted from the sample (for example, DNA or other biological material) and stored in the reagent storage unit The reagent thus prepared is sent to a downstream channel, and mixed and reacted at a reaction site in the fine channel, for example, a site where a gene amplification reaction (in the case of protein, antigen-antibody reaction or the like) is performed. 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 storage unit in the test chip communicates with the sample injection unit, and temporarily stores the sample and supplies the sample to the mixing unit. The specimen injection part that injects the specimen from the upper surface of the specimen storage part has a stopper made of an elastic material such as a rubbery material to prevent leakage, infection and contamination to the outside, and to ensure sealing performance, Or it is desirable to be covered with resin, such as polydimethylsiloxane (PDMS), and 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. In the former case, it is preferable that the needle hole is immediately closed when the needle is pulled out. Alternatively, another specimen injection mechanism may be installed.

  The sample injected into the sample storage unit is pretreated, for example, by mixing the sample and the treatment liquid, for example, in a sample pretreatment unit provided in advance in the flow path before mixing with the reagent. The Such a specimen pretreatment unit may include a separation filter, an adsorption resin, beads, and the like. Preferred sample pretreatments include analyte separation or concentration, 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 inspection chip 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 for gene testing may include various reagents used for gene amplification, probes used for detection, and coloring reagent, as well as pretreatment reagents used for the sample pretreatment if necessary.

  By supplying the driving liquid from the micropump, the specimen liquid and the reagent liquid are pushed out from each container and merged to start a reaction required for analysis such as gene amplification reaction, analyte trap or antigen-antibody reaction. The

  As a DNA amplification method, a PCR amplification method described in various documents including improvements and actively used in various fields can be used. In the PCR amplification method, it is necessary to manage the temperature by raising and lowering between three temperatures. However, a channel device that enables temperature control suitable for a microchip has already been proposed by the present inventors (Japanese Patent Application Laid-Open No. 2005-318867). 2004-108285). This device system may be applied to the amplification channel of the chip of the present invention. As a result, the heat cycle can be switched at high speed, and the micro flow path is a micro reaction cell with a small heat capacity. Therefore, DNA amplification can be performed in a much shorter time than the conventional method that is performed manually.

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

  A detection site for detecting an analyte, for example, an amplified gene, is provided downstream of the reaction site in the fine flow path of the test chip. 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 site on the microchannel is specific to biotin labeled on the probe substance or biotin labeled on the 5 'end of the primer used in the gene amplification reaction. Join. Thereby, the probe labeled with biotin or the amplified 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 a gene amplification reaction in the microchannel of the reaction site, the amplification reaction solution containing the gene amplified in the microchannel is mixed with a denaturation solution, and the amplified gene is single-stranded by denaturation treatment. And a probe DNA whose ends are fluorescently labeled with FITC (fluorescein isothiocyanate)
And are hybridized.

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 perspective view showing a micropump unit in one embodiment of the micro total analysis system of the present invention. FIG. 2 is a cross-sectional view of the micropump unit of FIG. FIG. 3 is a plan view of a test chip in one embodiment of the micro total analysis system of the present invention. FIG. 4 is a perspective view showing a micropump unit in another embodiment of the micro total analysis system of the present invention. FIG. 5 is a plan view showing one example of the arrangement of a plurality of micropumps and flow paths communicating with the micropumps in the micropump unit. FIG. 6 is a plan view showing another example of the arrangement of a plurality of micropumps and flow paths communicating therewith in the micropump unit. FIG. 7 is a cross-sectional view of the liquid feeding control unit (water repellent valve). FIG. 8 is a graph showing an example of a drive voltage waveform applied to the piezoelectric element of the piezo pump. FIG. 9 is a perspective view showing one embodiment of the micro total analysis system of the present invention. FIG. 10 is a diagram showing an internal configuration of the system main body in the micro integrated analysis system of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Micro comprehensive analysis system 2 Test | inspection chip 3 System main body 11 Micro pump unit 12, 12a-12i Micro pump 13 Chip connection part 14 Opening 15, 15a-15c Opening 16a Through-hole 16b Through-hole 17 Substrate 18 Substrate 19 Substrate 20, 20a- 20c flow path 21 piezoelectric element 22 pressurization chamber 23 first flow path 24 second flow path 25 first liquid chamber 26 second liquid chamber 31 pump connection portions 32a to 32j openings 33a to 33c reagent storage portions 34a and 34b end portion 35 Junction unit 36 Reagent mixing unit 37 Sample storage unit 38 Junction unit 39 Reaction unit 40 Detection unit 51 Liquid feed control unit (water repellent valve)
52 Liquid feed control passages 53a and 53b Flow path 54 Liquid 61 Driving liquid tank 62 Storage body 63 Chip insertion port 64 Display unit 65 Transport tray 66 Peltier element 67 Heater 68 Light source 69 Detector

Claims (7)

A series of fine flow paths are provided to detect a reaction by joining a plurality of reagents stored in advance in each position in the flow path and mixing them on the downstream side, and then mixing and reacting the mixed reagent and the sample. A test chip provided with a channel opening communicating with the micropump upstream of the position where the reagent is stored in the microchannel;
A driving liquid tank containing a driving liquid that pushes the reagent from the upstream side and feeds it in the downstream direction of the fine flow path of the inspection chip; and
A plurality of micropumps are provided at each position in the surface direction of one chip, the upstream side of each micropump is communicated with the driving liquid tank, and the flow path opening for communicating with the fine flow path of the inspection chip is provided downstream thereof. A provided micropump unit,
After the inspection chip is connected to the micro pump unit so that these flow path openings overlap, the driving liquid is sent to the micro flow path of the inspection chip by the plurality of micro pumps, and the plurality of reagents are moved downstream. These reagents are combined and mixed by extruding,
Thereafter, the mixed reagent and the specimen are combined and reacted to detect the reaction, thereby analyzing the target substance in the specimen.   All the micropumps in the micropump unit are communicated with one driving liquid tank, and the driving liquid stored in the driving liquid tank is sent from each micropump to the fine flow path of the inspection chip. The micro total analysis system according to claim 1.   Two or more micropumps are communicated with one reagent accommodated in the fine flow path of the test chip, and driving liquids from these micropumps are merged in the flow path provided in the micropump unit and merged. The micro total analysis system according to claim 1 or 2, wherein the reagent is pushed by the driving liquid and fed in the downstream direction of the fine channel.   The number of micropumps communicated with at least one of the plurality of reagents is different from the number of micropumps communicated with other reagents, and these reagents in a mixed reagent in which these reagents are combined and mixed The micro total analysis system according to claim 3, wherein mixing ratios of the reagents are different from each other. The micropump is
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 driven by voltage to change the internal pressure of the pressurizing chamber;
The micro total analysis system according to any one of claims 1 to 4, further comprising: The micro comprehensive analysis system includes:
A system main body in which a micro pump unit and a driving liquid tank are housed and integrated in one housing body, and the inspection chip, and
6. The micro total analysis system according to claim 1, wherein the target substance in the sample is analyzed in a state where the test chip is mounted on the system main body. The system main body is
A detection processing device for detecting a reaction in the inspection chip;
A control device for controlling the micropump unit and the detection processing device;
The micro total analysis system according to claim 6, further comprising:

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