WO2007099736A1 - Puce de micro-inspection, detecteur optique et systeme analytique micro-complet - Google Patents

Puce de micro-inspection, detecteur optique et systeme analytique micro-complet Download PDF

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Publication number
WO2007099736A1
WO2007099736A1 PCT/JP2007/051926 JP2007051926W WO2007099736A1 WO 2007099736 A1 WO2007099736 A1 WO 2007099736A1 JP 2007051926 W JP2007051926 W JP 2007051926W WO 2007099736 A1 WO2007099736 A1 WO 2007099736A1
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WO
WIPO (PCT)
Prior art keywords
micro
chip
channel
heating means
inspection chip
Prior art date
Application number
PCT/JP2007/051926
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English (en)
Japanese (ja)
Inventor
Yasuhiro Sando
Kusunoki Higashino
Youichi Aoki
Akihisa Nakajima
Yuushi Nobumoto
Original Assignee
Konica Minolta Medical & Graphic, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Konica Minolta Medical & Graphic, Inc. filed Critical Konica Minolta Medical & Graphic, Inc.
Priority to JP2008502680A priority Critical patent/JPWO2007099736A1/ja
Publication of WO2007099736A1 publication Critical patent/WO2007099736A1/fr

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Classifications

    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F04—POSITIVE - DISPLACEMENT MACHINES FOR LIQUIDS; PUMPS FOR LIQUIDS OR ELASTIC FLUIDS
    • F04B—POSITIVE-DISPLACEMENT MACHINES FOR LIQUIDS; PUMPS
    • F04B43/00—Machines, pumps, or pumping installations having flexible working members
    • F04B43/02—Machines, pumps, or pumping installations having flexible working members having plate-like flexible members, e.g. diaphragms
    • F04B43/04—Pumps having electric drive
    • F04B43/043—Micropumps
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00346—Heating or cooling arrangements
    • G01N2035/00356—Holding samples at elevated temperature (incubation)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis

Definitions

  • the present invention relates to a micro test chip, an optical detection device, and a micro total analysis system, and in particular, a micro test chip or a micro test chip that allows a reaction between an analyte and a reagent in a detection unit to proceed at an optimum temperature.
  • the present invention relates to an optical detection device, and a micro total analysis system capable of performing optical detection efficiently and with high accuracy by using such a micro inspection chip or optical detection device.
  • 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 connects these sections.
  • a specimen and reagent storage section a specimen and reagent storage section
  • a reagent mixing section a reaction section
  • a detection section a flow path that connects these sections.
  • - ⁇ In S, when an analyte is present in the detector, the reaction between the analyte and the reagent is optically detected.
  • the presence of the product detected by the extraction device that is, the product of the reaction between the analyte and the reagent, can be detected by, for example, visible light absorption analysis.
  • the reaction between the analyte and the reagent may take several steps, but it is desirable to carry out the reaction under optimum temperature conditions. Conventionally, however, such reactions have been performed at room temperature, and the progress of the reaction has been inefficient.
  • a separate heating member can be brought into contact with the microchip to raise the temperature to the optimum temperature.
  • the heating member is used as the detection unit. It was necessary to move the upper or lower surface force of the machine, resulting in complicated equipment.
  • Patent Document 1 Japanese Unexamined Patent Application Publication No. 2004-028589
  • Patent Document 2 Japanese Patent Application Laid-Open No. 2001-32099
  • Patent Document 3 Japanese Patent Laid-Open No. 2004-108285
  • Patent Document 4 Japanese Unexamined Patent Application Publication No. 2004-270537
  • the present invention has been made in view of the above-described problems, and a micro test chip or an optical detection device that enables a reaction between an analyte and a reagent in a detection unit to proceed at an optimum temperature.
  • the purpose is to provide.
  • Another object of the present invention is to provide a micro total analysis system capable of performing optical detection efficiently and with high accuracy by using such a micro inspection chip or an optical detection device.
  • a micro detection chip having a microchannel having at least a site (detection unit) for detecting a reaction between an analyte and a reagent by an optical detection device, and having a heating means on the chip surface, The detection unit is heated by the heating unit, and a micro inspection chip is provided.
  • the heating means is configured to have a heat generating resistance that can be heated by energization.
  • the optical path that passes through and is received by the light detector (detection optical path) is placed at a position that does not block the light path. It is desirable that Further, it is more preferable that the heating resistor is made of a transparent conductive film.
  • At least a light source, a photo detector, and a micro inspection chip having a micro flow path in which at least a site (detection unit) for optically detecting the reaction between the analyte and the reagent is present, and the detection unit is There is provided an optical detection device comprising a heating means capable of heating.
  • the heating means is configured to have a heat generating resistance that can be heated by energization. It is desirable to make contact with a position that does not block the optical path (detection optical path) that passes through and is received by the photodetector.
  • the heating resistor is more preferably made of a transparent conductive film.
  • a micro total analysis system comprising the micro test chip according to any one of claims 1 to 4 and a system device body,
  • the system apparatus main body is at least
  • a micropump unit including a chip connection having a channel opening for communicating with the chip and a micropump; an optical detection device; a function of the micropump unit; and a means for controlling the function of the optical detection device And a means for controlling the function of the heating means provided in the micro-inspection chip according to any one of claims 1 to 4.
  • a micro total analysis system equipped with a micro test chip and a system unit body
  • the system apparatus main body is at least
  • a chip connecting portion having a flow passage opening for communicating with the chip and a micropump;
  • a micropump unit including: the optical detection device according to any one of claims 5 to 8; means for controlling the function of the micropump unit and the function of the optical detection device;
  • a micro total analysis system is provided.
  • a micropump including a caloric pressure chamber connected to the flow path, an actuator that changes the internal pressure of the pressurizing chamber, and a drive device that drives the actuator is suitable.
  • the analyte and the reagent can be reacted at the detection unit at an appropriate temperature, and it is not necessary to move the member used for heating during the detection. . For this reason, it is possible to efficiently and accurately detect the analyte in the micro total analysis system with a simple mechanism.
  • FIG. 1 is a diagram showing a schematic configuration in one embodiment of a micro total analysis system.
  • FIG. 2 (a) is a sectional view showing an example of a piezo pump
  • FIG. 2 (b) is a top view thereof.
  • FIG. 3 (a) is a top view showing an aspect of installation of heating means (for example, a heating resistor) on the surface of the micro inspection chip.
  • FIG. 3 (b) is a top view showing a possible installation mode when a transparent conductive film is used as the heating resistor. Note that the fine channel in these figures is an enlargement of the area around the detection part in FIG.
  • FIG. 4 (a) is a cross-sectional view showing a mode in which heating means are installed on the upper and lower surfaces of the micro inspection chip.
  • FIG. 4 (b) is a cross-sectional view showing a possible installation mode when a transparent conductive film is used as the heating resistor.
  • FIG. 5 (a) is a cross-sectional view showing an aspect of installation of heating means in the optical detection device.
  • FIG. 5 (b) is a cross-sectional view showing a possible installation when a transparent conductive film is used as the heating resistor.
  • Fig. 6 is a diagram showing an embodiment of a configuration of a fine channel in a micro test chip.
  • FIG. 7 is a diagram showing a more specific embodiment of the fine flow path of the micro inspection chip. Explanation of symbols
  • micro test chip refers to a chip in a micro total analysis system that is used for various applications such as synthesis and test, especially for tests used for biological substances. Sometimes called a “chip”.
  • Flow path element refers to a functional component installed in a micro inspection chip.
  • the “fine channel” is a minute groove-like channel formed in the microphone opening inspection chip of the present invention, and communicates with this channel. Even in the case where the accommodating portion, the reaction portion, or the detection portion for the reagents, etc., are formed in a wide liquid reservoir shape having a large capacity, these portions are sometimes referred to as “fine channels”.
  • the fluid flowing in the fine channel is often a liquid, and specifically includes various reagents, sample liquid, denaturant liquid, cleaning liquid, and driving liquid.
  • Gene refers to DNA or RNA carrying genetic information that expresses a certain function, but may also be simply referred to as a chemical entity of DNA or RNA.
  • the target substance to be analyzed is sometimes called "analyte"
  • 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 part of the micro total analysis system of the present invention should be varied as long as they meet the spirit of the present invention. Can do.
  • FIG. 1 is a schematic diagram showing the configuration of an embodiment of a micro total analysis system of the present invention.
  • components other than the micro test chip 2 are integrated into the system unit body 1, and the micro test chip 2 is attached to and detached from the system unit body 1.
  • a series of analysis steps such as pretreatment, reaction, and detection of a specimen as a measurement sample are performed in the micro-inspection chip 2, for example, in a micro-channel formed in the micro-inspection chip 2.
  • the micro inspection chip 2 mounted on the chip transport tray is mounted on the system apparatus body 1. Samples and specimens to be analyzed may be accommodated in the micro test chip 2 in advance, or the micro test chip 2 may be attached to the system apparatus main body 1 for force storage.
  • a predetermined reaction and optical measurement based on the feeding, pretreatment, and mixing of samples and reagents are automatically performed as a series of continuous processes, and the measurement data is stored in the necessary conditions and recorded items. Desired form, stored in a file with U.
  • system apparatus body 1 and the micro inspection chip 2 will be further described.
  • the system apparatus body 1 includes, for example, a cooling unit 3, a heating unit 4, a micropump for liquid feeding, a driving liquid tank 10 and a micropump unit 9 having a chip connection portion, and their liquid feeding, temperature, and reaction.
  • a control device (not shown) related to each of these controls and an optical detection device 7 (not shown) including an optical detection system (light detector 5, light source 6, etc.) are provided as components.
  • These components are all downsized, and it is desirable that the system unit body 1 be in a form that is convenient to carry. As a result, it is possible to perform analysis by the micro total analysis system 100 without being restricted by place and time, and the workability and operability are also good.
  • the micropump is usually arranged inside the system apparatus main body 1, and for example, a plurality of micropumps are incorporated into the system apparatus main body as chip-shaped pump units formed by photolithography on a single substrate. .
  • a micropump unit 9 has a chip connection part having a flow path opening (port) for communicating with the micro inspection chip 2 and a connection part with the driving liquid tank 10.
  • the pump connection part of the micro test chip 2 and the chip connection part of the micro pump unit 9 are connected, and the flow paths of both are connected. It comes to communicate.
  • the micropump itself can be incorporated on the microphone port inspection 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 premised on repeated use, for example, a chip for chemical synthesis reaction.
  • micro pump various types of pumps such as a check valve type pump provided with a check valve in the inlet / outlet hole of the valve chamber provided with an actuator can be used. It is preferable to use a piezo pump capable of this.
  • this piezo pump has 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 resistance with respect to the change in the 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 pressurization chamber; A drive device for driving the eta, and by changing the drive voltage waveform, voltage value, and frequency of the actuator, the liquid feed direction and the liquid feed speed can be controlled! /.
  • the present inventors have already proposed such a micropump and a system using the micropump, and details thereof are described in Japanese Patent Laid-Open Nos. 2001-32099, 2004-108 285, and 2004-270537. Etc. can be referred to.
  • FIG. 2 (a) is a cross-sectional view showing an example of a piezo pump
  • FIG. 2 (b) is a top view thereof.
  • a substrate 42 on which a first liquid chamber 48, a first flow path 46, a pressurization chamber 45, a second flow path 47, and a second liquid chamber 49 are formed is laminated on the substrate 42.
  • the upper substrate 41, the diaphragm 43 laminated on the upper substrate 41, the piezoelectric element 44 laminated on the side facing the pressurizing chamber 45 of the diaphragm 43 functioning as an actuator, and the piezoelectric element 44 are driven.
  • a drive unit (not shown).
  • This drive unit and the two electrodes on the surface of the piezoelectric element 44 are connected by wiring using a flexible substrate or the like, and through the connection, a voltage of a specific waveform is applied to the piezoelectric element 44 by the drive circuit of the drive unit. Is applied.
  • the first liquid chamber 48 is provided with a port connected to the driving liquid tank 10, and the first liquid chamber “plays the role of lizano”.
  • the second liquid chamber 49 forms a flow path for the micropump unit 9, and the port of the chip connection part is located at the end of the flow path. Connected to the pump connection of micro inspection chip 2.
  • the optical detection device 7 can take a mode according to a detection method such as visible spectroscopy or fluorescence photometry.
  • a detection method such as visible spectroscopy or fluorescence photometry.
  • a light source 6 such as an LED
  • a photodetector 5 that receives transmitted light or reflected light (photo (photo) diode, etc.)
  • a lens, a photomultiplier, a CCD camera, or the like can be used as its constituent elements.
  • the cooling means 3 and the heating means 4 are arranged so as to abut against specific parts of the micro test chip (for example, a fine flow path in which a reaction process is performed), and cool or heat the parts.
  • a Peltier element can be used
  • various heaters, Peltier elements, or the like can be used.
  • control device The functions of components such as the micropump unit and the optical detection device are controlled by means such as a control device.
  • the control device is integrated with the component. However, it may be separately incorporated in the system apparatus main body 1.
  • a single control means may be configured to control and control the entire system in which functions of a plurality of components are integrated and controlled.
  • control device for the micropump controls the electric system to supply the micropump with a drive voltage in accordance with the order, capacity, timing, etc. of the liquid feeding appropriately.
  • control device for an optical detection device it is desirable to handle functions such as processing and recording of measurement data as well as control of the light irradiation method.
  • Various conditions for such operations can be set in advance as the contents of the program, and can be controlled according to software such as a microcomputer installed in the micro total analysis system 100.
  • the micro test chip 2 is equivalent to what is generally called a test chip, an analysis chip, a microreactor chip, and the like. Usually, the vertical and horizontal sizes of this chip are several tens of mm and the height is several mm.
  • Micro-inspection chip is a microfabrication technology in which micro-channels with channel elements or structures are functionally appropriate according to applications such as chemical analysis, various inspections, sample processing and separation, and chemical synthesis. It is arranged by. Desirably, necessary reagents are accommodated in advance in the microchannel of the chip in order to perform the above-described analysis quickly.
  • Such a micro test chip 2 is connected to the micro pump unit 9 via the pump connection portion, and the liquid such as the sample and the reagent is sent through the micro flow channel by the driving liquid sent from the micro pump unit 9. .
  • the above-described micro inspection chip 2 preferably has a structure having a basic substrate such as a groove forming substrate and a covering substrate as a structure. At least on the groove forming substrate, the pump connection part, the valve base part and the liquid storage part (respective storage parts such as reagent storage part and specimen storage part, reaction part, detection part, waste liquid storage part etc.), liquid feed control part, backflow A fine channel including a structure part such as a prevention part, a reagent quantification part, and a mixing 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 in close contact with each other, and may cover the entire surface of the groove forming substrate.
  • the fine flow path may be formed only on one side of the chip, or may be formed on both sides.
  • the micro inspection chip 2 is desired to be excellent in processability, non-water absorption, chemical resistance, heat resistance, inexpensiveness, and the like, considering the structure, application, detection method, etc. of the chip. Therefore, it is necessary to select an appropriate chip material.
  • 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 according to the characteristics of each material.
  • the micro test chip 2 for a large number of measurement specimens, particularly clinical specimens at risk of contamination and infection be of a disposable type. Therefore, it can be mass-produced and is lightweight, impact-resistant plastic resin that is easy to dispose of by incineration, and in particular, it has excellent transparency, mechanical properties and moldability, and fine processing is good! /, Polystyrene is preferred It is done. Polypropylene is also preferably used because it is excellent in chemical resistance such as acid and alkali with little protein adsorption and is inexpensive.
  • a resin having excellent heat resistance for example, polycarbonate.
  • the detection unit of the fine channel performs optical detection of a fluorescent substance or a product of a color reaction, it is necessary to use a light-transmitting material at least for the substrate at this part.
  • a light-transmitting material at least for the substrate at this part.
  • Alkali glass, quartz glass, and transparent plastics can be used as the light transmissive material, but transparent plastics are preferred.
  • the fine flow path of the micro inspection chip is formed on the substrate according to the flow path arrangement designed in advance according to the purpose.
  • the flow path of the fluid is, for example, a micrometer order formed with a width of several to several hundred m, preferably 10 to 500 ⁇ m, and a depth of 10 to about LOOO ⁇ m, preferably 10 to 300 ⁇ m. It is a fine channel with a width. If the flow path width is less than 5 / z m, the flow path resistance increases, which is inconvenient for fluid delivery and detection. The advantage of micro-scale space is diminished in flow paths exceeding 500 / z m.
  • a conventional microfabrication technique can be used as a method for forming a microchannel, but a photolithography technique is typically preferable. With this technology, the microstructure of photosensitive resin can be reduced. Transfer, removal of unnecessary portions, and the like are performed to form a fine channel.
  • a plastic having a good mechanical property and capable of accurately transferring a submicron structure is preferably used as the photosensitive resin used as the material for the groove-formed substrate at this time. Polystyrene, polydimethylsiloxane, etc. are excellent in shape transferability. If necessary, processing by injection molding, extrusion molding or the like may be used.
  • the detection part (the part for optically detecting the reaction between the specimen and the reagent) in the micro flow path of the micro test chip 2 is a heating provided on the surface of the micro test chip 2. Heated by means 4.
  • the heating means 4 can heat the detection unit to a desired temperature by the heat conduction of the part in the micro inspection chip 2.
  • the heating means 4 for example, a planar heating resistor that generates heat when energized using a separate energizing device, that is, a metal film such as chromium, gold, or platinum is used.
  • a metal film such as chromium, gold, or platinum is used.
  • the metal film can be patterned in a desired shape on the surface of the micro inspection chip 2 according to a known method such as vacuum deposition or sputtering. Heat generation is controlled by the resistance value determined by the thickness and line width of the patterned metal film, and the current value to be applied.
  • the heat-generating member made of a metal film on the surface of the micro inspection chip 2, the light emitted from the light source 6 (LED or the like) of the optical detection device 7 passes through the detection unit and is detected by the light detector. 5 It is not appropriate that the light path (detection light path) received by a photodiode (such as a photodiode) is blocked. Therefore, it is desirable to arrange the metal film so that it is not located in this detection optical path.
  • the position and shape of the metal film can be appropriately adjusted as long as the detection unit can be appropriately heated.
  • a mode in which the metal film 30a is arranged in a shape surrounding the detection unit 22 in a ring shape see FIG. 3 (a)
  • the metal film 30a may be disposed on either the upper surface (closer to the light source 6 or the side) or the lower surface (closer to the photodetector 5) or on both sides of the micro inspection chip (FIG. 4 ( see a)).
  • the transparent conductive film 30b As the material of the transparent conductive film 30b, known materials can be used, for example, ITO (addition of oxide tin to indium oxide), acid zinc (acid zinc, acid aluminum or acid gallium). ) And acid tin (tin oxide doped with acid antimony or fluorine).
  • ITO addition of oxide tin to indium oxide
  • acid zinc acid zinc, acid aluminum or acid gallium
  • acid tin tin oxide doped with acid antimony or fluorine
  • the conductive film obtained from these materials is transparent, and sufficient transmittance in the optical detection method is ensured in the visible light region. Therefore, the transparent conductive film 30b is used as the detection unit 22 of the micro inspection chip 2 and It is not necessary to form a film avoiding the detection optical path (see Fig. 3 (b) and Fig. 4 (b)). This simplifies the manufacture of micro inspection chips, and when appropriate temperature control of the detection unit 22 is required, the detection unit 22 can be directly heated, enabling temperature adjustment with higher accuracy
  • the detection unit 22 in the microchannel of the micro inspection chip 2 is heated by bringing the heating means 4 provided in the optical detection device 7 into contact with the micro inspection chip 2 and heating it. And heated to the desired temperature.
  • the light source 6 is disposed above the paper surface of the micro inspection chip 2, and light detection is performed below the paper surface on the opposite side across the chip. 5 is placed (see Fig. 1, Fig. 5 (a) and (b)). Further, a condensing lens 33 may be disposed between the light source 6 and the micro inspection chip 2. In such an embodiment, it is preferable to provide a heating means on the side of the photodetector 5 so as to contact the lower side of the micro inspection chip, but a heating means may be provided on the light source side.
  • the heating resistor that generates heat when energized can be suitably used also in the heating means provided in the optical detection device 7. Since the optical detector 5 of the optical detection device 7 and the micro inspection chip 2 cannot be separated from each other for reasons such as detection accuracy, the thin metal film 30a is preferable. If the condition regarding the thickness is satisfied, a member having high thermal conductivity (for example, a metal member such as aluminum) is connected to the metal film 30a, and the member is brought into contact with the micro inspection chip 2. It is also possible to use the heating means described above or a heating means using a Peltier element.
  • the micro inspection as described above is performed.
  • the metal film 30a may be disposed and brought into contact with a position that does not block the above-described detection optical path (see FIG. 5 (a)), and when the transparent conductive film 30b is used as a heating means, detection is performed.
  • the transparent conductive film 30b may be disposed on the optical path for use.
  • a transparent conductive film 30b is formed on the surface of a transparent glass plate or resin plate, and this plate is passed over the photodetector 5 so that it is in contact with the detection unit 22 of the micro inspection chip 2. It is possible to arrange the transparent electric membrane 30b (see FIG. 5 (b)).
  • a separate energization device is required in the micro total analysis system 100.
  • the energization apparatus can contact the heating resistor on the surface of the chip.
  • the energization device can contact the heating resistor of the optical detection device.
  • the surface of the micro inspection chip 2 or the heating means 4 provided in the optical detection device 7 is used. It is desirable that a temperature sensor is connected, and a controller having a memory storing a control program related to the heating operation is further connected to the temperature sensor. That is, a control device composed of a temperature sensor, a controller, an energization device, etc. controls the energization of the heating means 4 based on the measured temperature and according to the set temperature and heating timing determined by the control program. As a result, the temperature of the detector 22 is adjusted appropriately. Such a control device may be integrated with the optical detection device 7, or may be separately incorporated in the system device body 1! /.
  • the micro test chip 2 or the optical detection device 7 of the present invention can be used.
  • the temperature adjustment of the detection unit 22 will be described with reference to the case of detection by the following. It is not limited to cases only.
  • the micro test chip 2 for genetic testing first, DNA extracted from the sample or sample, or cDNA synthesized from the sample or RNA extracted from the sample or sample by reverse transcription reaction, and modified with piotin at the 5 'position.
  • the primer thus prepared is sent from these storage parts to the downstream fine flow path, and the gene is reacted with the amplification reagent in the reaction part to be amplified.
  • the amplification reaction solution containing the amplified gene and the denaturing solution are mixed in a fine channel, and the amplified gene is made into a single strand by denaturation treatment.
  • Step (1) Preliminarily send the above single-stranded DNA to the detection unit 22 where the piotin-affinity protein (avidin, streptavidin, etc.) is adsorbed and immobilized on the channel surface
  • the Piotin-affinity protein specifically binds to piotin labeled at the 5 'end of the primer used in the gene amplification reaction.
  • the single-stranded DNA labeled with piotin is trapped in the detection unit 22.
  • Step (2) A reagent containing a probe DNA fluorescently labeled with FITC (fluorescein isothiocyanate) is sent to the detection unit 22, and this probe DNA and the single strand obtained in the above step (1) are sent. Hybridize with DNA. As a result, piotin and FITC are bound to the single-stranded DNA.
  • FITC fluorescein isothiocyanate
  • Step (3) A colloidal gold reagent whose surface was modified with an anti-FITC antibody that specifically binds to FITC was sent to the detection unit 22, and was immobilized on the DNA immobilized on the surface of the microchannel. This gold colloid is adsorbed to the FIT C-modified probe based on the antigen-antibody reaction.
  • the detection unit 22 is irradiated with light from the light source 6, and from the amount of light detected by the photodetector 5, the gold roller bonded to the reaction product by the detection reaction described above is used.
  • the concentration of id can be measured.
  • a gold colloid with a particle size of about 20 nm has an absorption maximum near 560 ⁇ m. Therefore, when light (red light) having a wavelength in the vicinity of 560 nm is irradiated from the light source 6, the amount of light received by the photodetector 5 depends on the concentration of the gold colloid bound to the reaction product by the detection reaction described above. The concentration can be calculated because it decreases. This makes it possible to detect the presence and amount of the analyte.
  • step (1) is about 42 ° C
  • step (2) is about 50 ° C
  • step (3) is about 42 ° C.
  • Proteins such as streptavidin and anti-FITC antibodies are vulnerable to heat, so excessive heating must be avoided in steps (1) and (3), and the hybridization reaction in step (2) is around 50 ° C.
  • the reaction is most advanced. Therefore, in order to perform the detection reaction efficiently and detect the analyte with high accuracy, it is required to control the heating of the detection unit 22 so that the temperature becomes optimum in each step.
  • the micro inspection chip 2 provided with the heating means 4 on the surface according to the present invention or the optical detection device 7 provided with the heating means 4 has the temperature of the detection unit 22 of the micro inspection chip 2. Therefore, it is possible to perform genetic testing efficiently by performing each step of the detection reaction in the detection unit 22 under appropriate temperature conditions.
  • micro comprehensive analysis system 100 will be further described mainly using aspects in gene testing, but the embodiments are not limited to these, and various modifications can be made without departing from the scope of the invention. Can be changed.
  • the micro comprehensive analysis system 100 of the present invention can be suitably used particularly for testing (gene testing) for genes or nucleic acids (DNA, RNA).
  • One of the more specific modes of genetic testing is testing to determine whether a specimen contains a causative virus, bacterium, microorganism, etc. of an infectious disease or to identify those species that are present. . For example, if a gene is present in a sample by carrying out a gene amplification reaction using the micro test chip 2 using a gene (DNA or RNA having a predetermined base sequence) possessed by a specific bacterium as a primer, DNA or RNA is replicated and can be detected.
  • Another example of genetic testing is to determine the predisposition of a gene predisposing to a specific disease or the presence or absence of a genetic mutation involved in drug side effects by using the corresponding gene as a primer. It is an aspect.
  • Gene testing using the micro total analysis system 100 using the micro test chip of the present invention due to its configuration and analysis principle, has a much smaller amount of sample, less labor, and simple operation than conventional methods. A highly accurate result can be obtained. For this reason, various types of genetic analysis are performed in laboratory tests' diagnosis, pharmaceutical screening, pharmaceuticals, pesticides or various Safety of chemical substances' toxicity inspection, environmental analysis, food inspection, forensic medicine, science, brewing, fishery, livestock, agricultural production, agriculture and forestry, etc.
  • the micro flow path of the micro test chip is PCR (Polymerase chain reaction method or ICAN (Isothermal chimera primer mitiated nucleic acid amplification) method (Takara Bio Inc.), It is suitable for gene amplification reaction by registered trademark) and optical detection method using colloidal gold.
  • the basic flow path configuration is substantially the same for biological materials other than genetic tests. Normally, it is sufficient to change the specimen pretreatment unit, reagents, and probes. In that case, the arrangement and number of liquid feeding elements will change.
  • a person skilled in the art mounts the reagents necessary for the immunoassay method on the micro test chip, and modifies the types of analysis by making modifications including slight changes in flow path elements and specifications. It can be changed easily.
  • Biological substances other than genes mentioned here refer to various metabolites, hormones, proteins (including enzymes, antigens, etc.) and the like.
  • FIG. 6 shows an embodiment of the configuration of the fine channel in the micro test chip 2.
  • FIG. 7 shows a more specific embodiment of the fine flow path of the micro inspection chip 2.
  • an outline of the fine flow path will be described with reference to an example of the progress of gene testing in the micro test chip 2 of the embodiment of FIG.
  • the micropump 11 connected via the pump connection part 12 of the micro inspection chip 2 sends the driving liquid from the channel opening to the fine channel.
  • the reagent accommodated in the reagent accommodating portions 18a to 18c is sent to the flow paths 15a to 15d.
  • the specimen housed in the specimen housing section 20 or the analyte (for example, DNA) from which the specimen force is extracted is also sent by the driving liquid.
  • the amplified solution is combined with the denaturing solution contained in the denaturing solution storage unit 21b and the amplified DNA is subjected to single strand treatment, and the processing solution is sent to the detection unit 22a downstream of the reaction unit 15e. Liquid.
  • the treatment solution is combined with the probe DNA stored in the probe container 21d and the gold colloid stored in the gold colloid container 21e to bind the DNA, probe DNA, and gold colloid, and this reaction is performed in the detection unit 22a. Detection of biological material based on the product. Further, the 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.
  • the target specimen is a sample containing DNA or RNA.
  • DNA or RNA for example, whole blood, plasma, serum, buffy coat, urine, feces, saliva, sputum, biological samples, viruses , Bacteria, mold, yeast, animal and plant cells, and the like.
  • DNA or RNA isolated by these techniques may be used as the analyte.
  • Such a sample or analyte is temporarily stored in the sample storage unit of the micro test chip 2 at the time of testing, and then supplied to the mixing unit for testing.
  • the specimen is accommodated, for example, from a specimen injecting section that communicates with the specimen accommodating section provided on the upper surface of the specimen accommodating section.
  • This specimen injection part is formed with a rubber plug or other elastic material, or covered with a resin or reinforced film such as polydimethylsiloxane (PDMS)! / Is desirable.
  • PDMS polydimethylsiloxane
  • the specimen in the syringe is injected with one dollar inserted through a rubber stopper or through a pore with a lid.
  • the amount of specimen required is extremely small compared to the case of manual work performed using a conventional apparatus. For example, for a chip with a length and width of several centimeters, it is only necessary to inject a blood sample of about 2 to 3 mm 3 .
  • the DNA is 0.001 to 100 ng.
  • sample injected into the sample storage unit may be pretreated before mixing with the reagent, if necessary.
  • sample pretreatments include analyte separation or concentration, deproteinization, and the like.
  • DNA extraction treatment in which lysis is performed using a lysing agent such as a 1% SDS mixed solution, and the released DNA is adsorbed on the membrane surface of beads, adsorbent resin or filter.
  • a lysing agent such as a 1% SDS mixed solution
  • Such specimen pretreatment can be performed by providing a specimen pretreatment section in the fine channel.
  • a predetermined amount of necessary reagents according to the intended reaction method and detection method is sealed in the reagent container of the micro test chip 2 in advance. Therefore, it is not necessary to fill the required amount of reagent each time it is used.
  • the reagents can be accommodated at the time of manufacturing the micro test chip, for example, in the same manner as in the case of the sample. This can be done from the reagent injection part communicating with the reagent storage part provided on the upper surface of the medicine storage part, and then a sealing process is performed to prevent evaporation, leakage, air bubbles, contamination, denaturation, etc. .
  • Reagents necessary for analyzing a biological substance in a specimen are the same as those in the past.
  • a reagent containing an antibody (preferably a monoclonal antibody) against the antigen is used.
  • the antibody is preferably labeled with piotin and FIT C.
  • the reagents for genetic testing include various reagents used for gene amplification, probes used for detection, coloring reagents, and the like.
  • pretreatment reagents used for the sample pretreatment can also be used. included.
  • a cleaning solution, a denaturing solution, and the like are also stored as reagents in each storage unit.
  • the specimen, reagent, etc. stored in each container of the micro test chip 2 are pumped by the driving liquid that is also supplied with the micropump force, and these are combined and mixed. Reactions required for analysis such as gene amplification reactions, analyte traps or antigen-antibody reactions are initiated.
  • the mixing of the reagent and the reagent, or the mixing of the sample and the reagent may be performed at a desired ratio in a single mixing unit, or one or both may be divided to provide a plurality of merging units. Alternatively, mixing may be performed so as to finally obtain a desired mixing ratio.
  • the mode of the micro flow path for such merging and reaction is not particularly limited, and various forms and modes are conceivable.
  • two or more liquids containing reagents are merged at a merging section (flow path branching point) that is a slightly wider flow path, and then each liquid is diffusely mixed in a fine flow path downstream of the merging section.
  • a gene amplification reaction by PCR or the like is performed in a flow path (reaction part) having a space wider than the fine flow path, which is provided at the downstream end of the fine flow path. Can be.
  • a gene amplification method in the micro comprehensive analysis system 100 for example, a PCR method described in various documents including improved points and actively used in various fields can be used.
  • the PCR method requires a temperature control that raises and lowers between three temperatures. Use an appropriate device to control the temperature of the micro test chip 2.
  • Micro inspection In the micro-channel of the loop, the heat cycle can be switched at a high speed, and the gene amplification can be performed in a much shorter time than that performed manually.
  • the recently developed ICAN method is a suitable amplification technique for the system of the present invention because gene amplification can be performed in a short time at an arbitrary constant temperature of 50 to 65 ° C (Patent No. 3433929). .
  • a known optical detection method can be used, but an optical detection method using gold colloid is preferable.
  • a method to measure the fluorescence of fluorescent dyes for example, FITC, RITC, NBD, Cy3, Cy5, etc.
  • colloidal gold when colloidal gold is used, it can be measured with high sensitivity by visible light, and there are few interfering factors, data processing is easy, and instruments used for absorption analysis of visible light are more versatile than those of fluorescence analysis. It is preferable in terms of
  • the detection technique using gold colloid is as follows. In the detection section where a phytin affinity protein (for example, avidin) is immobilized on the surface of the microchannel, a single-stranded DNA amplified with piotin and a probe DNA complementary to this and labeled with FITC, The gold colloid bound with the anti-FIT C antibody is reacted, and the single-stranded DNA trapped in the detector is labeled with the gold colloid. Subsequently, when light is irradiated to this detection unit, if gold colloid exists in the detection unit (that is, DNA that is an analyte exists), a part of the light is absorbed by the gold colloid.
  • a phytin affinity protein for example, avidin
  • the analyte can be detected by measuring the amount of light transmitted.
  • an antibody specific to these analytes preferably a monoclonal antibody
  • genetic testing can be performed. It can be detected as in the case.
  • 10-35 X 10_ 9 can be prepared by dissolving piotin affinity proteins such as avidin and streptavidin in SSC buffer or physiological saline.
  • a solution with a concentration of 20-30 X 10 _9 g / mm 3 is prepared, and this is applied to the micro flow channel (detection part) formed on the surface of the polystyrene substrate during the manufacture of the micro inspection chip.
  • the biotin-affinity protein is adsorbed and immobilized.

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Abstract

La présente invention concerne une puce de micro-inspection, ou détecteur optique, qui active l'avancement, à la température la plus adéquate, d'une réaction entre un réactif et un analyte au point de détection d'un microcanal d'écoulement. Une puce de micro-inspection est fournie : elle dispose d'un microcanal d'écoulement où il existe au moins un site (site de détection) destiné à la détection d'une réaction entre le réactif et l'analyte au moyen d'un détecteur optique, la puce de micro-inspection fournie sur sa surface de puce par moyen de chauffage, étant caractérisée en ce que le site de détection est chauffé par ce moyen de chauffage. En outre, un détecteur optique caractérisé en ce qu'il inclut au moins une source lumineuse, un photodétecteur et des moyens de chauffage jouxtant une puce de micro-inspection à microcanal d'écoulement sachant qu'il existe au moins un site (site de détection) permettant la détection optique d'une réaction entre le réactif et l'analyte, le moyen de chauffage capable de chauffer le site de détection.
PCT/JP2007/051926 2006-03-03 2007-02-05 Puce de micro-inspection, detecteur optique et systeme analytique micro-complet WO2007099736A1 (fr)

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JP2010539503A (ja) * 2007-09-20 2010-12-16 イリネ ミクロシステムス、エセ．エレ． マイクロ流体デバイスおよび流体の凝固時間測定方法
WO2011122216A1 (fr) * 2010-03-31 2011-10-06 コニカミノルタオプト株式会社 Micropuce
JP2011247587A (ja) * 2010-05-21 2011-12-08 Enplas Corp 分析用具及びマイクロ分析システム
WO2012124579A1 (fr) * 2011-03-15 2012-09-20 Canon Kabushiki Kaisha Dispositif de trajectoire de fluide
JP2014020920A (ja) * 2012-07-18 2014-02-03 Canon Inc 発光検出用流路デバイス
JP2015025818A (ja) * 2007-09-10 2015-02-05 日本電気株式会社 マイクロチップの試料処理装置
JP2016065879A (ja) * 2010-03-31 2016-04-28 アボット ポイント オブ ケア インコーポレイテッド サンプルモーションを有する生体体液分析システム
JP2018512590A (ja) * 2015-04-07 2018-05-17 セル アイディー ピーティーイー リミテッドＣｅｌｌ Ｉｄ Ｐｔｅ Ｌｔｄ 流体チップ

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JP2015025818A (ja) * 2007-09-10 2015-02-05 日本電気株式会社 マイクロチップの試料処理装置
JP2010539503A (ja) * 2007-09-20 2010-12-16 イリネ ミクロシステムス、エセ．エレ． マイクロ流体デバイスおよび流体の凝固時間測定方法
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JP2016065879A (ja) * 2010-03-31 2016-04-28 アボット ポイント オブ ケア インコーポレイテッド サンプルモーションを有する生体体液分析システム
JP2011247587A (ja) * 2010-05-21 2011-12-08 Enplas Corp 分析用具及びマイクロ分析システム
WO2012124579A1 (fr) * 2011-03-15 2012-09-20 Canon Kabushiki Kaisha Dispositif de trajectoire de fluide
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JP2018512590A (ja) * 2015-04-07 2018-05-17 セル アイディー ピーティーイー リミテッドＣｅｌｌ Ｉｄ Ｐｔｅ Ｌｔｄ 流体チップ
US10638547B2 (en) 2015-04-07 2020-04-28 Cell Id Pte Ltd Digital PCR device
US10750577B2 (en) 2015-04-07 2020-08-18 Cell Id Pte Ltd Fluidic chip
US11477857B2 (en) 2015-04-07 2022-10-18 Cell Id Pte Ltd Fluidic chip

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