WO2018034108A1 - Dispositif d'examen et procédé d'examen - Google Patents

Dispositif d'examen et procédé d'examen Download PDF

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Publication number
WO2018034108A1
WO2018034108A1 PCT/JP2017/026704 JP2017026704W WO2018034108A1 WO 2018034108 A1 WO2018034108 A1 WO 2018034108A1 JP 2017026704 W JP2017026704 W JP 2017026704W WO 2018034108 A1 WO2018034108 A1 WO 2018034108A1
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Prior art keywords
liquid
reaction
inspection
pores
chip
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PCT/JP2017/026704
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English (en)
Japanese (ja)
Inventor
孝明 伊藤
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富士フイルム株式会社
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Publication of WO2018034108A1 publication Critical patent/WO2018034108A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01F—MEASURING VOLUME, VOLUME FLOW, MASS FLOW OR LIQUID LEVEL; METERING BY VOLUME
    • G01F23/00—Indicating or measuring liquid level or level of fluent solid material, e.g. indicating in terms of volume or indicating by means of an alarm
    • G01F23/22—Indicating or measuring liquid level or level of fluent solid material, e.g. indicating in terms of volume or indicating by means of an alarm by measuring physical variables, other than linear dimensions, pressure or weight, dependent on the level to be measured, e.g. by difference of heat transfer of steam or water
    • G01F23/28—Indicating or measuring liquid level or level of fluent solid material, e.g. indicating in terms of volume or indicating by means of an alarm by measuring physical variables, other than linear dimensions, pressure or weight, dependent on the level to be measured, e.g. by difference of heat transfer of steam or water by measuring the variations of parameters of electromagnetic or acoustic waves applied directly to the liquid or fluent solid material
    • G01F23/284—Electromagnetic waves
    • G01F23/292—Light, e.g. infrared or ultraviolet
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a test system and a test method for detecting a test substance such as an antigen, an antibody, or deoxyribonucleic acid.
  • the binding phenomenon between a test substance and a capture substance is optically determined.
  • a method of detecting is known. In this method, a test substance is bound to a capture substance fixed at a predetermined position and emits fluorescence upon receiving excitation light, or a label that catalyzes a substrate reaction to generate color, fluorescence, or chemiluminescence. Is applied to the test substance, and light generated due to the label is detected.
  • a method of detecting a fluorescence generated from a fluorescent label by attaching a fluorescent label to a binding substance such as an antibody that specifically binds to the test substance, a binding of an antibody that specifically binds to the test substance
  • a binding substance such as an antibody that specifically binds to the test substance
  • a binding of an antibody that specifically binds to the test substance There are known methods for detecting color development, fluorescence, chemiluminescence, etc. generated from a chromogenic substrate, fluorescent substrate, or chemiluminescent substrate that reacts with this enzyme as a catalyst. Identification becomes possible.
  • Patent Documents 1 to 3 propose a method in which a liquid is efficiently brought into contact with a capture substance and a test substance is bound to the capture substance. By this method, the measurement time can be greatly shortened.
  • Japanese Patent Application Laid-Open No. 2003-294755 discloses a light guide member for detecting a liquid level at the time of light detection or by receiving a light-transmitting liquid level position recognizing sensor provided with a specimen container made of a transparent body. A method for performing the position control of is disclosed.
  • International Publication No. 2011/027851 discloses a liquid level in a flow path in a liquid feeding system that promotes a reaction by flowing a sample liquid in a micro flow path having a detection region on which a capture substance is fixed.
  • a configuration is disclosed in which the position is detected and the pump connected to the microchannel is controlled according to the liquid level position.
  • an object of the present invention is to provide an inspection system and an inspection method capable of suppressing noise in a detection signal and suppressing the amount of waste liquid.
  • the inspection system of the present invention is a plate-shaped inspection having pores penetrating from one surface to the other surface, and a trapping substance that specifically binds to a specific substance is immobilized on the inner wall surface of the pore.
  • Chips A container for storing liquid; In the container, a support member that horizontally supports the inspection chip; A liquid holding unit capable of holding liquid on the upper surface of the inspection chip; and A liquid supply unit for supplying liquid to the pores from the lower surface of the inspection chip; A liquid level detection unit for detecting the position of the liquid level in the liquid holding unit; In an inspection system including a control unit that is connected to the liquid supply unit and the liquid level detection unit and controls the liquid supply operation by the liquid supply unit according to the position of the liquid level detected by the liquid level detection unit.
  • the reaction liquid reference position which is the arrival position of the reaction liquid surface
  • the cleaning liquid is supplied to the pores as a liquid
  • the cleaning liquid reference position which is the arrival setting position of the cleaning liquid level in the process, is set above the reaction liquid reference position
  • the reaction liquid reference position is preferably within 25 mm from the upper surface of the inspection chip, and the cleaning liquid reference position is preferably within 50 mm from the upper surface of the inspection chip.
  • the liquid supply unit also performs a discharge operation for discharging the liquid supplied to the pores from the pores.
  • control unit performs the discharge operation after a preset time has elapsed after stopping the supply operation by the liquid supply unit.
  • the liquid supply unit can be constituted by a pump that depressurizes and pressurizes the liquid holding unit.
  • the liquid supply unit may be constituted by a pump that depressurizes and pressurizes the inside of the container.
  • the liquid holding unit has light permeability and the liquid level detection unit is an optical camera.
  • a photodetector that is disposed opposite to one surface or the other surface of the inspection chip and detects light emitted from the inspection chip.
  • the capture substance provided in the inspection chip is an antigen, an antibody, or deoxyribonucleic acid (DNA).
  • the inspection chip is made of one or more of Si, SiO 2 , Al, Al 2 O 3 , stainless steel, and a resin material.
  • the reaction solution can be any one of a sample solution used for testing, a labeled solution containing a labeled substance, and a testing solution.
  • a first reaction liquid which is a sample liquid to be used for inspection, is supplied to the pores, and a specific substance in the sample liquid is removed. Binds to the capture substance, discharges the sample liquid from the pores, Performing a first cleaning process;
  • a labeling solution containing a labeling substance that specifically binds to a specific substance which is a second reaction solution, is supplied to the pores to bind the labeling substance to the specific substance, and the labeling solution Are discharged from the pores, Perform a second cleaning process
  • a test solution that is a third reaction solution is supplied to the pores, This is an inspection method for detecting light emitted from the inspection chip in a state where the inspection solution is retained in the pores.
  • an enzyme label is used as a labeling substance
  • a reaction solution containing a substrate that reacts catalyzed by the enzyme label is used as a test solution
  • the substrate in the test solution is used as emitted light.
  • Light generated by catalysis by an enzyme label may be detected.
  • the substrate include a chromogenic substrate, a fluorescent substrate, and a chemiluminescent substrate, and these substrates are appropriately selected according to the type of enzyme label.
  • the light emitted from the inspection chip differs depending on the substrate, and the detected light is light absorption (coloration), fluorescence, or chemiluminescence.
  • a substance containing a fluorescent label is used as the labeling substance, the inspection chip is irradiated with excitation light that excites the fluorescent label, and the fluorescence generated from the fluorescent label by irradiation of the excitation light is emitted as the emitted light. It may be detected.
  • the reaction liquid reference position which is the arrival setting position of the liquid surface of the reaction liquid
  • the cleaning liquid reference position which is the arrival setting position of the cleaning liquid level
  • the controller supplies the reaction liquid as a liquid.
  • the reaction liquid fills the pores, so that various binding reactions can be performed in the entire area of the pores. Also, since the liquid level position of the cleaning liquid is detected and raised to the cleaning liquid reference position set above the liquid level position of the reaction liquid, the reaction liquid can be sufficiently cleaned and noise in the detection signal is suppressed. be able to. Further, since the liquid level position of the cleaning liquid is detected and the supply of the cleaning liquid is stopped at the cleaning liquid reference position, the amount of waste liquid can be suppressed without using an excessive amount of cleaning liquid.
  • FIG. 1 is a schematic configuration diagram of an inspection system according to a first embodiment of the present invention. It is a figure which shows an example of the control sequence of the liquid level of a reaction liquid, and liquid supply operation
  • FIG. 1 is a schematic configuration diagram of an inspection system according to the first embodiment of the present invention.
  • the inspection system 1 of the present embodiment has pores 11 penetrating from one surface 10a to the other surface 10b, and a trapping substance that specifically binds to a specific substance is immobilized on the inner wall surface of the pore 11.
  • Plate-shaped inspection chip 10 a container 12 for storing liquid, and a support member 14 that horizontally supports the inspection chip 10 and holds the liquid on the upper surface 10 a of the inspection chip 10 in the container 12.
  • the support member 14 also serves as a liquid holding unit that holds liquid on the upper surface 10a of the test chip 10, but the liquid holding unit may be separated from the support member.
  • support horizontally means that the test chip 10 is supported so that the lower surface 10 b of the test chip 10 is parallel to the surface of the liquid. However, the liquid surface and the lower surface 10 b of the test chip 10 are supported. If the angle formed by is in the range of ⁇ 10 °, it is allowed. Further, in the present specification, the vertical direction coincides with the vertical direction, and the vertically upward surface of the inspection chip 10 in use in which the inspection chip 10 is horizontally disposed is the upper surface 10a, and the vertically downward surface is the lower surface 10b.
  • the inspection system 1 further closes the space 15 surrounded by the upper surface 10a of the inspection chip 10 and the support member 14 as a liquid supply unit that supplies liquid to the pores 11 from the lower surface 10b of the inspection chip 10, and the space 15
  • a control unit 20 is provided for controlling the liquid supply operation by the pump 16 in accordance with the detected position of the liquid level.
  • the control unit 20 is provided separately from the pump 16 in this example, but may be provided so as to be integrated with the pump.
  • the inspection system 1 also includes a cleaning liquid discharge unit 21 for discharging the cleaning liquid drain.
  • the cleaning liquid discharge unit 21 is provided with a waste liquid container 21a.
  • the inspection chip 10 is once removed from the container 24 together with the support member 14, and the waste liquid container 21a Then, the cleaning liquid in the pores 11 and in the liquid holding part 14b of the support member 14 is discharged into the waste liquid container 21a.
  • the inspection system 1 further includes an optical signal measurement unit 22 including a photodetector 22a disposed in the dark room 23 and a lens 22b that collects light from the inspection chip 10.
  • the inspection chip 10 is disposed below the photodetector 22 a, and the photodetector 22 a detects light emitted from the inspection chip 10 from vertically above the inspection chip 10.
  • the optical signal measuring unit 22 is installed at a location different from the component for supplying the reaction liquid or the cleaning liquid to the test chip 10.
  • the photodetector 22a is provided in the optical signal measurement unit 22 provided in a place different from the container 12 as in this example. Further, in this example, the light is detected from above the inspection chip 10 vertically, but the inspection chip 10 is disposed above the photodetector to detect light from the lower surface side of the inspection chip 10. It may be configured as follows.
  • inspection chip 10 is a plate-shaped base material by which the several pore 11 is arranged in two dimensions.
  • the inspection chip 10 is made of one or more materials selected from Si (silicon), SiO 2 (silicon oxide), Al (aluminum), Al 2 O 3 (alumina), stainless steel, and a resin material. It is preferable.
  • the thickness of the inspection chip 10 is not particularly limited, but is preferably about 100 ⁇ m to 2000 ⁇ m.
  • the opening shape of the pores 11 of the inspection chip 10 is not particularly limited, and may be a circle, an ellipse, or a polygon.
  • the pores 11 are generally columnar and the cross-sectional shape does not change, but the cross-sectional shape may partially change or the cross-sectional size may change.
  • the equivalent circle diameter of the opening on at least one surface of the pore 11 is about 1 ⁇ m to 100 ⁇ m. More preferably, it is 3 ⁇ m to 50 ⁇ m, and particularly preferably 5 ⁇ m to 30 ⁇ m.
  • the equivalent circle diameter refers to the diameter of a circle having an area equivalent to the area of the opening region.
  • the planar shape of the inspection chip 10 is not particularly limited, but is preferably a rectangle such as a square or a rectangle, or a circle.
  • the support member 14 includes an inspection chip receiving portion 14 a corresponding to the outer shape of the inspection chip 10.
  • the inspection chip receiving portion 14a and the inspection chip 10 of the support member 14 may be provided with an engagement portion for engaging both or a fitting portion for fitting each other.
  • the support member 14 includes a liquid holding portion 14b that holds a liquid on the upper surface 10a of the inspection chip 10 and a flange portion 14c that is locked to a part of the container 12 in order to support the inspection chip 10 in the container 12. It has.
  • the support member 14 can be fixed to the container 12 by locking the flange 14 c to a part of the upper surface of the container 12.
  • the form of the support member 14 is not limited to this example.
  • the liquid holding unit may be configured separately from the support member.
  • a pedestal for placing the inspection chip is provided on a part of the container 12 and the pedestal is supported by the pedestal.
  • a cylindrical member capable of holding a liquid, which is installed so as to be pressed against the upper surface of the inspection chip, may be provided as the liquid holding member.
  • maintenance part 14b among the supporting members 14 has a light transmittance for confirmation of the liquid level position by the optical camera 18.
  • FIG. The support member 14 and the inspection chip 10 may be detachable so that the support member 14 may be reused, or after the support member 14 and the inspection chip 10 are once connected and used, they are discarded as they are. Also good.
  • inspection chip 10 may be integrally formed.
  • the test substance (target molecule) to be tested in this test system 1 is mainly a biological molecule, such as proteins such as antigens and antibodies, saccharides, peptides, DNA, ribonucleic acid (RNA), peptides Examples thereof include nucleic acids (peptide nucleic acid: PNA).
  • the capture substance that is specifically fixed to the inner wall surface of the pore 11 and specifically binds to a specific substance is a substance that specifically binds to these test substances.
  • the inspection flow of the inspection method using the inspection system 1 configured as described above includes a reaction process for supplying a reaction liquid as liquid to the pores and a cleaning process for supplying a cleaning liquid as liquid to the pores.
  • the space 15 When the space 15 is depressurized by the pump 16 in a state where the inspection chip 10 is horizontally supported so that the lower surface 10b comes into contact with the liquid in the container 12, the liquid enters the pores 11 from the lower surface 10b side of the inspection chip 10. Is sucked. Further, when the space 15 is pressurized in a state where the liquid is contained in the pores 11, the liquid is pushed out from the pores 11 of the inspection chip 10 and discharged. By the operation of the pump 16, supply (suction) and discharge of the liquid into the pores 11 are performed. In this example, the decompression operation of the space 15 by the pump 16 corresponds to a liquid supply operation, and the pressurization operation of the space 15 corresponds to a liquid discharge operation.
  • the reaction liquid reference position in the first reaction process is set as h 11
  • the cleaning liquid reference position in the first cleaning process performed subsequent to the first reaction process is set as h 12
  • the second reaction process performed thereafter is performed.
  • the reaction liquid reference position is h 21
  • the cleaning liquid reference position in the second cleaning process is h 22
  • h 11 h 21
  • h 12 h 22 need not be satisfied.
  • a control sequence can be made common, it is preferable that a plurality of reaction processes or a plurality of cleaning processes in one inspection flow have the same reaction liquid reference position and cleaning liquid reference position, respectively.
  • the reaction liquid reference position h 1 is preferably within 25 mm from the inspection chip upper surface position h 0 . If the liquid level of the reaction liquid exceeds the position h 0 , the reaction in the pores is efficiently performed. In addition, by setting the reaction liquid reference position h 1 within 25 mm from the inspection chip upper surface position h 0, the amount of the reaction liquid can be suppressed, and the range to be cleaned can also be suppressed.
  • the cleaning liquid reference position h 2 is preferably within 50 mm from the inspection chip upper surface position h 0 . However, as previously described, the cleaning liquid reference position h 2 is located above the reaction liquid reference position h 1.
  • the cleaning liquid reference position h 2 is above the reaction liquid reference position h 1 , the cleaning liquid can be supplied to a position higher than the position where the reaction liquid is supplied. Therefore, the effect of removing the residual liquid of the reaction liquid is high. Further, by within 50mm of the cleaning liquid reference position h 2 from the test chip surface position h 0, it is possible to suppress the liquid amount of the cleaning liquid. According to this configuration, it is possible to achieve both sufficient cleaning and reduction of the amount of waste liquid.
  • Control unit 20 in the reaction process, when supplying the reaction liquid into the pores 11 stops the supply operation by the pump 16 when the liquid surface of the reaction solution reached the reaction liquid reference position h 1 or more, the cleaning process
  • the pump 16 is controlled so that the supply operation is stopped when the level of the cleaning liquid reaches or exceeds the cleaning liquid reference position h 2 .
  • the position of the liquid surface of the reaction liquid and the cleaning liquid is monitored by the optical camera 18, and the liquid surface position information is constantly sent to the control unit 20.
  • the control unit 20 can be configured by a personal computer, for example.
  • a program that causes the computer to function as the control unit 20 is incorporated in the computer.
  • FIG. 2 An example of the control sequence of the pump 16 in the reaction process is shown in FIG. In FIG. 2, the time change of the liquid level position and the time change of the depressurization control by the pump are shown together.
  • the control unit 20 starts the pressure reducing operation of the space 15 by the pump 16.
  • the reaction liquid is supplied (suctioned) into the pores 11 from the lower surface 10b of the test chip 10, and the liquid surface position gradually rises. Comparing the liquid level of the reaction solution to be detected by the optical camera 18 and the reaction solution reference position h 1, at time t 1 the liquid level reaches the reaction solution reference position h 1, stops the pressure reducing operation by the pump 16 And switch to pressurization.
  • the reaction solution In the pressurizing operation, it may be detected by the optical camera 18 that the reaction solution is completely discharged from the pores 11, or the time required for discharging is measured in advance to determine the pressurizing operation time. May be. By repeating this decompression and pressurization three times, the reaction in the pores 11 can be further promoted.
  • Control unit 20 first starts the depressurization operation of the space 15 by the pump 16 at time t 0.
  • the reaction liquid is supplied into the pores 11 from the lower surface 10b of the test chip 10, and the liquid surface position gradually rises. Comparing the liquid level of the reaction solution to be detected by the optical camera 18 and the reaction solution reference position h 1, at time t 1 the liquid level reaches the reaction solution reference position h 1, stops the pressure reducing operation by the pump 16 To do.
  • the decompression operation is stopped, and after maintaining the space 15 to atmospheric pressure for a predetermined time t, the operation is switched to the pressurization operation.
  • liquid level decreases gradually from the reaction solution reference position h 1 to the inspection chip upper surface position h 0, it is maintained at a position h 0 by the surface tension.
  • the pressurizing operation it may be detected by the optical camera 18 that the reaction solution is completely discharged from the pores 11, or the time required for discharging is measured in advance to determine the pressurizing operation time. May be.
  • the time t maintained at atmospheric pressure during switching from the decompression operation to the pressurization operation may be appropriately set in advance, and is set to about 30 minutes as an example.
  • the reaction in the pores 11 can be further promoted by repeating this decompression, holding under atmospheric pressure, and pressurization three times.
  • the reaction liquid when the reaction liquid is discharged by pressurizing the space 15, the reaction liquid may not be completely discharged from the pores 11. Further, in each of the above examples, the depressurization to pressurization are repeated three times. However, the number of times is not particularly limited, and may be only once, or may be twice or four or more times. .
  • FIG. 4A shows the time change of the liquid surface position in the cleaning process
  • FIG. 4B shows the flow of the cleaning process.
  • the cleaning process starts from a state in which the cleaning liquid is stored in the container 12 and the inspection chip 10 is not immersed in the cleaning liquid above the cleaning liquid and the lower surface 10b.
  • the control unit 20 starts the pressure reducing operation of the space 15 by the pump 16 (S11).
  • the lower surface 10b of the inspection chip 10 is immersed in a cleaning liquid (S12).
  • the space 15 is kept in a state where the lower surface 10b of the test chip 10 is not in contact with the cleaning solution in the container 12 in this way.
  • the pressure of the reduced pressure is made smaller than the reduced pressure in the case of the reaction process (large in absolute value), and the liquid level is raised to the cleaning liquid reference position h 2 located above the reaction liquid reference position h 1 .
  • Comparing the liquid level position and the cleaning liquid reference position h 2 of the cleaning liquid is detected by the optical camera 18 at time t 11 the liquid level reaches the cleaning liquid reference position h 2, to stop the depressurization operation by the pump 16 (S13 ).
  • the test chip 10 is removed from the container 12 together with the support member 14, moved to the cleaning liquid discharger 21, and set on the waste liquid container 21a (S14).
  • to start the pressurization of the space 15 by the pump 16 to discharge the cleaning liquid has accumulated in the pores 11 of the test chip 10 and the liquid holding portion 14b in a waste container 21a (S15).
  • FIG. 4A shows a change in the liquid surface position when the steps S11 to S15 are repeated three times.
  • the negative pressure in the space 15 is maintained and moved to the cleaning liquid discharge unit 21.
  • the space 15 is opened to atmospheric pressure, and the pores 11 of the test chip 10 are opened. You may make it move the test
  • FIGS. 2 to 4A schematically show the sequence, and the change in the liquid surface position at the time of depressurization is not necessarily limited to that shown by a linear function as shown in FIGS. 2 to 4A. .
  • FIG. 5 is a schematic configuration diagram of an inspection system 2 according to the second embodiment of the present invention.
  • differences from the inspection system 1 according to the first embodiment will be mainly described, and the same components are denoted by the same reference numerals and detailed description thereof will be omitted.
  • the shape of the container 24 that holds the liquid and the pump 16 that is the liquid supply unit are connected to the container 24, and the waste liquid container 21 b of the cleaning liquid discharge unit 21 is connected to the pump 16. It differs from the structure of the said inspection system 1 by the point.
  • the pump 16 can switch the connection between the container 12 and the waste liquid container 21b by the switching valve 16a.
  • the pump 16 pressurizes and depressurizes the space 25 of the container 24 to supply liquid to the pores 11 of the inspection chip 10 and discharge liquid from the pores 11. Further, the cleaning liquid is discharged from the pores 11 of the test chip 10 by reducing the pressure inside the waste liquid container 21 b of the cleaning liquid discharger 21.
  • the container 24 has a shape that can form a space 25 closed by the lower surface 10b of the inspection chip 10 and the container 24.
  • the flange portion 14 c of the support member 14 can be locked to a part of the upper surface of the container 24, and can support the inspection chip 10 in the container 24.
  • the waste liquid container 21 b has substantially the same shape as the container 24.
  • the inspection chip 10 When the space 25 is pressurized by the pump 16 in a state where the inspection chip 10 is horizontally supported so that the lower surface 10b comes into contact with the liquid in the container 12, the inspection chip 10 enters the pores 11 from the lower surface 10b side of the inspection chip 10. Liquid is pushed up. Further, when the space 25 is depressurized while the liquid is in the pores 11, the liquid is sucked downward and discharged from the pores 11 of the inspection chip 10. By the operation of the pump 16, the liquid is supplied to and discharged from the pores 11.
  • the pressurization operation of the space 25 by the pump 16 corresponds to the liquid supply operation
  • the decompression operation of the space 25 corresponds to the liquid discharge operation.
  • the relationship between the inspection chip upper surface position h 0 , the reaction liquid reference position h 1 and the cleaning liquid reference position h 2 in the inspection system 2 is the same as that in the inspection system 1 of the first embodiment, and the same effect can be obtained. .
  • Control unit 20 first at time t 20, to start the pressurizing operation of the space 25 by the pump 16.
  • the reaction liquid is supplied into the pores 11 from the lower surface 10b of the test chip 10, and the liquid surface position gradually rises. Comparing the liquid level of the reaction solution to be detected by the optical camera 18 and the reaction solution reference position h 1, at time t 21 the liquid level reaches the reaction solution reference position h 1, the pressurizing operation by the pump 16 Stop and switch to decompression.
  • the reaction solution In the depressurization operation, it may be detected by the optical camera 18 that the reaction solution is completely discharged from the pores 11, or the time required for the discharge may be measured in advance to determine the depressurization operation time. Good. By repeating this pressurization and depressurization three times, the reaction in the pores 11 can be further promoted.
  • Control unit 20 first starts a pressurizing operation of the space 25 by the pump 16 at time t 20.
  • the reaction liquid is supplied into the pores 11 from the lower surface 10b of the test chip 10, and the liquid surface position gradually rises. Comparing the liquid level of the reaction solution to be detected by the optical camera 18 and the reaction solution reference position h 1, at time t 21 the liquid level reaches the reaction solution reference position h 1, the pressurizing operation by the pump 16 Stop.
  • the pressurizing operation is stopped, and after maintaining the space 25 to atmospheric pressure for a predetermined time t, the operation is switched to the depressurizing operation.
  • the depressurization operation it may be detected by the optical camera 18 that the reaction solution is completely discharged from the pores 11, or the time required for the discharge may be measured in advance to determine the depressurization operation time. Good.
  • the time t maintained at the atmospheric pressure during the switching from the pressurizing operation to the pressurizing operation may be appropriately set in advance, and is set to about 30 minutes as an example.
  • control sequence in the reaction process of the present embodiment is the same except that the decompression and pressurization are reversed from those in the first embodiment.
  • the cleaning process may be performed by reversing the timing of pressure reduction and pressurization in the first embodiment as in the case of the reaction process.
  • the cleaning liquid is supplied to the pores 11 and the liquid holding unit 14b, and after stopping the pressurizing operation of the pump 16, the inspection chip 10 is moved to the cleaning liquid discharging unit 21 while maintaining the liquid level position. , Drain the cleaning solution.
  • the space (the space of the liquid holding portion 14b of the support member 14) 15a of the upper surface 10a of the test chip 10 is closed, and the cleaning liquid is not discharged from the pores 11 of the test chip 10 due to natural fall. It is necessary to move the inspection chip 10 in a state.
  • the support member 14 is provided with a lid for closing the space 15a, and the space 15a can be switched between an open state and a closed state by opening and closing the lid.
  • the space 15a is opened when supplying the cleaning liquid, the pump is stopped after supplying the cleaning liquid, and the opening of the support member is covered to close the space 15a.
  • the space 25 is opened to atmospheric pressure, the inspection chip 10 is taken out of the container 12 together with the support member 14, and is set in the waste liquid container 21b.
  • the lid of the opening of the support member 14 is opened to open the space 15a to atmospheric pressure. At this time, a part of the cleaning liquid is discharged from the pores 11 and a part thereof is maintained in the pores 11.
  • the connection of the pump 16 is switched to the waste liquid container 21b side and the pressure inside the waste liquid container 21b is reduced, all the cleaning liquid in the pores 11 can be discharged.
  • the light emitted from the test chip and detected by the photodetector is, for example, fluorescence generated by excitation of a label attached to the test substance, or binding of an antibody that specifically binds to the test substance.
  • the substance is given the same label as above, and light is emitted from the label, or an enzyme is labeled on a binding substance such as an antibody that specifically binds to the test substance, and light emission by the reaction using this enzyme as a catalyst (in the following) , Or “chemiluminescence”)).
  • chemiluminescence if the test substance generates autofluorescence, no label is necessary, and autofluorescence may be detected.
  • the optical signal includes absorbance (colorimetric) in addition to fluorescence and chemiluminescence light.
  • FIG. 8 is a diagram showing an inspection flow
  • FIG. 9 is a diagram schematically showing reactions in the first to third reaction processes in the inspection flow.
  • the first reaction liquid in the first reaction process is a sample liquid used for the test.
  • Specific examples of the sample liquid include plasma or serum.
  • the sample liquid is put in the container 12, and the test chip 10 is set so that the lower surface 10b is immersed in the surface of the sample liquid in the container 12.
  • the space 15 is decompressed by the pump 16 to supply the sample liquid into the pores 11. Sucked into the pores 11, when the liquid level of the sample fluid to rise to the upper surface 10a side of the test chip 10 is detected by the optical camera 18, and reaches the reaction solution reference position h 1, return the space 15 to the atmospheric pressure, Hold for 30 minutes.
  • the space 15 is pressurized by the pump 16 to discharge the sample liquid in the pores 11 (S2).
  • the steps S1-S2 may be repeated a plurality of times.
  • the specific binding reaction between the specific substance in the sample liquid and the capture substance fixed to the inner wall in the pore 11 is promoted three times.
  • a first cleaning process is performed (S3).
  • the cleaning liquid is put into the container 12, and the inspection chip 10 is set so that the lower surface 10 b is immersed in the surface of the cleaning liquid in the container 12 while reducing the space 15 by the pump 16.
  • the cleaning liquid is sucked up in the pores 11, to detect the liquid level of the cleaning liquid rises to the upper surface 10a side of the test chip 10 in the optical camera 18, when it reaches the cleaning liquid reference position h 2, the pressure reducing operation by the pump 16 Stop temporarily.
  • the inspection chip 10 is removed together with the support member 14 from the container 12, moved to the cleaning liquid discharger 21, and set on the waste liquid container 21a.
  • the pump 16 is switched to a pressurizing operation to pressurize the space 15 and discharge the cleaning liquid in the pores 11.
  • Step S3 may be repeated a plurality of times.
  • the cleaning effect is increased by repeating three times.
  • the second reaction solution in the second reaction process is a labeling solution containing a labeling substance.
  • the labeling solution is put in the container 12, and the inspection chip 10 is set so that the lower surface 10b is immersed in the surface of the labeling solution in the container 12.
  • the space 15 is decompressed by the pump 16 to supply the labeling solution into the pores 11. Sucked into the pores 11, when the liquid level of the labeling solution to rise to the upper surface 10a side of the test chip 10 is detected by the optical camera 18, and reaches the reaction solution reference position h 1, return the space 15 to the atmospheric pressure, Hold for 30 minutes.
  • the space 15 is pressurized by the pump 16 to discharge the label solution in the pores 11 (S5).
  • Steps S4 to S5 may be repeated a plurality of times.
  • the binding reaction of the labeling substance to the specific substance specifically bonded to the capturing substance fixed to the inner wall in the pore 11 is promoted three times.
  • the second cleaning process is similar to the first cleaning process.
  • the third reaction solution in the third reaction process is a test solution.
  • the inspection solution is put in the container 12 and the inspection chip 10 is set so that the lower surface 10 b is immersed in the surface of the inspection solution in the container 12.
  • the space 15 is decompressed by the pump 16, and the inspection solution is supplied into the pores 11. It sucked into the pores 11, when the liquid surface of the test solution to rise to the upper surface 10a side of the test chip 10 is detected by the optical camera 18, and reaches the reaction solution reference position h 1, returning the space 15 to the atmospheric pressure . Even if the space 15 is returned to atmospheric pressure, the sample liquid remains in the pores 11.
  • the inspection chip 10 is removed from the container 12 with the inspection solution held in the pores 11 and set in the photodetector 22 a of the optical signal measurement unit 22.
  • the inspection solution kept in the pores 11 of the inspection chip 10
  • the light emitted from the inspection chip 10 is detected by the photodetector 22a (S9), and the inspection process ends.
  • FIG. 9 is a diagram schematically showing the reaction in each of the above reaction processes.
  • a trapping substance 30 such as an allergen is fixed to the inner wall surface 11a of the pore 11 of the test chip 10 (S0).
  • a specimen liquid containing a test substance for example, a specific IgE antibody that specifically binds to the allergen
  • the test substance 32 is captured by the capture substance 30. Specifically bind to.
  • a labeling solution containing the substance 35 is supplied to the pores 11 to bind the labeling substance 35 to the test substance 32.
  • Label F is an enzyme label that functions as a catalyst for chemiluminescent substrates such as luminol, lophine, lucigenin and oxalate.
  • a test solution containing a luminescent substrate that undergoes a luminescence reaction using the label F as a catalyst is supplied into the pores 11, and the label F Is used as a catalyst to cause a luminescence reaction.
  • a light signal from a luminescence reaction that reacts with the label F as a catalyst is detected by the photodetector 22a.
  • the specimen solution or the remaining solution of the labeling solution is washed, and the test substance 32 nonspecifically adsorbed in the pores 11 or Since the labeling substance is removed, noise in the measurement signal can be suppressed.
  • reaction liquid containing a luminol-based chemiluminescent substrate in which HRP functions as a catalyst is converted into an ALP (alkaline phosphatase) enzyme.
  • ALP alkaline phosphatase
  • the luminol reaction solution contains at least a luminol substrate and a hydrogen peroxide solution.
  • the enzyme label catalyzes the oxidation of luminol in the presence of hydrogen peroxide.
  • the reaction solution preferably contains a sensitizer that sensitizes chemiluminescence.
  • a color reaction (absorption) reaction or fluorescence may be detected using a reaction solution containing not only the chemiluminescent substrate but also a luminescent substrate or a fluorescent substrate.
  • the label F is not limited to the enzyme label as described above.
  • the fluorescence measurement may be performed without supplying the inspection solution into the pores 11 after the second cleaning process.
  • the fluorescence measurement may be performed in a state where the buffer solution is supplied into the pores 11 as a test solution and the pores 11 are filled with the buffer solution.
  • the inspection chip is irradiated with light having a wavelength for exciting the fluorescent label as excitation light, and fluorescence from the label excited by the excitation light is detected.
  • the optical signal measurement unit is provided with an excitation light irradiation unit.
  • the inspection system of the present invention by using the inspection system of the present invention, it is possible to detect noise with high accuracy while suppressing noise, and it is possible to suppress the amount of waste liquid generated.

Abstract

L'invention concerne un système d'examen, une position de référence de liquide de réaction dans un processus de réaction dans lequel des pores sont alimentés en liquide de réaction étant définie au-dessus de la surface supérieure d'une puce de test, une position de référence de solution de nettoyage dans un processus de nettoyage dans lequel des pores sont alimentés en solution de nettoyage étant définie au-dessus de la position de référence de liquide de réaction, et une unité de commande effectuant une commande de telle sorte que pendant l'alimentation en liquide de réaction sous la forme d'un liquide, l'opération d'alimentation par une unité d'alimentation en liquide est arrêtée lorsque le niveau de surface du liquide de réaction devient supérieur ou égal à la position de référence de liquide de réaction et que, pendant l'alimentation en solution de nettoyage sous la forme d'un liquide, l'opération d'alimentation est arrêtée lorsque le niveau de surface de la solution de nettoyage devient supérieur ou égal à la position de référence de solution de nettoyage.
PCT/JP2017/026704 2016-08-16 2017-07-24 Dispositif d'examen et procédé d'examen WO2018034108A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020157239A (ja) * 2019-03-27 2020-10-01 ヤマト科学株式会社 反応容器の液面制御装置

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0627111A (ja) * 1992-07-09 1994-02-04 Olympus Optical Co Ltd 反応容器
JP2005148048A (ja) * 2003-04-25 2005-06-09 Jsr Corp バイオチップおよびバイオチップキットならびにその製造方法および使用方法
JP2006153889A (ja) * 2001-03-21 2006-06-15 Olympus Corp 生化学的検査方法
WO2011027851A1 (fr) * 2009-09-07 2011-03-10 コニカミノルタホールディングス株式会社 Système d'alimentation en liquide pour micropuce, dispositif de détection d'échantillons et procédé d'alimentation en liquide pour système d'alimentation en liquide d'une micropuce

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0627111A (ja) * 1992-07-09 1994-02-04 Olympus Optical Co Ltd 反応容器
JP2006153889A (ja) * 2001-03-21 2006-06-15 Olympus Corp 生化学的検査方法
JP2005148048A (ja) * 2003-04-25 2005-06-09 Jsr Corp バイオチップおよびバイオチップキットならびにその製造方法および使用方法
WO2011027851A1 (fr) * 2009-09-07 2011-03-10 コニカミノルタホールディングス株式会社 Système d'alimentation en liquide pour micropuce, dispositif de détection d'échantillons et procédé d'alimentation en liquide pour système d'alimentation en liquide d'une micropuce

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020157239A (ja) * 2019-03-27 2020-10-01 ヤマト科学株式会社 反応容器の液面制御装置

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