WO2016132945A1 - Reaction method and reaction device - Google Patents

Reaction method and reaction device Download PDF

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Publication number
WO2016132945A1
WO2016132945A1 PCT/JP2016/053672 JP2016053672W WO2016132945A1 WO 2016132945 A1 WO2016132945 A1 WO 2016132945A1 JP 2016053672 W JP2016053672 W JP 2016053672W WO 2016132945 A1 WO2016132945 A1 WO 2016132945A1
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WO
WIPO (PCT)
Prior art keywords
tip
pipette
pipette tip
position information
reaction
Prior art date
Application number
PCT/JP2016/053672
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French (fr)
Japanese (ja)
Inventor
正貴 松尾
野田　哲也
Original Assignee
コニカミノルタ株式会社
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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Application filed by コニカミノルタ株式会社 filed Critical コニカミノルタ株式会社
Priority to JP2017500607A priority Critical patent/JPWO2016132945A1/en
Publication of WO2016132945A1 publication Critical patent/WO2016132945A1/en

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    • 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
    • G01N35/02—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 plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • 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
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Definitions

  • the present invention uses a pipette tip attached to a pipette nozzle and a reaction chip having a recess and a seal sealing the opening of the recess to react two or more substances in the reaction chip.
  • the present invention relates to a method and a reaction apparatus.
  • SPR surface plasmon resonance
  • a detection chip having a prism made of a dielectric material, a metal film disposed on the prism, and a flow path member that is disposed on the metal film and forms a liquid flow path is used.
  • a capturing body for capturing the substance to be detected is disposed on the metal film.
  • the flow path member has an injection part for injecting a liquid such as a specimen containing a substance to be detected into the liquid flow path, and a discharge part for discharging the liquid from the liquid flow path.
  • the inlet of the inlet and the outlet of the outlet are formed in a complementary shape with respect to the tip of the pipette tip.
  • the tip of the pipette tip when the tip of the pipette tip is inserted into the inlet or outlet, the tip of the pipette tip and the inlet or outlet are fitted. Thereby, the tip of the pipette tip is arranged at a fixed position with respect to the bottom surface of the liquid channel, and the amount of liquid in the liquid channel can be controlled with high accuracy.
  • the tip of the pipette chip is arranged near the flow path in the detection chip or the bottom of the well, and the pipette chip This is done by repeatedly sucking the liquid in and discharging the liquid out of the pipette tip.
  • the pipette chip it is necessary to accurately control the positional relationship between the liquid channel or the bottom of the well and the tip of the pipette tip.
  • reaction chip when performing a plurality of reaction steps including a liquid removal step in the detection chip (reaction chip), from the viewpoint of improving the accuracy of the detection result and stabilizing the reaction efficiency, the liquid flow path in the liquid removal step or The amount of liquid remaining in the well needs to be minimized and constant. Also in this case, it is necessary to control the position of the tip of the pipette tip with high accuracy.
  • a strain gauge or a load cell as a method for detecting the position information of the tip of such a pipette tip.
  • the strain gauge is arranged between a pipette nozzle to which a pipette tip is attached and a pipette drive unit. Then, the position information of the tip of the pipette tip is acquired based on the output of the strain gauge when the pipette attached with the pipette tip is brought into contact with the reference portion that is the positioning reference of the tip of the pipette tip.
  • a pressure-sensitive sensor as another method for acquiring the position information of the tip of the pipette tip.
  • the position information of the tip of the pipette tip is acquired based on the output of the pressure sensitive sensor when the tip of the pipette tip is brought into contact with the pressure sensitive sensor.
  • a detection chip in which an inlet and an outlet are sealed with a seal may be used.
  • This detection tip may be used by piercing the seal with a pipette tip.
  • the object of the present invention is to accurately control the position of the tip of the pipette tip without increasing the manufacturing cost of the pipette tip and the reaction tip even when the seal of the reaction tip is broken by the pipette tip.
  • a reaction method uses a pipette tip attached to a pipette nozzle and a reaction tip having a recess and a seal that seals the opening of the recess.
  • a reaction method of reacting two or more substances in the reaction chip, the first step of breaking through the seal of the reaction chip with the pipette tip, and the tip of the pipette tip after the first step A second step of acquiring the position information of the second step, and after the second step, the pipette tip is operated based on the position information of the tip of the pipette tip to react two or more substances in the reaction tip. 3 steps.
  • a reaction apparatus uses a reaction chip having a recess and a seal that seals the opening of the recess, and breaks the seal with a pipette chip.
  • a reaction apparatus for reacting two or more substances in the reaction chip afterwards a chip holder for holding the reaction chip, a pipette having a pipette nozzle to which the pipette chip can be attached and detached, and a pipette for moving the pipette
  • a position information acquisition unit that acquires position information of a tip of the pipette tip.
  • the position information acquisition unit is configured such that the pipette tip moved by the pipette movement unit performs the sealing once.
  • the pipette moving unit After the information acquisition unit acquires the position information of the tip of the pipette tip, the pipette is used once or twice or more based on the position information of the tip of the pipette tip to react two or more substances in the reaction tip. Move.
  • the position of the tip of the pipette tip is accurately controlled without increasing the manufacturing cost of the pipette tip and the reaction tip.
  • Two or more substances can be appropriately reacted in the inside.
  • the presence or amount of a substance to be detected can be detected with high accuracy.
  • FIG. 1 is a schematic diagram showing the configuration of the SPFS apparatus according to the first embodiment.
  • 2A to 2C are diagrams showing the configuration of the detection chip.
  • FIG. 3 is a schematic cross-sectional view of another form of the detection chip.
  • FIG. 4 is a flowchart showing the operation of the SPFS apparatus according to the first embodiment.
  • FIG. 5 is a flowchart showing the content of the step of acquiring the position information of the tip of the pipette tip.
  • 6A is a diagram for explaining a process of breaking through a seal that closes an injection port
  • FIG. 6B is a diagram for explaining a process of breaking through a seal that closes a recess
  • FIG. It is a figure for demonstrating the process of attracting
  • FIG. 7A is a diagram illustrating a partial configuration of the SPFS apparatus according to the second embodiment
  • FIG. 7B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip
  • FIG. 8A is a diagram illustrating a partial configuration of the SPFS apparatus according to the third embodiment
  • FIG. 8B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip.
  • FIG. 9A is a diagram illustrating a configuration of a part of the SPFS device according to the fourth embodiment
  • FIG. 9B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip.
  • FIG. 9A is a diagram illustrating a configuration of a part of the SPFS device according to the fourth embodiment
  • FIG. 9B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip.
  • FIG. 10A is a diagram illustrating a partial configuration of the SPFS apparatus according to the fifth embodiment
  • FIG. 10B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip
  • FIG. 11A is a diagram illustrating a partial configuration of the SPFS device according to the sixth embodiment
  • FIG. 11B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip.
  • SPFS apparatus surface plasmon excitation enhanced fluorescence analyzer
  • FIG. 1 is a schematic diagram showing a configuration of a surface plasmon excitation enhanced fluorescence analyzer (SPFS apparatus) 100 according to Embodiment 1 of the present invention.
  • SPFS apparatus surface plasmon excitation enhanced fluorescence analyzer
  • the SPFS device (detection device) 100 includes a liquid feeding unit 110 including a pipette 111 and a pipette moving unit 112, a transport unit 120 including a tip holder 121, a position information acquisition unit 130, a light The irradiation unit 140, the light detection unit 150, and the control unit 160 are included.
  • the SPFS device 100 is used with the detection chip (reaction chip) 10 mounted on the chip holder 121. Therefore, the detection chip 10 will be described first, and then each component of the SPFS device 100 will be described.
  • FIG. 2 is a diagram illustrating a configuration of the detection chip 10.
  • 2A is a plan view of the detection chip 10
  • FIG. 2B is a cross-sectional view taken along line AA shown in FIG. 2A
  • FIG. 2C is a cross-sectional view taken along line BB shown in FIG. 2A.
  • FIG. 3 is a schematic cross-sectional view showing another form of the detection chip 10.
  • the detection chip 10 includes a prism 20 including an incident surface 21, a film formation surface 22 and an output surface 23, a metal film 30, a flow path including a reaction region 41 and a reagent storage region 42. It has a lid 40 and a seal 50.
  • the metal film 30 and the flow path lid 40 are disposed on the film formation surface 22 of the prism 20.
  • the prism 20, the metal film 30, and the channel lid 40 form a channel 60 through which liquid flows.
  • the flow path 60 is disposed directly or via the metal film 30 on the film formation surface 22 of the prism 20.
  • the detection chip 10 may be a reusable chip or a disposable chip. In the present embodiment, the detection chip 10 is a disposable chip.
  • liquid flowing through the flow path examples include a sample containing a substance to be detected (for example, blood, serum, plasma, urine, nasal fluid, saliva, semen, etc.) or a label containing a capturing substance labeled with a fluorescent substance.
  • a substance to be detected for example, blood, serum, plasma, urine, nasal fluid, saliva, semen, etc.
  • a label containing a capturing substance labeled with a fluorescent substance for example, blood, serum, plasma, urine, nasal fluid, saliva, semen, etc.
  • the prism 20 is made of an insulator that is transparent to the excitation light ⁇ . As described above, the prism 20 has the entrance surface 21, the film formation surface 22, and the exit surface 23. The incident surface 21 allows the excitation light ⁇ from the light irradiation unit 140 to enter the prism 20. A metal film 30 is disposed on the film formation surface 22. In the present embodiment, the excitation light ⁇ incident on the inside of the prism 20 is applied to the metal film 30 where the substance to be detected is captured. The excitation light ⁇ is reflected on the back surface of the metal film 30 to become reflected light ⁇ . More specifically, the excitation light ⁇ is reflected at the interface (deposition surface 22) between the prism 20 and the metal film 30 to become reflected light ⁇ . The emission surface 23 emits the reflected light ⁇ to the outside of the prism 20.
  • the shape of the prism 20 is not particularly limited.
  • the prism 20 is a pillar having a trapezoidal bottom surface.
  • the surface corresponding to one base of the trapezoid is the film formation surface 22, the surface corresponding to one leg is the incident surface 21, and the surface corresponding to the other leg is the emission surface 23.
  • the trapezoid serving as the bottom surface is preferably an isosceles trapezoid. Thereby, the entrance surface 21 and the exit surface 23 are symmetric, and the S wave component of the excitation light ⁇ is less likely to stay in the prism 20.
  • the incident surface 21 is formed so that the excitation light ⁇ does not return to the light irradiation unit 140.
  • the light source of the excitation light ⁇ is a laser diode (hereinafter also referred to as “LD”)
  • LD laser diode
  • the angle of the incident surface 21 is set so that the excitation light ⁇ does not enter the incident surface 21 perpendicularly in the scanning range centered on the enhancement angle.
  • the “enhancement angle” means scattered light having the same wavelength as the excitation light ⁇ emitted above the detection chip 10 when the incident angle of the excitation light ⁇ with respect to the metal film 30 is scanned (hereinafter referred to as “plasmon scattered light”). This means the angle of incidence when the amount of ⁇ is maximized.
  • the angle between the incident surface 21 and the film formation surface 22 and the angle between the film formation surface 22 and the emission surface 23 are both about 80 °.
  • the enhancement angle is generally determined by the design of the detection chip 10.
  • the design factors are the refractive index of the prism 20, the refractive index of the metal film 30, the film thickness of the metal film 30, the extinction coefficient of the metal film 30, the wavelength of the excitation light ⁇ , and the like.
  • the enhancement angle is shifted by the substance to be detected trapped on the metal film 30, but the amount is less than several degrees.
  • the prism 20 has a considerable amount of birefringence.
  • the material of the prism 20 include insulating resin and glass.
  • the material of the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.
  • the metal film 30 is disposed so as to be exposed to at least a part of the flow path 60 on the film formation surface 22 of the prism 20.
  • the metal film 30 causes an interaction (SPR) between the photon of the excitation light ⁇ incident on the film formation surface 22 under total reflection conditions and the free electrons in the metal film 30, and is locally on the surface of the metal film 30.
  • SPR interaction
  • In-situ light commonly referred to as “evanescent light” or “near-field light” can be generated.
  • the material of the metal film 30 is not particularly limited as long as it is a metal capable of generating SPR.
  • Examples of the material of the metal film 30 include gold, silver, copper, aluminum, and alloys thereof.
  • the metal film 30 is a gold thin film.
  • the method for forming the metal film 30 is not particularly limited. Examples of the method for forming the metal film 30 include sputtering, vapor deposition, and plating.
  • the thickness of the metal film 30 is not particularly limited, but is preferably in the range of 30 to 70 nm.
  • a capturing body for capturing a substance to be detected is fixed on the surface of the metal film 30.
  • the capturing body is uniformly fixed to a predetermined region on the metal film 30.
  • the region where the capturing body is fixed serves as a reaction field where a primary reaction and a secondary reaction described later occur.
  • the capturing body fixed to the metal film 30 is exposed in the flow path 60.
  • the type of capturing body is not particularly limited as long as it can capture the substance to be detected.
  • the capturing body is an antibody or a fragment thereof that can specifically bind to the substance to be detected.
  • the channel lid 40 is disposed on the film formation surface 22.
  • the flow path lid 40 has the reaction region 41 and the reagent storage region 42.
  • the reaction region 41 is a region for performing a primary reaction and a secondary reaction described later.
  • the reagent storage area 42 is an area in which a labeling solution used for the secondary reaction, a cleaning solution used for cleaning after each reaction, and the like are stored.
  • a channel groove 43 to be the channel 60 is formed on the back surface of the reaction region 41 in the channel lid 40.
  • a first through hole 44 serving as the injection portion 70 and a second through hole 45 serving as the storage portion 80 are opened on the front surface and the back surface of the reaction region 41, respectively.
  • Both ends of the flow channel 43 are connected to the first through hole 44 and the second through hole 45, respectively.
  • a recess 46 opened on the surface is formed in the reagent storage area 42.
  • the number of the recessed parts 46 is not specifically limited. In the present embodiment, the number of recesses 46 is two.
  • a labeling solution used for the secondary reaction, a cleaning solution, and the like are stored in the recess 46.
  • the channel groove 43, the first through hole 44, and the second through hole 45 become the channel 60, the injection unit 70, and the storage unit 80, respectively, by stacking the prism 20, the metal film 30, and the channel lid 40 in this order. .
  • the openings of the injection part 70, the storage part 80 and the four recesses 46 are respectively sealed with seals 50.
  • the channel lid 40 is preferably made of a material that is transparent to the fluorescence ⁇ emitted from the metal film 30 and the plasmon scattered light ⁇ .
  • An example of the material of the flow path lid 40 includes a resin.
  • the flow path cover 40 may be formed of an opaque material as long as the portion from which the fluorescent ⁇ and the plasmon scattered light ⁇ are extracted is transparent to the fluorescent ⁇ and the plasmon scattered light ⁇ .
  • the channel lid 40 is bonded to the prism 20 or the metal film 30 by, for example, adhesion using a double-sided tape or an adhesive, laser welding, ultrasonic welding, or pressure bonding using a clamp member.
  • the seal 50 closes all the openings of the injection part 70, the storage part 80, and the four recesses 46.
  • the material of the seal 50 can seal the opening of the injection part 70, the storage part 80, and the recess 46, respectively, and the seal 50 that seals the opening of the injection part 70 and the recess 46 can be pierced by the pipette tip 170.
  • the material of the seal 50 include an aluminum foil, an aluminum deposited film, low-density polyethylene, an elastomer material such as polyurethane, and the like.
  • the seal 50 may be a laminated seal in which a plurality of materials are laminated. In the present embodiment, the material of the seal 50 is low density polyethylene.
  • the seal 50 is configured so that a force necessary for breaking through with the pipette tip 170 is 10 N or less.
  • the force required to pierce with the pipette tip 170 varies depending on the material of the seal 50, the recess 46, the opening of the injection portion 70, the area of the opening of the storage portion 80, the shape of the tip of the pipette tip 170, and the like.
  • the force required to pierce with the pipette tip 170 can be measured using a push-pull gauge or the like.
  • the opening area of the recess 46 is 12 mm 2
  • the outer diameter of the tip of the pipette tip 170 is 1 mm (without chamfering)
  • the required force is about 7N. Note that any one of the openings of the injection unit 70, the storage unit 80, and the recess 46 may not be blocked by the seal 50.
  • the detection chip 10 ′ may have a well 60 ′ instead of the flow path 60.
  • the opening of the well 60 ′ is sealed with a seal 50.
  • liquid is injected or removed from the opening of the well 60 '.
  • the excitation light ⁇ enters the prism 20 at the incident surface 21.
  • the excitation light ⁇ that has entered the prism 20 is applied to the metal film 30 at a total reflection angle (an angle at which SPR occurs).
  • a total reflection angle an angle at which SPR occurs.
  • This localized field light excites a fluorescent substance that labels the substance to be detected present on the metal film 30 and emits fluorescence ⁇ .
  • the SPFS device 100 detects the presence or amount of the substance to be detected by measuring the amount of fluorescence ⁇ emitted from the fluorescent substance.
  • the SPFS device 100 includes the liquid feeding unit 110, the transport unit 120, the position information acquisition unit 130, the light irradiation unit 140, the light detection unit 150, and the control unit 160.
  • the detection chip 10 can be held by the chip holder 121 of the transport unit 120.
  • the liquid feeding unit 110 includes a pipette 111, a pipette moving unit 112, and a liquid feeding pump drive mechanism 113.
  • the liquid feeding unit 110 injects a specimen into the flow path 60 of the detection chip 10 held by the chip holder 121, or a liquid such as a labeling solution or a cleaning liquid stored in the reagent storage region 42 of the detection chip 10 in the reaction region. 41 in the channel 60. Further, the liquid feeding unit 110 also discharges the liquid from the flow path 60 and stirs the liquid in the flow path 60.
  • the liquid feeding unit 110 is used in a state where the pipette tip 170 is attached to the pipette nozzle 116 of the pipette 111. It should be noted that the pipette tip 170 is preferably replaceable from the viewpoint of preventing contamination of impurities.
  • the pipette 111 sucks the liquid when injecting the liquid into the flow path 60 or removing the liquid from the flow path 60.
  • the pipette 111 includes a syringe 114, a plunger 115 that can reciprocate inside the syringe 114, and a pipette nozzle 116 connected to the syringe 114. Further, the pipette 111 can quantitatively suck and discharge the liquid by the reciprocating motion of the plunger 115. Thereby, the pipette 111 can inject liquid into the flow path 60 or remove the liquid from the flow path 60. Moreover, the pipette 111 can stir the liquid in the flow path 60 by repeating suction and discharge of the liquid.
  • the pipette moving unit 112 moves the pipette nozzle 116 in order to suck the liquid into the pipette tip 170, discharge the liquid from the pipette tip 170, or break the seal 50.
  • the pipette moving unit 112 freely moves the pipette nozzle 116 in the axial direction (for example, the vertical direction) of the pipette nozzle 116.
  • the pipette moving unit 112 includes, for example, a solenoid actuator and a stepping motor.
  • the pipette moving unit 112 moves the seal 50 to be broken through the detection chip 10 to the position directly below the pipette nozzle 116 by the transport unit 120, and then seals the pipette nozzle 116 (pipette tip 170).
  • the seal 50 is broken with the pipette tip 170.
  • the distance between the seal 50 and the bottom surface of the flow path 60 see FIG. 6A; d1
  • the distance between the seal 50 and the bottom surface of the recess 46 see FIG. 6B; d3
  • the recess 46 are stored. Since the depth of the liquid (see FIG. 6B; d4) is recorded in advance, the tip of the pipette tip 170 does not come into contact with the metal film 30 in the flow path 60 or the liquid stored in the recess 46. .
  • the liquid feed pump drive mechanism 113 moves the plunger 115 to suck the external liquid into the pipette tip 170 or discharge the liquid inside the pipette 111 to the outside.
  • the liquid feed pump drive mechanism 113 includes a device for reciprocating the plunger 115 such as a stepping motor.
  • the stepping motor is preferable from the viewpoint of managing the remaining liquid amount of the detection chip 10 because it can manage the liquid feeding amount and the liquid feeding speed of the pipette 111.
  • the liquid feed pump drive mechanism 113 is preferably not driven during the process in which the pipette tip 170 breaks through the seal 50.
  • the liquid feeding unit 110 sucks various liquids from the recess 46 and injects them into the flow channel 60 of the detection chip 10.
  • the reciprocating operation of the plunger 115 with respect to the syringe 114 is repeated, so that the liquid is flown in the flow channel 60 in the detection chip 10. It reciprocates and the liquid in the flow path 60 is stirred.
  • the liquid in the channel 60 is again sucked by the pipette 111 and discharged to a waste liquid tank or the like not shown.
  • reaction with various liquids, washing, and the like can be performed, and a detection target substance labeled with a fluorescent substance can be arranged in the reaction field in the flow path 60.
  • the transport unit 120 transports the detection chip 10 to the detection position or the liquid feeding position, and holds the detection chip 10.
  • the “detection position” is a position where the light irradiation unit 140 irradiates the detection chip 10 with the excitation light ⁇ , and the light detection unit 150 detects the fluorescence ⁇ or the plasmon scattered light ⁇ generated accordingly.
  • the “liquid feeding position” is a position where the liquid feeding unit 110 injects liquid into the flow channel 60 of the detection chip 10 or removes the liquid in the flow channel 60 of the detection chip 10.
  • the transport unit 120 includes a chip holder 121 and a transport stage 122.
  • the chip holder 121 is fixed to the transfer stage 122 and holds the detection chip 10 in a detachable manner.
  • the shape of the chip holder 121 is not particularly limited as long as it can hold the detection chip 10 and does not interfere with the optical paths of the excitation light ⁇ , fluorescence ⁇ , and plasmon scattered light ⁇ .
  • the shape of the chip holder 121 is configured so that the detection chip 10 can be held with the flow path lid 40 interposed therebetween.
  • the transfer stage 122 moves the chip holder 121 in one direction and in the opposite direction (left and right direction on the paper surface of FIG. 1).
  • the transport stage 122 also has a shape that does not interfere with the optical paths of the excitation light ⁇ , fluorescence ⁇ , and plasmon scattered light ⁇ .
  • the transfer stage 122 is driven by, for example, a stepping motor.
  • the position information acquisition unit 130 acquires position information on the tip of the pipette tip 170.
  • the configuration of the position information acquisition unit 130 is not particularly limited as long as the position information of the tip of the pipette tip 170 can be acquired.
  • Examples of the position information acquisition unit 130 include a strain sensor, a pneumatic sensor 131, a capacitance sensor, a power supply and ammeter, a pressure sensor, and the like.
  • the position information acquisition unit 130 includes an air pressure sensor 131.
  • the air pressure sensor 131 is connected between the pipette nozzle 116 and the syringe 114.
  • the position information of the tip of the pipette tip 170 is obtained when gas is sucked or discharged from the tip of the pipette tip 170 while changing the distance between the tip of the pipette tip 170 and the reference portion 180 (see FIG. 6A).
  • the change in the air pressure in the pipette tip 170 is measured by the air pressure sensor 131.
  • the “reference portion” means a reference position at the tip of the pipette tip 170.
  • the reference unit 180 is not particularly limited as long as its position is specified with high accuracy, and may be a part of the detection chip 10 or a part of the SPFS device 100.
  • Examples of the reference unit 180 included in the detection chip 10 include the flow path lid 40, the seal 50, the prism 20, and the like.
  • examples of the reference unit 180 included in the SPFS apparatus 100 include a transport stage 122, a chip holder 121, and a base that supports the transport stage 122 in the transport unit 120 (portion located below the pipette nozzle 116). There may be. The suction or discharge of gas at the tip of the pipette tip 170 may be performed continuously or intermittently.
  • the light irradiation unit 140 irradiates the excitation light ⁇ toward the incident surface 21 of the detection chip 10 held by the chip holder 121. At the time of measuring the fluorescence ⁇ or the plasmon scattered light ⁇ , the light irradiation unit 140 emits only the P wave toward the incident surface 21 so that the incident angle with respect to the metal film 30 is an angle that causes SPR. .
  • the “excitation light” is light that directly or indirectly excites the fluorescent material.
  • the excitation light ⁇ is light that generates localized field light on the surface of the metal film 30 that excites the fluorescent material when the metal film 30 is irradiated through the prism 20 at an angle at which SPR occurs.
  • the light irradiation unit 140 includes a light source unit 141, an angle adjustment mechanism 142, and a light source control unit 143.
  • the light source unit 141 emits the collimated excitation light ⁇ having a constant wavelength and light amount so that the shape of the irradiation spot on the back surface of the metal film 30 is substantially circular.
  • the light source unit 141 includes, for example, a light source of excitation light ⁇ , a beam shaping optical system, an APC mechanism, and a temperature adjustment mechanism (all not shown).
  • the type of the light source is not particularly limited, and is, for example, a laser diode (LD).
  • Other examples of light sources include light emitting diodes, mercury lamps, and other laser light sources.
  • the light emitted from the light source is not a beam, the light emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like.
  • the light emitted from the light source is not monochromatic light, the light emitted from the light source is converted into monochromatic light by a diffraction grating or the like.
  • the light emitted from the light source is not linearly polarized light, the light emitted from the light source is converted into linearly polarized light by a polarizer or the like.
  • the beam shaping optical system includes, for example, a collimator, a band pass filter, a linear polarization filter, a half-wave plate, a slit, and a zoom means.
  • the beam shaping optical system may include all of these or a part thereof.
  • the collimator collimates the excitation light ⁇ emitted from the light source.
  • the band-pass filter turns the excitation light ⁇ emitted from the light source into narrowband light having only the center wavelength. This is because the excitation light ⁇ from the light source has a slight wavelength distribution width.
  • the linear polarization filter turns the excitation light ⁇ emitted from the light source into completely linearly polarized light.
  • the half-wave plate adjusts the polarization direction of the excitation light ⁇ so that the P-wave component is incident on the metal film 30.
  • the slit and zoom means adjust the beam diameter, contour shape, and the like of the excitation light ⁇ so that the shape of the irradiation spot on the back surface of the metal film 30 is a circle of a predetermined size.
  • the APC mechanism controls the light source so that the output of the light source is constant. More specifically, the APC mechanism detects the amount of light branched from the excitation light ⁇ with a photodiode (not shown) or the like.
  • the APC mechanism controls the input energy by a regression circuit, thereby controlling the output of the light source to be constant.
  • the angle adjustment mechanism 142 adjusts the incident angle of the excitation light ⁇ with respect to the metal film 30 (the interface between the prism 20 and the metal film 30 (film formation surface 22)). In order to irradiate the excitation light ⁇ at a predetermined incident angle toward a predetermined position of the metal film 30 via the prism 20, the angle adjustment mechanism 142 relatively places the optical axis of the excitation light ⁇ and the chip holder 121. Rotate.
  • the angle adjusting mechanism 142 rotates the light source unit 141 around an axis (axis perpendicular to the paper surface of FIG. 1) orthogonal to the optical axis of the excitation light ⁇ .
  • the position of the rotation axis is set so that the position of the irradiation spot on the metal film 30 hardly changes even when the incident angle is scanned.
  • the angle at which the amount of plasmon scattered light ⁇ is maximum is the enhancement angle.
  • High intensity fluorescence ⁇ can be measured by setting the incident angle of the excitation light ⁇ to the enhancement angle or an angle in the vicinity thereof.
  • the basic incident condition of the excitation light ⁇ is determined by the material and shape of the prism 20 of the detection chip 10, the film thickness of the metal film 30, the refractive index of the liquid in the flow channel 60, etc. The optimum incident condition varies slightly depending on the type and amount of the light and the shape error of the prism 20. For this reason, it is preferable to obtain an optimal enhancement angle for each measurement.
  • the light source control unit 143 controls various devices included in the light source unit 141 to control the emission of the excitation light ⁇ from the light source unit 141.
  • the light source control unit 143 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
  • the light detection unit 150 detects the amount of fluorescence ⁇ emitted from the vicinity of the surface of the metal film 30 on the flow path 60 side when the light irradiation unit 140 irradiates the metal film 30 of the detection chip 10 with the excitation light ⁇ . To do. Further, as necessary, the light detection unit 150 also detects plasmon scattered light ⁇ generated by irradiation of the excitation light ⁇ to the metal film 30.
  • the light detection unit 150 includes a light receiving unit 151, a position switching mechanism 152, and a sensor control unit 153.
  • the light receiving unit 151 is disposed in the normal direction to the surface of the metal film 30 of the detection chip 10.
  • the light receiving unit 151 includes a first lens 154, an optical filter 155, a second lens 156, and a light receiving sensor 157.
  • the first lens 154 is, for example, a condensing lens, and condenses light emitted from the metal film 30.
  • the second lens 156 is an imaging lens, for example, and forms an image of the light collected by the first lens 154 on the light receiving surface of the light receiving sensor 157.
  • the optical path between the first lens 154 and the second lens 156 is substantially parallel.
  • the optical filter 155 is disposed between the first lens 154 and the second lens 156.
  • the optical filter 155 guides only the fluorescence component to the light receiving sensor 157 and removes the excitation light component (plasmon scattered light ⁇ ) in order to detect the fluorescence ⁇ with a high S / N ratio.
  • Examples of the optical filter 155 include an excitation light reflection filter, a short wavelength cut filter, and a band pass filter.
  • the optical filter 155 is, for example, a filter including a multilayer film that reflects a predetermined light component, or a color glass filter that absorbs a predetermined light component.
  • the light receiving sensor 157 detects fluorescence ⁇ and plasmon scattered light ⁇ .
  • the light receiving sensor 157 has a high sensitivity capable of detecting weak fluorescence ⁇ from a very small amount of a substance to be detected.
  • the light receiving sensor 157 is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).
  • the position switching mechanism 152 switches the position of the optical filter 155 on or off the optical path in the light receiving unit 151. Specifically, when the light receiving sensor 157 detects the fluorescence ⁇ , the optical filter 155 is disposed on the optical path of the light receiving unit 151, and when the light receiving sensor 157 detects the plasmon scattered light ⁇ , the optical filter 155 is placed on the light receiving unit 151. Placed outside the optical path.
  • the sensor control unit 153 controls detection of an output value of the light receiving sensor 157, management of sensitivity of the light receiving sensor 157 based on the detected output value, change of sensitivity of the light receiving sensor 157 for obtaining an appropriate output value, and the like.
  • the sensor control unit 153 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
  • the controller 160 controls the liquid feed pump drive mechanism 113, the transport stage 122, the angle adjustment mechanism 142, the light source controller 143, the position switching mechanism 152, and the sensor controller 153.
  • the control unit 160 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
  • FIG. 4 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 100.
  • FIG. 5 is a flowchart showing the contents of the step (step S120 in FIG. 4) of acquiring the position information of the tip of the pipette tip 170.
  • 6A is a diagram for explaining a process of breaking through the seal 50 blocking the injection portion 70
  • FIG. 6B is a diagram for explaining a process of breaking through the seal 50 closing the recess 46
  • 6C is a diagram for explaining a step of sucking the liquid from the recess 46.
  • the primary antibody is immobilized on the metal film 30 as a capturing body.
  • a secondary antibody labeled with a fluorescent substance is used as a capturing body used for fluorescent labeling.
  • the injection part 70, the storage part 80, and the concave part 46 are each closed by a seal 50.
  • the reference portion 180 is the bottom surface of the flow path 60.
  • step S110 preparation for measurement is performed (step S110). Specifically, the detection chip 10 is prepared, and the detection chip 10 is installed in the chip holder 121 at the set position of the detection chip 10. A pipette tip 170 is attached to the tip of the pipette nozzle 116.
  • step S120 the position information of the tip of the pipette tip 170 is acquired (step S120).
  • the seal 50 having the maximum force required to pierce is pierced by the pipette tip 170 (step S121).
  • the control unit 160 operates the transport stage 122 to move the detection chip 10 to the liquid feeding position.
  • the control unit 160 drives the pipette moving unit 112 to break through the pipette tip 170 with the seal 50 having the maximum force required to pierce among the plurality of seals 50 to be pierced by the pipette tip 170.
  • the seal 50 having the maximum force necessary for piercing is the seal 50 closing the opening of the injection portion 70.
  • the distance d1 between the back surface of the seal 50 and the bottom surface of the flow path 60 is stored in advance (FIG. 6A). Therefore, the pipette moving unit 112 controls so that the tip of the pipette tip 170 does not contact the bottom surface of the flow path 60.
  • the distance between the seal 50 and the bottom surface of the recess 46 (see FIG. 6B; d3) and the depth of the liquid stored in the recess 46 (see FIG. 6B; d4) are stored in advance. Yes. Therefore, when the seal 50 having the maximum force required to pierce among the plurality of seals 50 to be pierced by the pipette tip 170 is the seal 50 closing the opening of the recess 46, the pipette moving unit 112 has the pipette tip 112. Control is performed so that the tip of 170 does not come into contact with the liquid stored in the recess 46.
  • the control unit 160 drives the pipette moving unit 112 to move the tip of the pipette tip 170 toward the bottom surface (reference unit 180) of the flow channel 60.
  • the controller 160 drives the liquid feed pump drive mechanism 113 to advance the plunger 115 relative to the syringe 114. That is, the control unit 160 brings the tip of the pipette tip 170 closer to the reference unit 180 while continuously ejecting air from the tip of the pipette tip 170.
  • the control unit 160 acquires position information of the tip of the pipette tip 170 with respect to the reference unit 180 when the air pressure sensor 131 detects the air pressure.
  • the tip of the pipette tip 170 may be brought close to the reference portion 180 while air is intermittently ejected from the tip of the pipette tip 170. Further, the tip of the pipette tip 170 may be brought close to the reference portion 180 while sucking air from the tip of the pipette tip 170 continuously or intermittently. Even in these cases, the position information of the tip of the pipette tip 170 with respect to the reference portion 180 can be obtained with high accuracy.
  • the step of breaking through the seal 50 is preferably performed before performing an operation in which the position accuracy of the pipette tip 170 is most required in all steps included in the detection operation (reaction method) of the SPFS device 100. .
  • the pipette tip 170 has the seal 50 having the maximum force required for piercing. You may make it break through.
  • the operation for which the position accuracy of the pipette tip 170 is most required is a step of removing the specimen from the flow path 60 in the primary reaction.
  • step S120 of acquiring the position information of the tip of the pipette tip is performed before the primary reaction (step S140).
  • step S120 the step of acquiring the position information of the tip of the pipette tip 170 (step S120) may be performed before the cleaning liquid is removed from the metal film 30.
  • a plurality of seals 50 may be broken.
  • the other seal 50 may be pierced, or after piercing through the other seal 50, the force required to pierce is the maximum.
  • the seal 50 may be pierced.
  • the position information of the tip of the pipette tip 170 is acquired after breaking the seal 50 once or twice or more.
  • the incident angle of the excitation light ⁇ is determined (step S130). Specifically, the control unit 160 operates the transfer stage 122 to move the detection chip 10 to the detection position. The controller 160 drives the sensor controller 153 to detect the plasmon scattered light ⁇ by the light receiving sensor 157 while driving the angle adjusting mechanism 142 to scan the incident angle of the excitation light ⁇ .
  • the angle at which the amount of plasmon scattered light ⁇ is maximized is defined as the incident angle (enhancement angle) of the excitation light ⁇ .
  • tip of the pipette tip 170 and the process (process S130) which determines the incident angle of excitation light (alpha) is not limited to this.
  • the step of determining the incident angle of the excitation light ⁇ step S130
  • the step of acquiring the position information of the tip of the pipette tip 170 step S120) may be performed.
  • step S140 the detection target substance in the specimen is reacted with the primary antibody (primary reaction; step S140).
  • the control unit 160 operates the transport stage 122 to move the container in which the specimen is stored directly below the pipette tip 170. Then, the tip of the pipette tip 170 is moved toward the container in which the specimen is stored, and the specimen is inhaled into the pipette tip 170.
  • the controller 160 operates the transfer stage 122 to move the detection chip 10 to the liquid feeding position. Then, the control unit 160 drives the pipette moving unit 112 to move the tip of the pipette tip 170 into the injection unit 70 and injects the sample into the flow channel 60.
  • the distance (see FIG. 6A; d2) between the tip of the pipette tip 170 and the bottom surface of the flow path 60 can be controlled with high accuracy. Therefore, the distance between the tip of the pipette tip 170 and the bottom surface of the flow path 60 is too large, and the specimen may adhere to the side wall of the injection unit 70. Therefore, the bottom surface (metal film 30) of the flow path 60 is not damaged.
  • a substance to be detected exists in the sample, at least a part of the substance to be detected binds to the primary antibody. After the primary reaction, the specimen is removed from the flow path 60.
  • the tip of the pipette tip 170 is brought close to the bottom surface of the channel 60 based on the position information of the tip of the pipette tip 170. Then, the sample is removed from the flow path 60 by inhaling the sample into the pipette tip 170.
  • the container in which the specimen is stored may be disposed on the detection chip 10.
  • an accommodation hole for accommodating the container is formed in the flow path lid 40 of the detection chip 10.
  • the types of the specimen and the substance to be detected are not particularly limited.
  • the specimen include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, semen, and diluted solutions thereof.
  • substances to be detected include nucleic acids (such as DNA and RNA), proteins (such as polypeptides and oligopeptides), amino acids, carbohydrates, lipids, and modified molecules thereof.
  • the specimen may be reciprocated in the flow path 60.
  • the tip of the pipette tip 170 is brought close to the bottom surface of the channel 60 based on the positional information of the tip of the pipette tip 170 as in the step of injecting the sample into the channel 60.
  • the plunger 115 is reciprocated while the position of the tip of the pipette tip 170 is fixed. Accordingly, the sample can be reciprocated in the flow path 60 by repeatedly inhaling and discharging the sample with the pipette tip 170. After reciprocating the sample in the flow channel 60, the sample is removed from the flow channel 60 by inhaling the sample into the pipette tip 170.
  • the metal film 30 is cleaned with a cleaning solution such as a buffer solution.
  • the controller 160 moves the tip of the pipette tip 170 toward the cleaning liquid in the recess 46 and causes the cleaning liquid to be sucked into the pipette tip 170.
  • the distance between the tip of the pipette tip 170 and the surface of the cleaning liquid (see FIG. 6C; d5) and the tip of the pipette tip 170 and the recess 46 The distance from the bottom surface (see FIG. 6C; d6) can also be controlled with high accuracy. Therefore, the cleaning liquid can be appropriately sucked into the pipette tip 170.
  • the control unit 160 drives the pipette moving unit 112 to move the tip of the pipette tip 170 into the injection unit 70 and injects the cleaning liquid into the flow channel 60.
  • the washing liquid containing the substance that has not bound to the primary antibody is removed from the flow path 60.
  • the control unit 160 drives the pipette moving unit 112 to move the tip of the pipette tip 170 to the injection unit 70.
  • the tip of the pipette tip 170 is brought close to the bottom surface of the channel 60 to remove the cleaning liquid from the channel 60.
  • the tip of the pipette tip 170 is brought close to the prism 20 (metal film 30) based on the position information of the tip of the pipette tip 170 described above, the amount of liquid remaining in the flow path 60 is minimized. be able to.
  • the position of the tip of the pipette tip 170 when removing the cleaning liquid is preferably the same as the position of the tip of the pipette tip 170 in the step of removing the specimen from the flow path 60. Thereby, the amount of liquid remaining in the flow path 60 can be made constant.
  • the target substance captured on the metal film 30 is labeled with a fluorescent substance (secondary reaction; step S150).
  • the control unit 160 moves the tip of the pipette tip 170 toward the concave portion 46 in which a liquid (labeling liquid) containing a capturing body labeled with a fluorescent substance is stored, and labels the pipette tip 170 in the pipette tip 170. Inhale fluid.
  • the position information of the pipette tip 170 is specified with high accuracy, the distance between the tip of the pipette tip 170 and the surface of the labeling liquid (see d5 in FIG.
  • the control unit 160 drives the pipette moving unit 112 to move the tip of the pipette tip 170 into the injection unit 70 and injects the labeling solution into the flow channel 60.
  • the detection target substance captured on the metal film 30 is labeled with a fluorescent substance by an antigen-antibody reaction. Thereafter, the labeling liquid in the flow path 60 is removed, and the inside of the flow path 60 is cleaned with a cleaning liquid.
  • the position of the tip of the pipette tip 170 when removing the labeling liquid in the flow channel 60 is determined based on the position information of the tip of the pipette tip 170 described above. As a result, the amount of liquid remaining in the flow path 60 can be kept to a minimum and constant.
  • the order of the primary reaction (step S140) and the secondary reaction (step S150) is not limited to this.
  • the liquid containing these complexes is deposited on the metal film 30. May be provided. Further, the specimen and the labeling solution may be provided on the metal film 30 at the same time.
  • the pipette 111 is moved once or twice or more based on the position information of the tip of the pipette chip 170.
  • a substance to be detected is detected (step S160). Specifically, the control unit 160 operates the transfer stage 122 to move the detection chip 10 to the detection position.
  • the sensor control unit 153 is driven to drive the metal film 30 (while the light source control unit 143 is driven to irradiate the excitation light ⁇ to a predetermined position of the metal film 30 at the incident angle (enhancement angle) determined in step S130.
  • the light receiving sensor 157 is controlled so as to detect the intensity of the fluorescence ⁇ emitted from the surface of the metal film 30 and the vicinity thereof.
  • control part 160 may measure a blank value before a secondary reaction (process S150).
  • the excitation light ⁇ is irradiated onto the metal film 30 at an enhancement angle, and the detection value of the light receiving sensor 157 is set as a blank value.
  • the amount of fluorescence ⁇ indicating the amount of the substance to be detected in the sample is calculated by subtracting the blank value from the detected value of fluorescence ⁇ .
  • the SPFS device 100 acquires the position information of the tip of the pipette tip 170 after breaking through the seal 50 with the pipette tip 170, the position information of the tip of the pipette tip 170 is obtained.
  • the position of the tip end of the pipette tip 170 can be controlled with high accuracy without the position of the tip end of the pipette tip 170 being shifted after obtaining the above.
  • the precision of a detection result can be improved by making constant the residual liquid amount in the flow path 60.
  • the SPFS device according to the second embodiment differs from the SPFS device 100 according to the first embodiment in that the configuration of the position information acquisition unit 230 is different. Therefore, the same components as those of the SPFS device 100 according to the first embodiment are denoted by the same reference numerals, description thereof is omitted, and different portions are mainly described.
  • FIG. 7A is a diagram illustrating a partial configuration of the SPFS apparatus according to the second embodiment.
  • the position information acquisition unit 230 includes a strain sensor 231.
  • the strain sensor 231 is disposed on the pipette nozzle 116.
  • the position information of the tip of the pipette tip 170 is acquired by measuring the strain of the pipette nozzle 116 when the tip of the pipette tip 170 and the reference unit 180 come into contact with each other by the strain sensor 231.
  • the pipette tip 170 and the pipette nozzle 116 to which the pipette tip 170 is attached are distorted.
  • the contact between the tip of the pipette tip 170 and the reference portion 180 can be detected. Therefore, the position information of the tip of the pipette tip 170 can be acquired.
  • FIG. 7B is a flowchart showing a process of acquiring the position information of the tip of the pipette tip 170.
  • the force required to break through the plurality of seals 50 that the pipette tip 170 should break through is maximized.
  • the seal 50 is broken with the pipette tip 170 (step S121).
  • the distortion of the pipette nozzle 116 is measured (step S222).
  • the control unit 160 operates the pipette moving unit 112 to move the tip of the pipette tip 170 toward the bottom surface (reference unit 180) of the channel 60.
  • the pipette tip 170 and the pipette nozzle 116 to which the pipette tip 170 is attached are distorted.
  • the position information of the tip of the pipette tip 170 is acquired by detecting the contact between the tip of the pipette tip 170 and the reference portion 180 by the strain sensor 231 disposed in the pipette nozzle 116.
  • the SPFS device according to the second embodiment has the same effects as the SPFS device 100 according to the first embodiment.
  • the SPFS apparatus 100 according to the third embodiment is different from the SPFS apparatus 100 according to the first embodiment in that the configuration of the position information acquisition unit 330 is different. Therefore, the same components as those of the SPFS device 100 according to Embodiment 1 are denoted by the same reference numerals, and the description thereof is omitted.
  • FIG. 8A is a diagram illustrating a partial configuration of the SPFS apparatus according to the third embodiment.
  • the position information acquisition unit 330 has a capacitance sensor 331.
  • the position where the capacitance sensor 331 is disposed is not particularly limited. In the present embodiment, the capacitance sensor 331 is disposed on the transfer stage 122. Acquisition of the position information of the tip of the pipette tip 170 is performed between the tip of the pipette tip 170 and the reference portion 180 when the tip of the pipette tip 170 is brought close to the reference portion 180 (detection surface of the capacitance sensor 331). The change in the electrostatic capacity is measured by the electrostatic capacity sensor 331.
  • FIG. 8B is a flowchart showing a process of acquiring position information of the tip of the pipette tip 170.
  • the force required to pierce out of the plurality of seals 50 to be pierced by the pipette tip 170 is maximized.
  • the seal 50 is broken with the pipette tip 170 (step S121).
  • step S322 the change in capacitance between the tip of the pipette tip 170 and the reference portion is measured (step S322).
  • the control unit 160 operates the pipette moving unit 112 to move the tip of the pipette tip 170 toward the detection surface (reference unit 180) of the capacitance sensor 331.
  • the capacitance between the tip of the pipette tip 170 and the reference portion 180 changes.
  • position information of the tip of the pipette tip 170 is acquired.
  • the SPFS device according to the third embodiment has the same effects as the SPFS device 100 according to the first embodiment.
  • the SPFS apparatus according to the fourth embodiment differs from the SPFS apparatus 100 according to the first embodiment in that the configuration of the position information acquisition unit 430 is different. Therefore, the same components as those of the SPFS device 100 according to the first embodiment are denoted by the same reference numerals, description thereof is omitted, and different portions are mainly described.
  • FIG. 9A is a diagram illustrating a partial configuration of the SPFS apparatus according to the fourth embodiment.
  • the position information acquisition unit 430 includes a power source 431 and an ammeter 432.
  • the power source 431 and the ammeter 432 are connected to the tip of the pipette tip 170 and the reference unit 180, respectively.
  • the pipette tip 170 uses a conductive pipette tip containing carbon or the like.
  • the position information of the tip of the pipette tip 170 is acquired by applying a voltage between the tip of the pipette tip 170 and the reference portion 180 (metal film 30) by the power source 431, and using the ammeter 432 for the tip of the pipette tip 170 and the reference. This is done by measuring the impedance characteristic between the portion 180 (metal film 30).
  • FIG. 9B is a flowchart showing a process of acquiring position information of the tip of the pipette tip 170.
  • the force required to break through the plurality of seals 50 that the pipette tip 170 should break through is maximized.
  • the seal 50 is broken with the pipette tip 170 (step S121).
  • the control unit 160 operates the pipette moving unit 112 to move the tip of the pipette tip 170 toward the bottom surface (reference unit 180) of the channel 60.
  • a voltage is applied between the tip of the pipette tip 170 and the reference portion 180 (metal film 30) by the power source 431, and the pipette tip 170 is drawn by the ammeter 432.
  • the position information of the tip of the pipette tip 170 is obtained by measuring the impedance characteristic between the tip of the pipe and the reference portion 180 (metal film 30).
  • the SPFS device according to the fourth embodiment has the same effects as the SPFS device 100 according to the first embodiment.
  • the SPFS device according to the fifth embodiment is different from the SPFS device 100 according to the first embodiment in that the configuration of the position information acquisition unit 530 is different. Therefore, the same components as those of the SPFS device 100 according to the first embodiment are denoted by the same reference numerals, description thereof is omitted, and different portions are mainly described.
  • FIG. 10A is a diagram illustrating a partial configuration of the SPFS apparatus according to the fifth embodiment.
  • the position information acquisition unit 530 includes a pressure-sensitive sensor 531.
  • the position where the pressure-sensitive sensor 531 is disposed is not particularly limited. In the present embodiment, the pressure-sensitive sensor 531 is disposed on the transfer stage 122.
  • the position information of the tip of the pipette tip 170 is acquired by measuring the pressure when the tip of the pipette tip 170 comes into contact with the reference portion 180 (detection surface of the pressure sensor 531) by the pressure sensor 531.
  • the pressure-sensitive sensor 531 detects a predetermined pressure. Thereby, the contact between the tip of the pipette tip 170 and the reference portion 180 can be detected.
  • FIG. 10B is a flowchart showing a process of acquiring the position information of the tip of the pipette tip 170.
  • the force required to break through the plurality of seals 50 that the pipette tip 170 should break through is maximized.
  • the seal 50 is broken with the pipette tip 170 (step S121).
  • step S522 the contact pressure between the tip of the pipette tip 170 and the reference portion 180 is measured (step S522).
  • the control unit 160 operates the pipette moving unit 112 to move the tip of the pipette tip 170 toward the bottom surface (reference unit 180) of the channel 60.
  • the pressure-sensitive sensor 531 detects the pressure at that time. At this time, the pressure sensor 531 detects a predetermined pressure to acquire position information of the tip of the pipette tip 170.
  • the SPFS device according to the fifth embodiment has the same effect as the SPFS device 100 according to the first embodiment.
  • the SPFS device according to the sixth embodiment differs from the SPFS device 100 according to the first embodiment in that the configuration of the position information acquisition unit 630 is different. Therefore, the same components as those of the SPFS device 100 according to the first embodiment are denoted by the same reference numerals, description thereof is omitted, and different portions are mainly described.
  • FIG. 11A is a diagram illustrating a partial configuration of the SPFS apparatus according to the sixth embodiment.
  • the position information acquisition unit 630 includes an optical sensor 631.
  • the optical sensor 631 is not particularly limited as long as it can optically acquire the position information of the tip of the pipette tip 170.
  • An example of the optical sensor 631 may be a method of detecting by the presence / absence of laser light passing between the light emitting element and the light receiving element, or a method of detecting by imaging with a CCD camera.
  • the optical sensor 631 is a method of detecting by imaging with a CCD camera.
  • the position where the optical sensor 631 is arranged is not particularly limited. In the present embodiment, the optical sensor 631 is fixed to the transfer stage 122 via a support member.
  • Acquisition of the position information of the tip of the pipette tip 170 is performed by imaging when the tip of the pipette tip 170 enters the imaging region of the optical sensor 631.
  • the optical sensor 631 detects and images the tip of the pipette tip 170. Thereby, the position information of the tip of the pipette tip 170 can be acquired.
  • FIG. 11B is a flowchart showing a process of acquiring the position information of the tip of the pipette tip 170.
  • the force required for breaking through the plurality of seals 50 that the pipette tip 170 should break through is maximized.
  • the seal 50 is broken with the pipette tip 170 (step S121).
  • the tip position of the pipette tip 170 is detected (step S622).
  • the control unit 160 operates the pipette moving unit 112 to move the tip of the pipette tip 170 toward the imaging area of the optical sensor 631.
  • the optical sensor 631 detects that the tip of the pipette tip 170 has entered the imaging area of the optical sensor 631, captures the tip of the pipette tip 170, and acquires positional information of the tip of the pipette tip 170.
  • the SPFS device according to the sixth embodiment has the same effect as the SPFS device 100 according to the first embodiment.
  • the position information of the tip of the pipette tip 170 may be acquired in a state where the labeling solution or the cleaning solution is sucked into the pipette tip 170.
  • the acquisition of the position information of the tip of the pipette tip 170 and the injection operation of the labeling liquid and the cleaning liquid into the flow channel 60 can be performed simultaneously, and the detection time can be shortened. Further, in Embodiments 1 to 4 and 6, the SPFS apparatus is not contaminated.
  • the detection apparatus including the reaction apparatus according to the present invention has been described.
  • a configuration for detecting a substance to be detected for example, the conveyance unit 120, the light irradiation unit 140, and the light detection unit 150).
  • Etc. may not be included.
  • the detection method may not include the detection step (step S160).
  • the present invention is applied to the SPFS device 100 using SPR as a detection device including a reaction device.
  • the present invention is applied to an SPR device (detecting reflected light ⁇ ) using SPR. May be.
  • the same effect as in the first to sixth embodiments can be obtained.
  • the detection apparatus performs measurement by the total reflection measurement (ATR) method or the like without using SPR, but the same effects as in the first to sixth embodiments can be obtained.
  • ATR total reflection measurement
  • the reaction method and reaction apparatus according to the present invention can measure a substance to be detected with high reliability. Therefore, it is expected to contribute to the development, spread and development of a very simple quantitative immunoassay system.

Abstract

In this reaction method, a pipette tip attached to a pipette nozzle and a reaction tip having a recess and a seal sealing the opening of the recess are used to cause two or more substances to react within the reaction tip. First, the seal of the reaction tip is pierced by the pipette tip (step 1). Then, after step 1, position information for the leading end of the pipette tip is acquired (step 2). After step 2, the pipette tip is manipulated on the basis of the position information for the leading end of the pipette tip, and two or more substances are made to react within the reaction tip (step 3).

Description

反応方法および反応装置Reaction method and reaction apparatus
　本発明は、ピペットノズルに装着されたピペットチップと、凹部および前記凹部の開口を封止しているシールを有する反応チップとを使用して、前記反応チップ内において２以上の物質を反応させる反応方法および反応装置に関する。 The present invention uses a pipette tip attached to a pipette nozzle and a reaction chip having a recess and a seal sealing the opening of the recess to react two or more substances in the reaction chip. The present invention relates to a method and a reaction apparatus.
　臨床検査などにおいて、タンパク質やＤＮＡなどの微量の被検出物質を高感度かつ定量的に検出できれば、患者の状態を迅速に把握して治療を行うことが可能となる。このため、微量の被検出物質を高感度かつ定量的に検出できる方法および装置が求められている。 In clinical examinations and the like, if a very small amount of a substance to be detected such as protein or DNA can be detected with high sensitivity and quantitativeness, it becomes possible to quickly grasp and treat a patient. Therefore, there is a need for a method and apparatus that can detect a minute amount of a substance to be detected with high sensitivity and quantitatively.
　被検出物質を高感度に検出できる方法として、表面プラズモン共鳴（Surface plasmon resonance：以下「ＳＰＲ」ともいう）を利用する検出方法が知られている（例えば、特許文献１参照）。 As a method for detecting a substance to be detected with high sensitivity, a detection method using surface plasmon resonance (hereinafter also referred to as “SPR”) is known (see, for example, Patent Document 1).
　特許文献１に記載の検出方法では、誘電体からなるプリズムと、プリズム上に配置された金属膜と、金属膜上に配置され、液体流路を構成する流路部材とを有する検出チップを使用する。金属膜上には、被検出物質を捕捉するための捕捉体が配置されている。流路部材は、被検出物質を含む検体などの液体を液体流路内に注入するための注入部と、液体を液体流路内から排出するための排出部とを有する。注入部の注入口および排出部の排出口は、ピペットチップの先端に対して相補的な形状に形成されている。したがって、ピペットチップの先端部を注入口または排出口に挿入させると、ピペットチップの先端部と、注入口または排出口とが嵌合する。これにより、液体流路の底面に対して、ピペットチップの先端が一定の位置に配置され、液体流路内の液体の量を高精度に制御することができる。 In the detection method described in Patent Document 1, a detection chip having a prism made of a dielectric material, a metal film disposed on the prism, and a flow path member that is disposed on the metal film and forms a liquid flow path is used. To do. A capturing body for capturing the substance to be detected is disposed on the metal film. The flow path member has an injection part for injecting a liquid such as a specimen containing a substance to be detected into the liquid flow path, and a discharge part for discharging the liquid from the liquid flow path. The inlet of the inlet and the outlet of the outlet are formed in a complementary shape with respect to the tip of the pipette tip. Therefore, when the tip of the pipette tip is inserted into the inlet or outlet, the tip of the pipette tip and the inlet or outlet are fitted. Thereby, the tip of the pipette tip is arranged at a fixed position with respect to the bottom surface of the liquid channel, and the amount of liquid in the liquid channel can be controlled with high accuracy.
　特許文献１に記載の検出方法では、液体流路の金属膜上に被検出物質を含む検体を提供すると、被検出物質は、捕捉体に捕捉される。この状態で、表面プラズモン共鳴が生じるように、プリズムを介して金属膜に対して入射光を照射する。そして、入射光の反射光を検出部で検出することで、被検出物質を検出している。 In the detection method described in Patent Document 1, when a specimen containing a substance to be detected is provided on a metal film in a liquid channel, the substance to be detected is captured by a capturing body. In this state, incident light is irradiated to the metal film through the prism so that surface plasmon resonance occurs. And the to-be-detected substance is detected by detecting the reflected light of incident light in a detection part.
特開２００８－２３２９５１号公報JP 2008-232951 A
　一般的に、検出チップ（反応チップ）内で２種類の液体（例えば、試薬および検体）を混合する場合、ピペットチップの先端を検出チップ内の流路またはウェルの底面近傍に配置し、ピペットチップ内への液体の吸入およびピペットチップ外への液体の排出を繰り返すことにより行う。この場合、攪拌効果を安定させる観点から、液体流路またはウェルの底面と、ピペットチップの先端との位置関係を精度よく制御する必要がある。また、検出チップ（反応チップ）内で液体の除去工程を含む複数の反応工程を行う場合、検出結果の精度を向上させるとともに反応効率を安定化させる観点から、液体の除去工程における液体流路またはウェル内の液体残量を最小限に、かつ一定にする必要がある。この場合も、ピペットチップの先端の位置を精度よく制御する必要がある。 Generally, when two kinds of liquids (for example, a reagent and a sample) are mixed in a detection chip (reaction chip), the tip of the pipette chip is arranged near the flow path in the detection chip or the bottom of the well, and the pipette chip This is done by repeatedly sucking the liquid in and discharging the liquid out of the pipette tip. In this case, from the viewpoint of stabilizing the stirring effect, it is necessary to accurately control the positional relationship between the liquid channel or the bottom of the well and the tip of the pipette tip. In addition, when performing a plurality of reaction steps including a liquid removal step in the detection chip (reaction chip), from the viewpoint of improving the accuracy of the detection result and stabilizing the reaction efficiency, the liquid flow path in the liquid removal step or The amount of liquid remaining in the well needs to be minimized and constant. Also in this case, it is necessary to control the position of the tip of the pipette tip with high accuracy.
　特許文献１に記載の検出方法では、ピペットチップの先端と注入口または排出口とを嵌合させるため、検出チップ内におけるピペットチップの先端の位置を精度よく制御することができる。しかしながら、特許文献１に記載の検出方法では、ピペットチップの先端と注入口または排出口とを嵌合させる必要があるため、ピペットチップおよび検出チップを高精度に作製する必要があり、これらの製造コストが高くなるという問題があった。また、ピペットチップを軸方向（ｚ方向）だけでなく、当該軸方向に直交する方向（ｘ方向およびｙ方向）にも高精度に移動させる必要があり、検出装置の製造コストも高くなるという問題があった。 In the detection method described in Patent Document 1, since the tip of the pipette tip and the inlet or outlet are fitted, the position of the tip of the pipette tip in the detection tip can be accurately controlled. However, in the detection method described in Patent Document 1, since it is necessary to fit the tip of the pipette tip and the inlet or outlet, it is necessary to manufacture the pipette tip and the detection tip with high accuracy. There was a problem of high costs. Further, it is necessary to move the pipette tip with high accuracy not only in the axial direction (z direction) but also in the direction orthogonal to the axial direction (x direction and y direction), and the manufacturing cost of the detection device increases. was there.
　一方、ピペットチップの先端の位置を精度よく制御するために、ピペットチップを操作する前にピペットチップの先端の位置を検出することが考えられる。このようにピペットチップの先端の位置を検出することで、ピペットチップおよび検出チップの製造コストを増大させることなく、ピペットチップの先端の位置を精度よく制御することが可能となる。 On the other hand, in order to accurately control the position of the tip of the pipette tip, it is conceivable to detect the position of the tip of the pipette tip before operating the pipette tip. By detecting the position of the tip of the pipette tip in this way, the position of the tip of the pipette tip can be accurately controlled without increasing the manufacturing costs of the pipette tip and the detection tip.
　このようなピペットチップの先端の位置情報を検出する方法として、歪みゲージやロードセルを使用することが考えられる。たとえば、歪みゲージを使用する方法では、ピペットチップを装着するピペットノズルとピペットの駆動部との間に歪みゲージを配置する。そして、ピペットチップを装着したピペットを、ピペットチップの先端の位置決め基準となる基準部に接触させたときの歪みゲージの出力に基づいて、ピペットチップの先端の位置情報を取得する。 It is conceivable to use a strain gauge or a load cell as a method for detecting the position information of the tip of such a pipette tip. For example, in a method using a strain gauge, the strain gauge is arranged between a pipette nozzle to which a pipette tip is attached and a pipette drive unit. Then, the position information of the tip of the pipette tip is acquired based on the output of the strain gauge when the pipette attached with the pipette tip is brought into contact with the reference portion that is the positioning reference of the tip of the pipette tip.
　また、ピペットチップの先端部の位置情報を取得する別の方法として、感圧センサーを用いることも考えられる。この方法では、ピペットチップの先端を感圧センサーに接触させたときの感圧センサーの出力に基づいて、ピペットチップの先端の位置情報を取得する。 Also, it is conceivable to use a pressure-sensitive sensor as another method for acquiring the position information of the tip of the pipette tip. In this method, the position information of the tip of the pipette tip is acquired based on the output of the pressure sensitive sensor when the tip of the pipette tip is brought into contact with the pressure sensitive sensor.
　ところで、微量の被検出物質を高感度かつ定量的に検出するために、また汚染防止の観点から、注入口や排出口がシールによって封止されている検出チップを使用する場合がある。この検出チップは、ピペットチップでシールを突き破って使用されることがある。 Incidentally, in order to detect a very small amount of a substance to be detected with high sensitivity and quantitatively, and from the viewpoint of preventing contamination, a detection chip in which an inlet and an outlet are sealed with a seal may be used. This detection tip may be used by piercing the seal with a pipette tip.
　しかしながら、前述した歪みゲージや感圧センサーなどを用いてピペットチップの先端の位置を検出する場合、ピペットチップの先端の位置を検出しても、シールを突き破るときにピペットチップの先端の位置がずれてしまうため、ピペットチップの先端の位置を高精度に制御することができないおそれがある。 However, when the position of the tip of the pipette tip is detected using the strain gauge or the pressure sensor described above, the position of the tip of the pipette tip is shifted when the seal is broken even if the position of the tip of the pipette tip is detected. Therefore, there is a possibility that the position of the tip of the pipette tip cannot be controlled with high accuracy.
　そこで、本発明の目的は、反応チップのシールをピペットチップにより突き破る場合であっても、ピペットチップおよび反応チップの製造コストを増大させることなく、ピペットチップの先端の位置を精度よく制御して、反応チップ内において２以上の物質を適切に反応させることができる反応方法および反応装置を提供することである。 Therefore, the object of the present invention is to accurately control the position of the tip of the pipette tip without increasing the manufacturing cost of the pipette tip and the reaction tip even when the seal of the reaction tip is broken by the pipette tip. To provide a reaction method and a reaction apparatus capable of appropriately reacting two or more substances in a reaction chip.
　上記課題を解決するため、本発明の一実施の形態に係る反応方法は、ピペットノズルに装着されたピペットチップと、凹部および前記凹部の開口を封止しているシールを有する反応チップとを使用して、前記反応チップ内において２以上の物質を反応させる反応方法であって、前記反応チップの前記シールを前記ピペットチップで突き破る第１工程と、前記第１工程の後、前記ピペットチップの先端の位置情報を取得する第２工程と、前記第２工程の後、前記ピペットチップの先端の位置情報に基づいて前記ピペットチップを操作して、前記反応チップ内において２以上の物質を反応させる第３工程と、を有する。 In order to solve the above-described problem, a reaction method according to an embodiment of the present invention uses a pipette tip attached to a pipette nozzle and a reaction tip having a recess and a seal that seals the opening of the recess. A reaction method of reacting two or more substances in the reaction chip, the first step of breaking through the seal of the reaction chip with the pipette tip, and the tip of the pipette tip after the first step A second step of acquiring the position information of the second step, and after the second step, the pipette tip is operated based on the position information of the tip of the pipette tip to react two or more substances in the reaction tip. 3 steps.
　また、上記課題を解決するため、本発明の一実施の形態に係る反応装置は、凹部および前記凹部の開口を封止しているシールを有する反応チップを使用し、ピペットチップで前記シールを突き破った後に前記反応チップ内において２以上の物質を反応させる反応装置であって、前記反応チップを保持するチップホルダーと、前記ピペットチップを着脱可能なピペットノズルを有するピペットと、前記ピペットを移動させるピペット移動部と、前記ピペットチップの先端の位置情報を取得する位置情報取得部と、を有し、前記位置情報取得部は、前記ピペット移動部により移動させられた前記ピペットチップが前記シールを１回または２回以上突き破った後に、前記ピペットチップの先端の位置情報を取得し、前記ピペット移動部は、前記位置情報取得部が前記ピペットチップの先端の位置情報を取得した後に、前記反応チップ内において２以上の物質を反応させるために前記ピペットチップの先端の位置情報に基づいて１回または２回以上前記ピペットを移動させる。 In order to solve the above problems, a reaction apparatus according to an embodiment of the present invention uses a reaction chip having a recess and a seal that seals the opening of the recess, and breaks the seal with a pipette chip. A reaction apparatus for reacting two or more substances in the reaction chip afterwards, a chip holder for holding the reaction chip, a pipette having a pipette nozzle to which the pipette chip can be attached and detached, and a pipette for moving the pipette And a position information acquisition unit that acquires position information of a tip of the pipette tip. The position information acquisition unit is configured such that the pipette tip moved by the pipette movement unit performs the sealing once. Or, after breaking through two or more times, obtain the position information of the tip of the pipette tip, the pipette moving unit After the information acquisition unit acquires the position information of the tip of the pipette tip, the pipette is used once or twice or more based on the position information of the tip of the pipette tip to react two or more substances in the reaction tip. Move.
　本発明によれば、反応チップのシールをピペットチップにより突き破る場合であっても、ピペットチップおよび反応チップの製造コストを増大させることなく、ピペットチップの先端の位置を精度よく制御して、反応チップ内において２以上の物質を適切に反応させることができる。たとえば、本発明によれば、被検出物質の存在または量を高い精度で検出することができる。 According to the present invention, even when the seal of the reaction tip is pierced by the pipette tip, the position of the tip of the pipette tip is accurately controlled without increasing the manufacturing cost of the pipette tip and the reaction tip. Two or more substances can be appropriately reacted in the inside. For example, according to the present invention, the presence or amount of a substance to be detected can be detected with high accuracy.
図１は、実施の形態１に係るＳＰＦＳ装置の構成を示す模式図である。FIG. 1 is a schematic diagram showing the configuration of the SPFS apparatus according to the first embodiment. 図２Ａ～Ｃは、検出チップの構成を示す図である。2A to 2C are diagrams showing the configuration of the detection chip. 図３は、検出チップの他の形態の断面模式図である。FIG. 3 is a schematic cross-sectional view of another form of the detection chip. 図４は、実施の形態１に係るＳＰＦＳ装置の動作を示すフローチャートである。FIG. 4 is a flowchart showing the operation of the SPFS apparatus according to the first embodiment. 図５は、ピペットチップの先端の位置情報を取得する工程の内容を示すフローチャートである。FIG. 5 is a flowchart showing the content of the step of acquiring the position information of the tip of the pipette tip. 図６Ａは、注入口を塞いでいるシールを突き破る工程を説明するための図であり、図６Ｂは、凹部を塞いでいるシールを突き破る工程を説明するための図であり、図６Ｃは、凹部から液体を吸引する工程を説明するための図である。6A is a diagram for explaining a process of breaking through a seal that closes an injection port, FIG. 6B is a diagram for explaining a process of breaking through a seal that closes a recess, and FIG. It is a figure for demonstrating the process of attracting | sucking a liquid from. 図７Ａは、実施の形態２に係るＳＰＦＳ装置の一部の構成を示す図であり、図７Ｂは、ピペットチップの先端の位置情報を取得する工程を示すフローチャートである。FIG. 7A is a diagram illustrating a partial configuration of the SPFS apparatus according to the second embodiment, and FIG. 7B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip. 図８Ａは、実施の形態３に係るＳＰＦＳ装置の一部の構成を示す図であり、図８Ｂは、ピペットチップの先端の位置情報を取得する工程を示すフローチャートである。FIG. 8A is a diagram illustrating a partial configuration of the SPFS apparatus according to the third embodiment, and FIG. 8B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip. 図９Ａは、実施の形態４に係るＳＰＦＳ装置の一部の構成を示す図であり、図９Ｂは、ピペットチップの先端の位置情報を取得する工程を示すフローチャートである。FIG. 9A is a diagram illustrating a configuration of a part of the SPFS device according to the fourth embodiment, and FIG. 9B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip. 図１０Ａは、実施の形態５に係るＳＰＦＳ装置の一部の構成を示す図であり、図１０Ｂは、ピペットチップの先端の位置情報を取得する工程を示すフローチャートである。FIG. 10A is a diagram illustrating a partial configuration of the SPFS apparatus according to the fifth embodiment, and FIG. 10B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip. 図１１Ａは、実施の形態６に係るＳＰＦＳ装置の一部の構成を示す図であり、図１１Ｂは、ピペットチップの先端の位置情報を取得する工程を示すフローチャートである。FIG. 11A is a diagram illustrating a partial configuration of the SPFS device according to the sixth embodiment, and FIG. 11B is a flowchart illustrating a process of acquiring position information of the tip of the pipette tip.
　以下、本発明の実施の形態１について、図面を参照して詳細に説明する。ここでは、本発明に係る反応装置を含み、検体に含まれる被検出物質の存在または量を検出する表面プラズモン励起増強蛍光分析装置（ＳＰＦＳ装置）について説明する。 Hereinafter, Embodiment 1 of the present invention will be described in detail with reference to the drawings. Here, a surface plasmon excitation enhanced fluorescence analyzer (SPFS apparatus) that includes the reaction apparatus according to the present invention and detects the presence or amount of a substance to be detected contained in a specimen will be described.
　［実施の形態１］
　図１は、本発明の実施の形態１に係る表面プラズモン励起増強蛍光分析装置（ＳＰＦＳ装置）１００の構成を示す模式図である。 [Embodiment 1]
FIG. 1 is a schematic diagram showing a configuration of a surface plasmon excitation enhanced fluorescence analyzer (SPFS apparatus) 100 according to Embodiment 1 of the present invention.
　図１に示されるように、ＳＰＦＳ装置（検出装置）１００は、ピペット１１１およびピペット移動部１１２を含む送液部１１０と、チップホルダー１２１を含む搬送部１２０と、位置情報取得部１３０と、光照射部１４０と、光検出部１５０と、制御部１６０とを有する。ＳＰＦＳ装置１００は、チップホルダー１２１に検出チップ（反応チップ）１０を装着した状態で使用される。そこで、検出チップ１０について先に説明し、その後にＳＰＦＳ装置１００の各構成部材について説明する。 As shown in FIG. 1, the SPFS device (detection device) 100 includes a liquid feeding unit 110 including a pipette 111 and a pipette moving unit 112, a transport unit 120 including a tip holder 121, a position information acquisition unit 130, a light The irradiation unit 140, the light detection unit 150, and the control unit 160 are included. The SPFS device 100 is used with the detection chip (reaction chip) 10 mounted on the chip holder 121. Therefore, the detection chip 10 will be described first, and then each component of the SPFS device 100 will be described.
　（検出チップの構成）
　図２は、検出チップ１０の構成を示す図である。図２Ａは、検出チップ１０の平面図であり、図２Ｂは、図２Ａに示されるＡ－Ａ線の断面図であり、図２Ｃは、図２Ａに示されるＢ－Ｂ線の断面図である。図３は、検出チップ１０の他の形態を示す断面模式図である。 (Configuration of detection chip)
FIG. 2 is a diagram illustrating a configuration of the detection chip 10. 2A is a plan view of the detection chip 10, FIG. 2B is a cross-sectional view taken along line AA shown in FIG. 2A, and FIG. 2C is a cross-sectional view taken along line BB shown in FIG. 2A. . FIG. 3 is a schematic cross-sectional view showing another form of the detection chip 10.
　図２Ａ～Ｃに示されるように、検出チップ１０は、入射面２１、成膜面２２および出射面２３を含むプリズム２０と、金属膜３０と、反応領域４１および試薬貯留領域４２を含む流路蓋４０と、シール５０とを有する。金属膜３０および流路蓋４０は、プリズム２０の成膜面２２上に配置されている。プリズム２０、金属膜３０および流路蓋４０により、液体が流れる流路６０が形成される。流路６０は、プリズム２０の成膜面２２上に直接または金属膜３０を介して配置されている。検出チップ１０は、再利用可能なチップであってもよいし、使い捨てのチップであってもよい。本実施の形態では、検出チップ１０は、使い捨てのチップである。また、流路を流れる液体の例には、被検出物質を含む検体（例えば、血液や血清、血漿、尿、鼻孔液、唾液、精液など）や、蛍光物質で標識された捕捉体を含む標識液、洗浄液などが含まれる。 As shown in FIGS. 2A to 2C, the detection chip 10 includes a prism 20 including an incident surface 21, a film formation surface 22 and an output surface 23, a metal film 30, a flow path including a reaction region 41 and a reagent storage region 42. It has a lid 40 and a seal 50. The metal film 30 and the flow path lid 40 are disposed on the film formation surface 22 of the prism 20. The prism 20, the metal film 30, and the channel lid 40 form a channel 60 through which liquid flows. The flow path 60 is disposed directly or via the metal film 30 on the film formation surface 22 of the prism 20. The detection chip 10 may be a reusable chip or a disposable chip. In the present embodiment, the detection chip 10 is a disposable chip. Examples of the liquid flowing through the flow path include a sample containing a substance to be detected (for example, blood, serum, plasma, urine, nasal fluid, saliva, semen, etc.) or a label containing a capturing substance labeled with a fluorescent substance. Liquid and cleaning liquid.
　プリズム２０は、励起光αに対して透明な絶縁体からなる。前述したように、プリズム２０は、入射面２１、成膜面２２および出射面２３を有する。入射面２１は、光照射部１４０からの励起光αをプリズム２０の内部に入射させる。成膜面２２上には、金属膜３０が配置されている。本実施の形態では、プリズム２０の内部に入射した励起光αは、被検出物質が捕捉される金属膜３０に照射される。励起光αは、金属膜３０の裏面で反射して反射光βとなる。より具体的には、励起光αは、プリズム２０と金属膜３０との界面（成膜面２２）で反射して反射光βとなる。出射面２３は、反射光βをプリズム２０の外部に出射させる。 The prism 20 is made of an insulator that is transparent to the excitation light α. As described above, the prism 20 has the entrance surface 21, the film formation surface 22, and the exit surface 23. The incident surface 21 allows the excitation light α from the light irradiation unit 140 to enter the prism 20. A metal film 30 is disposed on the film formation surface 22. In the present embodiment, the excitation light α incident on the inside of the prism 20 is applied to the metal film 30 where the substance to be detected is captured. The excitation light α is reflected on the back surface of the metal film 30 to become reflected light β. More specifically, the excitation light α is reflected at the interface (deposition surface 22) between the prism 20 and the metal film 30 to become reflected light β. The emission surface 23 emits the reflected light β to the outside of the prism 20.
　プリズム２０の形状は、特に限定されない。本実施の形態では、プリズム２０の形状は、底面が台形の柱体である。台形の一方の底辺に対応する面が成膜面２２であり、一方の脚に対応する面が入射面２１であり、他方の脚に対応する面が出射面２３である。底面となる台形は、等脚台形であることが好ましい。これにより、入射面２１と出射面２３とが対称になり、励起光αのＳ波成分がプリズム２０内に滞留しにくくなる。 The shape of the prism 20 is not particularly limited. In the present embodiment, the prism 20 is a pillar having a trapezoidal bottom surface. The surface corresponding to one base of the trapezoid is the film formation surface 22, the surface corresponding to one leg is the incident surface 21, and the surface corresponding to the other leg is the emission surface 23. The trapezoid serving as the bottom surface is preferably an isosceles trapezoid. Thereby, the entrance surface 21 and the exit surface 23 are symmetric, and the S wave component of the excitation light α is less likely to stay in the prism 20.
　入射面２１は、励起光αが光照射部１４０に戻らないように形成される。励起光αの光源がレーザーダイオード（以下「ＬＤ」ともいう）である場合、励起光αがＬＤに戻ると、ＬＤの励起状態が乱れてしまい、励起光αの波長や出力が変動してしまう。そこで、増強角を中心とする走査範囲において、励起光αが入射面２１に垂直に入射しないように、入射面２１の角度が設定される。ここで「増強角」とは、金属膜３０に対する励起光αの入射角を走査した場合に、検出チップ１０の上方に放出される励起光αと同一波長の散乱光（以下「プラズモン散乱光」という）γの光量が最大となるときの入射角を意味する。本実施の形態では、入射面２１と成膜面２２との角度および成膜面２２と出射面２３との角度は、いずれも約８０°である。 The incident surface 21 is formed so that the excitation light α does not return to the light irradiation unit 140. When the light source of the excitation light α is a laser diode (hereinafter also referred to as “LD”), when the excitation light α returns to the LD, the excitation state of the LD is disturbed, and the wavelength and output of the excitation light α change. . Therefore, the angle of the incident surface 21 is set so that the excitation light α does not enter the incident surface 21 perpendicularly in the scanning range centered on the enhancement angle. Here, the “enhancement angle” means scattered light having the same wavelength as the excitation light α emitted above the detection chip 10 when the incident angle of the excitation light α with respect to the metal film 30 is scanned (hereinafter referred to as “plasmon scattered light”). This means the angle of incidence when the amount of γ is maximized. In the present embodiment, the angle between the incident surface 21 and the film formation surface 22 and the angle between the film formation surface 22 and the emission surface 23 are both about 80 °.
　なお、検出チップ１０の設計により、増強角が概ね決まる。設計要素は、プリズム２０の屈折率や、金属膜３０の屈折率、金属膜３０の膜厚、金属膜３０の消衰係数、励起光αの波長などである。金属膜３０上に捕捉された被検出物質によって増強角がシフトするが、その量は数度未満である。 Note that the enhancement angle is generally determined by the design of the detection chip 10. The design factors are the refractive index of the prism 20, the refractive index of the metal film 30, the film thickness of the metal film 30, the extinction coefficient of the metal film 30, the wavelength of the excitation light α, and the like. The enhancement angle is shifted by the substance to be detected trapped on the metal film 30, but the amount is less than several degrees.
　一方で、プリズム２０は、複屈折特性を少なからず有する。プリズム２０の材料の例には、絶縁性の樹脂およびガラスが含まれる。プリズム２０の材料は、好ましくは、屈折率が１．４～１．６であり、かつ複屈折が小さい樹脂である。 On the other hand, the prism 20 has a considerable amount of birefringence. Examples of the material of the prism 20 include insulating resin and glass. The material of the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.
　金属膜３０は、プリズム２０の成膜面２２上の流路６０の少なくとも一部に露出するように配置されている。金属膜３０により、成膜面２２に全反射条件で入射した励起光αの光子と、金属膜３０中の自由電子との間で相互作用（ＳＰＲ）が生じ、金属膜３０の表面上に局在場光（一般に「エバネッセント光」または「近接場光」とも呼ばれる）を生じさせることができる。 The metal film 30 is disposed so as to be exposed to at least a part of the flow path 60 on the film formation surface 22 of the prism 20. The metal film 30 causes an interaction (SPR) between the photon of the excitation light α incident on the film formation surface 22 under total reflection conditions and the free electrons in the metal film 30, and is locally on the surface of the metal film 30. In-situ light (commonly referred to as “evanescent light” or “near-field light”) can be generated.
　金属膜３０の材料は、ＳＰＲを生じさせることができる金属であれば特に限定されない。金属膜３０の材料の例には、金、銀、銅、アルミ、これらの合金が含まれる。本実施の形態では、金属膜３０は、金薄膜である。金属膜３０の形成方法は、特に限定されない。金属膜３０の形成方法の例には、スパッタリング、蒸着、メッキが含まれる。金属膜３０の厚みは、特に限定されないが、３０～７０ｎｍの範囲内であることが好ましい。 The material of the metal film 30 is not particularly limited as long as it is a metal capable of generating SPR. Examples of the material of the metal film 30 include gold, silver, copper, aluminum, and alloys thereof. In the present embodiment, the metal film 30 is a gold thin film. The method for forming the metal film 30 is not particularly limited. Examples of the method for forming the metal film 30 include sputtering, vapor deposition, and plating. The thickness of the metal film 30 is not particularly limited, but is preferably in the range of 30 to 70 nm.
　また、特に図示しないが、金属膜３０の表面には、被検出物質を捕捉するための捕捉体が固定されている。金属膜３０に捕捉体を固定することで、被検出物質を選択的に検出することが可能となる。本実施の形態では、金属膜３０上の所定の領域に、捕捉体が均一に固定されている。捕捉体が固定されている領域は、後述する１次反応および２次反応が起こる反応場となる。金属膜３０に固定されている捕捉体は、流路６０内に露出している。捕捉体の種類は、被検出物質を捕捉することができれば特に限定されない。本実施の形態では、捕捉体は、被検出物質に特異的に結合可能な抗体またはその断片である。 Although not particularly illustrated, a capturing body for capturing a substance to be detected is fixed on the surface of the metal film 30. By fixing the capturing body to the metal film 30, it becomes possible to selectively detect the substance to be detected. In the present embodiment, the capturing body is uniformly fixed to a predetermined region on the metal film 30. The region where the capturing body is fixed serves as a reaction field where a primary reaction and a secondary reaction described later occur. The capturing body fixed to the metal film 30 is exposed in the flow path 60. The type of capturing body is not particularly limited as long as it can capture the substance to be detected. In the present embodiment, the capturing body is an antibody or a fragment thereof that can specifically bind to the substance to be detected.
　流路蓋４０は、成膜面２２上に配置されている。前述したように、流路蓋４０は、反応領域４１および試薬貯留領域４２を有する。反応領域４１は、後述する１次反応や２次反応を行うための領域である。また、試薬貯留領域４２は、２次反応に使用する標識液や、各反応後の洗浄に使用される洗浄液などが貯留される領域である。流路蓋４０における反応領域４１の裏面には、流路６０となる流路溝４３が形成されている。また、反応領域４１の表面と裏面とには、注入部７０となる第１貫通孔４４と、貯留部８０となる第２貫通孔４５とがそれぞれ開口している。流路溝４３の両端は、第１貫通孔４４および第２貫通孔４５にそれぞれ接続されている。試薬貯留領域４２には、表面に開口した凹部４６が形成されている。凹部４６の数は、特に限定されない。本実施の形態では、凹部４６の数は、２個である。凹部４６には、２次反応に使用する標識液や、洗浄液などが貯留されている。流路溝４３、第１貫通孔４４および第２貫通孔４５は、プリズム２０、金属膜３０および流路蓋４０をこの順に積層することでそれぞれ流路６０、注入部７０および貯留部８０となる。注入部７０、貯留部８０および４個の凹部４６の開口は、それぞれシール５０で封止されている。 The channel lid 40 is disposed on the film formation surface 22. As described above, the flow path lid 40 has the reaction region 41 and the reagent storage region 42. The reaction region 41 is a region for performing a primary reaction and a secondary reaction described later. The reagent storage area 42 is an area in which a labeling solution used for the secondary reaction, a cleaning solution used for cleaning after each reaction, and the like are stored. A channel groove 43 to be the channel 60 is formed on the back surface of the reaction region 41 in the channel lid 40. In addition, a first through hole 44 serving as the injection portion 70 and a second through hole 45 serving as the storage portion 80 are opened on the front surface and the back surface of the reaction region 41, respectively. Both ends of the flow channel 43 are connected to the first through hole 44 and the second through hole 45, respectively. In the reagent storage area 42, a recess 46 opened on the surface is formed. The number of the recessed parts 46 is not specifically limited. In the present embodiment, the number of recesses 46 is two. In the recess 46, a labeling solution used for the secondary reaction, a cleaning solution, and the like are stored. The channel groove 43, the first through hole 44, and the second through hole 45 become the channel 60, the injection unit 70, and the storage unit 80, respectively, by stacking the prism 20, the metal film 30, and the channel lid 40 in this order. . The openings of the injection part 70, the storage part 80 and the four recesses 46 are respectively sealed with seals 50.
　流路蓋４０は、金属膜３０上から放出される蛍光δおよびプラズモン散乱光γに対して透明な材料からなることが好ましい。流路蓋４０の材料の例には、樹脂が含まれる。流路蓋４０は、蛍光δおよびプラズモン散乱光γを外部に取り出す部分が蛍光δおよびプラズモン散乱光γに対して透明であれば、他の部分は不透明な材料で形成されていてもよい。流路蓋４０は、例えば、両面テープや接着剤などによる接着や、レーザー溶着、超音波溶着、クランプ部材を用いた圧着などによりプリズム２０または金属膜３０に接合されている。 The channel lid 40 is preferably made of a material that is transparent to the fluorescence δ emitted from the metal film 30 and the plasmon scattered light γ. An example of the material of the flow path lid 40 includes a resin. The flow path cover 40 may be formed of an opaque material as long as the portion from which the fluorescent δ and the plasmon scattered light γ are extracted is transparent to the fluorescent δ and the plasmon scattered light γ. The channel lid 40 is bonded to the prism 20 or the metal film 30 by, for example, adhesion using a double-sided tape or an adhesive, laser welding, ultrasonic welding, or pressure bonding using a clamp member.
　シール５０は、注入部７０、貯留部８０および４個の凹部４６の全ての開口を塞いでいる。シール５０の材料は、注入部７０、貯留部８０および凹部４６の開口をそれぞれ封止し、かつ注入部７０および凹部４６の開口をそれぞれ封止するシール５０についてはピペットチップ１７０で突き破ることができれば特に限定されない。シール５０の材料の例には、アルミニウム箔、アルミ蒸着フィルム、低密度ポリエチレン、ポリウレタン等のエラストマー材料などが含まれる。また、シール５０は、複数の材料が積層された積層シールであってもよい。本実施の形態では、シール５０の材料は低密度ポリエチレンである。また、シール５０は、ピペットチップ１７０で突き破るために必要な力が１０Ｎ以下となるように構成されることが好ましい。ピペットチップ１７０で突き破るために必要な力は、シール５０の材料、凹部４６、注入部７０の開口、貯留部８０の開口の面積、ピペットチップ１７０の先端の形状などによって変化する。ピペットチップ１７０で突き破るために必要な力は、プッシュプルゲージなどを使用して測定することができる。たとえば、シール５０の材料が低密度ポリエチレンであり、凹部４６の開口の面積が１２ｍｍ２であり、ピペットチップ１７０の先端の外径が１ｍｍ（面取り加工なし）である場合、ピペットチップ１７０で突き破るために必要な力は、７Ｎ程度である。なお、注入部７０、貯留部８０および凹部４６の開口のうち、いずれかの開口は、シール５０によって塞がれていなくてもよい。 The seal 50 closes all the openings of the injection part 70, the storage part 80, and the four recesses 46. The material of the seal 50 can seal the opening of the injection part 70, the storage part 80, and the recess 46, respectively, and the seal 50 that seals the opening of the injection part 70 and the recess 46 can be pierced by the pipette tip 170. There is no particular limitation. Examples of the material of the seal 50 include an aluminum foil, an aluminum deposited film, low-density polyethylene, an elastomer material such as polyurethane, and the like. The seal 50 may be a laminated seal in which a plurality of materials are laminated. In the present embodiment, the material of the seal 50 is low density polyethylene. Moreover, it is preferable that the seal 50 is configured so that a force necessary for breaking through with the pipette tip 170 is 10 N or less. The force required to pierce with the pipette tip 170 varies depending on the material of the seal 50, the recess 46, the opening of the injection portion 70, the area of the opening of the storage portion 80, the shape of the tip of the pipette tip 170, and the like. The force required to pierce with the pipette tip 170 can be measured using a push-pull gauge or the like. For example, when the material of the seal 50 is low density polyethylene, the opening area of the recess 46 is 12 mm 2 , and the outer diameter of the tip of the pipette tip 170 is 1 mm (without chamfering), the pipette tip 170 breaks through. The required force is about 7N. Note that any one of the openings of the injection unit 70, the storage unit 80, and the recess 46 may not be blocked by the seal 50.
　なお、図３に示されるように、検出チップ１０’は、流路６０に代えてウェル６０’を有していてもよい。この検出チップ１０’では、ウェル６０’の開口がシール５０によって封止されている。また、ウェル６０’の開口から液体を注入したり、除去したりする。 In addition, as shown in FIG. 3, the detection chip 10 ′ may have a well 60 ′ instead of the flow path 60. In this detection chip 10 ′, the opening of the well 60 ′ is sealed with a seal 50. Also, liquid is injected or removed from the opening of the well 60 '.
　図１に示されるように、励起光αは、入射面２１でプリズム２０内に入射する。プリズム２０内に入射した励起光αは、金属膜３０に全反射角度（ＳＰＲが生じる角度）で照射される。このように金属膜３０に対して励起光αをＳＰＲが生じる角度で照射することで、金属膜３０上に局在場光を発生させることができる。この局在場光により、金属膜３０上に存在する被検出物質を標識する蛍光物質が励起され、蛍光δが放出される。ＳＰＦＳ装置１００は、蛍光物質から放出された蛍光δの光量を測定することで、被検出物質の存在または量を検出する。 As shown in FIG. 1, the excitation light α enters the prism 20 at the incident surface 21. The excitation light α that has entered the prism 20 is applied to the metal film 30 at a total reflection angle (an angle at which SPR occurs). By irradiating the metal film 30 with the excitation light α at an angle at which SPR occurs in this way, localized field light can be generated on the metal film 30. This localized field light excites a fluorescent substance that labels the substance to be detected present on the metal film 30 and emits fluorescence δ. The SPFS device 100 detects the presence or amount of the substance to be detected by measuring the amount of fluorescence δ emitted from the fluorescent substance.
（ＳＰＦＳ装置の構成）
　次に、本実施の形態に係るＳＰＦＳ装置１００の各構成部材について説明する。前述のとおり、ＳＰＦＳ装置１００は、送液部１１０、搬送部１２０、位置情報取得部１３０、光照射部１４０、光検出部１５０および制御部１６０を有する。検出チップ１０は、搬送部１２０のチップホルダー１２１に保持されうる。 (Configuration of SPFS device)
Next, each component of the SPFS device 100 according to the present embodiment will be described. As described above, the SPFS device 100 includes the liquid feeding unit 110, the transport unit 120, the position information acquisition unit 130, the light irradiation unit 140, the light detection unit 150, and the control unit 160. The detection chip 10 can be held by the chip holder 121 of the transport unit 120.
　送液部１１０は、ピペット１１１、ピペット移動部１１２および送液ポンプ駆動機構１１３を有する。送液部１１０は、チップホルダー１２１に保持された検出チップ１０の流路６０内に検体を注入したり、検出チップ１０の試薬貯留領域４２に貯留された標識液や洗浄液などの液体を反応領域４１の流路６０内に移動させたりする。また、送液部１１０は、流路６０から液体を排出したり、流路６０内の液体を攪拌したりもする。送液部１１０は、ピペット１１１のピペットノズル１１６にピペットチップ１７０を装着した状態で使用される。なお、不純物の混入などを防止する観点から、ピペットチップ１７０は、交換可能であることが好ましい。 The liquid feeding unit 110 includes a pipette 111, a pipette moving unit 112, and a liquid feeding pump drive mechanism 113. The liquid feeding unit 110 injects a specimen into the flow path 60 of the detection chip 10 held by the chip holder 121, or a liquid such as a labeling solution or a cleaning liquid stored in the reagent storage region 42 of the detection chip 10 in the reaction region. 41 in the channel 60. Further, the liquid feeding unit 110 also discharges the liquid from the flow path 60 and stirs the liquid in the flow path 60. The liquid feeding unit 110 is used in a state where the pipette tip 170 is attached to the pipette nozzle 116 of the pipette 111. It should be noted that the pipette tip 170 is preferably replaceable from the viewpoint of preventing contamination of impurities.
　ピペット１１１は、流路６０に液体を注入したり、流路６０から液体を除去したりする際に液体を吸入する。ピペット１１１は、シリンジ１１４と、シリンジ１１４内を往復動作可能なプランジャー１１５と、シリンジ１１４に接続されたピペットノズル１１６とを有する。また、ピペット１１１は、プランジャー１１５の往復運動によって、液体の吸入および排出を定量的に行うことができる。これによりピペット１１１は、流路６０に液体を注入したり、流路６０から液体を除去したりすることができる。また、ピペット１１１は、液体の吸引および排出を繰り返すことで、流路６０内の液体を攪拌することができる。 The pipette 111 sucks the liquid when injecting the liquid into the flow path 60 or removing the liquid from the flow path 60. The pipette 111 includes a syringe 114, a plunger 115 that can reciprocate inside the syringe 114, and a pipette nozzle 116 connected to the syringe 114. Further, the pipette 111 can quantitatively suck and discharge the liquid by the reciprocating motion of the plunger 115. Thereby, the pipette 111 can inject liquid into the flow path 60 or remove the liquid from the flow path 60. Moreover, the pipette 111 can stir the liquid in the flow path 60 by repeating suction and discharge of the liquid.
　ピペット移動部１１２は、ピペットチップ１７０内への液体の吸入、ピペットチップ１７０内からの液体の排出、またはシール５０を突き破るために、ピペットノズル１１６を移動させる。ピペット移動部１１２は、例えば、ピペットノズル１１６をピペットノズル１１６の軸方向（例えば垂直方向）に自在に動かす。ピペット移動部１１２は、例えば、ソレノイドアクチュエーターおよびステッピングモーターを含む。 The pipette moving unit 112 moves the pipette nozzle 116 in order to suck the liquid into the pipette tip 170, discharge the liquid from the pipette tip 170, or break the seal 50. For example, the pipette moving unit 112 freely moves the pipette nozzle 116 in the axial direction (for example, the vertical direction) of the pipette nozzle 116. The pipette moving unit 112 includes, for example, a solenoid actuator and a stepping motor.
　ピペットチップ１７０でシール５０を突き破る場合、ピペット移動部１１２は、搬送部１２０によって検出チップ１０の突き破るべきシール５０をピペットノズル１１６の直下に移動させた後、ピペットノズル１１６（ピペットチップ１７０）をシール５０に向かって移動させて、ピペットチップ１７０でシール５０を突き破る。このとき、シール５０と流路６０の底面との間の距離（図６Ａ；ｄ１参照）、シール５０と凹部４６の底面との間の距離（図６Ｂ；ｄ３参照）および凹部４６に貯留された液体の深さ（図６Ｂ；ｄ４参照）は、予め記録されているため、ピペットチップ１７０の先端が、流路６０内の金属膜３０や、凹部４６に貯留された液体に接触することがない。 When the pipette tip 170 breaks through the seal 50, the pipette moving unit 112 moves the seal 50 to be broken through the detection chip 10 to the position directly below the pipette nozzle 116 by the transport unit 120, and then seals the pipette nozzle 116 (pipette tip 170). The seal 50 is broken with the pipette tip 170. At this time, the distance between the seal 50 and the bottom surface of the flow path 60 (see FIG. 6A; d1), the distance between the seal 50 and the bottom surface of the recess 46 (see FIG. 6B; d3), and the recess 46 are stored. Since the depth of the liquid (see FIG. 6B; d4) is recorded in advance, the tip of the pipette tip 170 does not come into contact with the metal film 30 in the flow path 60 or the liquid stored in the recess 46. .
　送液ポンプ駆動機構１１３は、プランジャー１１５を移動させて、外部の液体をピペットチップ１７０内に吸入させたり、ピペット１１１内の液体を外部に排出させたりする。送液ポンプ駆動機構１１３は、ステッピングモーターなどのプランジャー１１５を往復運動させるための装置を含む。ステッピングモーターは、ピペット１１１の送液量や送液速度を管理できるため、検出チップ１０の残液量を管理する観点から好ましい。なお、送液ポンプ駆動機構１１３は、ピペットチップ１７０がシール５０を突き破る工程中には駆動しないことが好ましい。 The liquid feed pump drive mechanism 113 moves the plunger 115 to suck the external liquid into the pipette tip 170 or discharge the liquid inside the pipette 111 to the outside. The liquid feed pump drive mechanism 113 includes a device for reciprocating the plunger 115 such as a stepping motor. The stepping motor is preferable from the viewpoint of managing the remaining liquid amount of the detection chip 10 because it can manage the liquid feeding amount and the liquid feeding speed of the pipette 111. The liquid feed pump drive mechanism 113 is preferably not driven during the process in which the pipette tip 170 breaks through the seal 50.
　前述のとおり、送液部１１０は、凹部４６より各種液体を吸引し、検出チップ１０の流路６０内に注入する。このとき、ピペットチップ１７０の先端が流路６０内において流路の底面と近接した状態で、シリンジ１１４に対するプランジャー１１５の往復動作を繰り返すことで、検出チップ１０中の流路６０内を液体が往復し、流路６０内の液体が攪拌される。これにより、液体の濃度の均一化や、流路６０内における反応（例えば、１次反応および２次反応）の促進などを実現することができる。 As described above, the liquid feeding unit 110 sucks various liquids from the recess 46 and injects them into the flow channel 60 of the detection chip 10. At this time, in the state where the tip of the pipette tip 170 is close to the bottom surface of the flow channel in the flow channel 60, the reciprocating operation of the plunger 115 with respect to the syringe 114 is repeated, so that the liquid is flown in the flow channel 60 in the detection chip 10. It reciprocates and the liquid in the flow path 60 is stirred. As a result, it is possible to achieve a uniform concentration of the liquid and promotion of reactions (for example, primary reaction and secondary reaction) in the flow channel 60.
　流路６０内の液体は、再びピペット１１１で吸引され、図外の廃液タンクなどに排出される。これらの動作の繰り返しにより、各種液体による反応や洗浄などを実施し、流路６０内の反応場に、蛍光物質で標識された被検出物質を配置することができる。 The liquid in the channel 60 is again sucked by the pipette 111 and discharged to a waste liquid tank or the like not shown. By repeating these operations, reaction with various liquids, washing, and the like can be performed, and a detection target substance labeled with a fluorescent substance can be arranged in the reaction field in the flow path 60.
　搬送部１２０は、検出チップ１０を検出位置または送液位置に搬送するとともに、検出チップ１０を保持する。ここで「検出位置」とは、光照射部１４０が検出チップ１０に励起光αを照射し、それに伴い発生する蛍光δまたはプラズモン散乱光γを光検出部１５０が検出する位置である。また、「送液位置」とは、送液部１１０が検出チップ１０の流路６０内に液体を注入するか、または検出チップ１０の流路６０内の液体を除去する位置である。搬送部１２０は、チップホルダー１２１および搬送ステージ１２２を含む。 The transport unit 120 transports the detection chip 10 to the detection position or the liquid feeding position, and holds the detection chip 10. Here, the “detection position” is a position where the light irradiation unit 140 irradiates the detection chip 10 with the excitation light α, and the light detection unit 150 detects the fluorescence δ or the plasmon scattered light γ generated accordingly. The “liquid feeding position” is a position where the liquid feeding unit 110 injects liquid into the flow channel 60 of the detection chip 10 or removes the liquid in the flow channel 60 of the detection chip 10. The transport unit 120 includes a chip holder 121 and a transport stage 122.
　チップホルダー１２１は、搬送ステージ１２２に固定されており、検出チップ１０を着脱可能に保持する。チップホルダー１２１の形状は、検出チップ１０を保持することが可能であり、かつ励起光α、蛍光δおよびプラズモン散乱光γの光路を妨げなければ特に限定されない。本実施の形態では、チップホルダー１２１の形状は、流路蓋４０を挟み込んで検出チップ１０を保持できるように構成されている。 The chip holder 121 is fixed to the transfer stage 122 and holds the detection chip 10 in a detachable manner. The shape of the chip holder 121 is not particularly limited as long as it can hold the detection chip 10 and does not interfere with the optical paths of the excitation light α, fluorescence δ, and plasmon scattered light γ. In the present embodiment, the shape of the chip holder 121 is configured so that the detection chip 10 can be held with the flow path lid 40 interposed therebetween.
　搬送ステージ１２２は、チップホルダー１２１を一方向およびその逆方向（図１の紙面における左右方向）に移動させる。搬送ステージ１２２も、励起光α、蛍光δおよびプラズモン散乱光γの光路を妨げない形状である。搬送ステージ１２２は、例えば、ステッピングモーターなどで駆動される。 The transfer stage 122 moves the chip holder 121 in one direction and in the opposite direction (left and right direction on the paper surface of FIG. 1). The transport stage 122 also has a shape that does not interfere with the optical paths of the excitation light α, fluorescence δ, and plasmon scattered light γ. The transfer stage 122 is driven by, for example, a stepping motor.
　位置情報取得部１３０は、ピペットチップ１７０の先端の位置情報を取得する。位置情報取得部１３０の構成は、ピペットチップ１７０の先端の位置情報を取得することができれば特に限定されない。位置情報取得部１３０の例には、歪みセンサーや、空気圧センサー１３１や、静電容量センサーや、電源および電流計や、感圧センサーなどが含まれる。本実施の形態では、位置情報取得部１３０は、空気圧センサー１３１を含んでいる。空気圧センサー１３１は、ピペットノズル１１６とシリンジ１１４との間に接続されている。 The position information acquisition unit 130 acquires position information on the tip of the pipette tip 170. The configuration of the position information acquisition unit 130 is not particularly limited as long as the position information of the tip of the pipette tip 170 can be acquired. Examples of the position information acquisition unit 130 include a strain sensor, a pneumatic sensor 131, a capacitance sensor, a power supply and ammeter, a pressure sensor, and the like. In the present embodiment, the position information acquisition unit 130 includes an air pressure sensor 131. The air pressure sensor 131 is connected between the pipette nozzle 116 and the syringe 114.
　本実施の形態では、ピペットチップ１７０の先端の位置情報は、ピペットチップ１７０の先端と基準部１８０（図６Ａ参照）との間隔を変えつつ、ピペットチップ１７０の先端から気体を吸引または排出したときのピペットチップ１７０内の空気圧の変化を空気圧センサー１３１により測定することで取得される。ここで「基準部」とは、ピペットチップ１７０の先端の基準位置となるものを意味する。基準部１８０は、その位置が高精度に特定されていれば特に限定されず、検出チップ１０の一部であってもよいし、ＳＰＦＳ装置１００の一部であってもよい。検出チップ１０に含まれる基準部１８０の例には、流路蓋４０やシール５０、プリズム２０などが含まれる。一方、ＳＰＦＳ装置１００に含まれる基準部１８０の例には、搬送ステージ１２２や、チップホルダー１２１、搬送部１２０において搬送ステージ１２２を支持する基台（ピペットノズル１１６の下方に位置する部分）などであってもよい。ピペットチップ１７０の先端における気体の吸引または排出は、連続的に行われてもよいし、間欠的に行われてもよい。 In the present embodiment, the position information of the tip of the pipette tip 170 is obtained when gas is sucked or discharged from the tip of the pipette tip 170 while changing the distance between the tip of the pipette tip 170 and the reference portion 180 (see FIG. 6A). The change in the air pressure in the pipette tip 170 is measured by the air pressure sensor 131. Here, the “reference portion” means a reference position at the tip of the pipette tip 170. The reference unit 180 is not particularly limited as long as its position is specified with high accuracy, and may be a part of the detection chip 10 or a part of the SPFS device 100. Examples of the reference unit 180 included in the detection chip 10 include the flow path lid 40, the seal 50, the prism 20, and the like. On the other hand, examples of the reference unit 180 included in the SPFS apparatus 100 include a transport stage 122, a chip holder 121, and a base that supports the transport stage 122 in the transport unit 120 (portion located below the pipette nozzle 116). There may be. The suction or discharge of gas at the tip of the pipette tip 170 may be performed continuously or intermittently.
　光照射部１４０は、チップホルダー１２１に保持された検出チップ１０の入射面２１に向かって励起光αを照射する。蛍光δまたはプラズモン散乱光γの測定時には、光照射部１４０は、金属膜３０に対する入射角がＳＰＲを生じさせる角度となるように、金属膜３０に対するＰ波のみを入射面２１に向けて出射する。ここで「励起光」とは、蛍光物質を直接または間接的に励起させる光である。たとえば、励起光αは、プリズム２０を介して金属膜３０にＳＰＲが生じる角度で照射されたときに、蛍光物質を励起させる局在場光を金属膜３０の表面上に生じさせる光である。光照射部１４０は、光源ユニット１４１、角度調整機構１４２および光源制御部１４３を含む。 The light irradiation unit 140 irradiates the excitation light α toward the incident surface 21 of the detection chip 10 held by the chip holder 121. At the time of measuring the fluorescence δ or the plasmon scattered light γ, the light irradiation unit 140 emits only the P wave toward the incident surface 21 so that the incident angle with respect to the metal film 30 is an angle that causes SPR. . Here, the “excitation light” is light that directly or indirectly excites the fluorescent material. For example, the excitation light α is light that generates localized field light on the surface of the metal film 30 that excites the fluorescent material when the metal film 30 is irradiated through the prism 20 at an angle at which SPR occurs. The light irradiation unit 140 includes a light source unit 141, an angle adjustment mechanism 142, and a light source control unit 143.
　光源ユニット１４１は、コリメートされ、かつ波長および光量が一定の励起光αを、金属膜３０の裏面における照射スポットの形状が略円形となるように出射する。光源ユニット１４１は、例えば、励起光αの光源、ビーム整形光学系、ＡＰＣ機構および温度調整機構（いずれも不図示）を含む。 The light source unit 141 emits the collimated excitation light α having a constant wavelength and light amount so that the shape of the irradiation spot on the back surface of the metal film 30 is substantially circular. The light source unit 141 includes, for example, a light source of excitation light α, a beam shaping optical system, an APC mechanism, and a temperature adjustment mechanism (all not shown).
　光源の種類は、特に限定されず、例えばレーザーダイオード（ＬＤ）である。光源の他の例には、発光ダイオード、水銀灯、その他のレーザー光源が含まれる。光源から出射される光がビームでない場合は、光源から出射される光は、レンズや鏡、スリットなどによりビームに変換される。また、光源から出射される光が単色光でない場合は、光源から出射される光は、回折格子などにより単色光に変換される。さらに、光源から出射される光が直線偏光でない場合は、光源から出射される光は、偏光子などにより直線偏光の光に変換される。 The type of the light source is not particularly limited, and is, for example, a laser diode (LD). Other examples of light sources include light emitting diodes, mercury lamps, and other laser light sources. When the light emitted from the light source is not a beam, the light emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like. When the light emitted from the light source is not monochromatic light, the light emitted from the light source is converted into monochromatic light by a diffraction grating or the like. Furthermore, when the light emitted from the light source is not linearly polarized light, the light emitted from the light source is converted into linearly polarized light by a polarizer or the like.
　ビーム整形光学系は、例えば、コリメーターやバンドパスフィルター、直線偏光フィルター、半波長板、スリット、ズーム手段などを含む。ビーム整形光学系は、これらのすべてを含んでいてもよいし、一部を含んでいてもよい。コリメーターは、光源から出射された励起光αをコリメートする。バンドパスフィルターは、光源から出射された励起光αを中心波長のみの狭帯域光にする。光源からの励起光αは、若干の波長分布幅を有しているためである。直線偏光フィルターは、光源から出射された励起光αを完全な直線偏光の光にする。半波長板は、金属膜３０にＰ波成分が入射するように励起光αの偏光方向を調整する。スリットおよびズーム手段は、金属膜３０の裏面における照射スポットの形状が所定サイズの円形となるように、励起光αのビーム径や輪郭形状などを調整する。ＡＰＣ機構は、光源の出力が一定となるように光源を制御する。より具体的には、ＡＰＣ機構は、励起光αから分岐させた光の光量を不図示のフォトダイオードなどで検出する。そして、ＡＰＣ機構は、回帰回路で投入エネルギーを制御することで、光源の出力を一定に制御する。 The beam shaping optical system includes, for example, a collimator, a band pass filter, a linear polarization filter, a half-wave plate, a slit, and a zoom means. The beam shaping optical system may include all of these or a part thereof. The collimator collimates the excitation light α emitted from the light source. The band-pass filter turns the excitation light α emitted from the light source into narrowband light having only the center wavelength. This is because the excitation light α from the light source has a slight wavelength distribution width. The linear polarization filter turns the excitation light α emitted from the light source into completely linearly polarized light. The half-wave plate adjusts the polarization direction of the excitation light α so that the P-wave component is incident on the metal film 30. The slit and zoom means adjust the beam diameter, contour shape, and the like of the excitation light α so that the shape of the irradiation spot on the back surface of the metal film 30 is a circle of a predetermined size. The APC mechanism controls the light source so that the output of the light source is constant. More specifically, the APC mechanism detects the amount of light branched from the excitation light α with a photodiode (not shown) or the like. The APC mechanism controls the input energy by a regression circuit, thereby controlling the output of the light source to be constant.
　角度調整機構１４２は、金属膜３０（プリズム２０と金属膜３０との界面（成膜面２２））に対する励起光αの入射角を調整する。角度調整機構１４２は、プリズム２０を介して金属膜３０の所定の位置に向けて所定の入射角で励起光αを照射するために、励起光αの光軸とチップホルダー１２１とを相対的に回転させる。 The angle adjustment mechanism 142 adjusts the incident angle of the excitation light α with respect to the metal film 30 (the interface between the prism 20 and the metal film 30 (film formation surface 22)). In order to irradiate the excitation light α at a predetermined incident angle toward a predetermined position of the metal film 30 via the prism 20, the angle adjustment mechanism 142 relatively places the optical axis of the excitation light α and the chip holder 121. Rotate.
　たとえば、角度調整機構１４２は、光源ユニット１４１を励起光αの光軸と直交する軸（図１の紙面に対して垂直な軸）を中心として回動させる。このとき、入射角を走査しても金属膜３０上での照射スポットの位置がほとんど変化しないように、回転軸の位置を設定する。回転中心の位置を、入射角の走査範囲の両端における２つの励起光αの光軸の交点近傍（成膜面２２上の照射位置と入射面２１との間）に設定することで、照射位置のズレを極小化できる。 For example, the angle adjusting mechanism 142 rotates the light source unit 141 around an axis (axis perpendicular to the paper surface of FIG. 1) orthogonal to the optical axis of the excitation light α. At this time, the position of the rotation axis is set so that the position of the irradiation spot on the metal film 30 hardly changes even when the incident angle is scanned. By setting the position of the center of rotation near the intersection of the optical axes of the two excitation lights α at both ends of the scanning range of the incident angle (between the irradiation position on the film forming surface 22 and the incident surface 21), the irradiation position Can be minimized.
　前述のとおり、金属膜３０に対する励起光αの入射角のうち、プラズモン散乱光γの光量が最大となる角度が増強角である。励起光αの入射角を増強角またはその近傍の角度に設定することで、高強度の蛍光δを測定することが可能となる。検出チップ１０のプリズム２０の材料および形状、金属膜３０の膜厚、流路６０内の液体の屈折率などにより、励起光αの基本的な入射条件が決まるが、流路６０内の蛍光物質の種類および量、プリズム２０の形状誤差などにより、最適な入射条件はわずかに変動する。このため、測定ごとに最適な増強角を求めることが好ましい。 As described above, of the incident angles of the excitation light α to the metal film 30, the angle at which the amount of plasmon scattered light γ is maximum is the enhancement angle. High intensity fluorescence δ can be measured by setting the incident angle of the excitation light α to the enhancement angle or an angle in the vicinity thereof. The basic incident condition of the excitation light α is determined by the material and shape of the prism 20 of the detection chip 10, the film thickness of the metal film 30, the refractive index of the liquid in the flow channel 60, etc. The optimum incident condition varies slightly depending on the type and amount of the light and the shape error of the prism 20. For this reason, it is preferable to obtain an optimal enhancement angle for each measurement.
　光源制御部１４３は、光源ユニット１４１に含まれる各種機器を制御して、光源ユニット１４１からの励起光αの出射を制御する。光源制御部１４３は、例えば、演算装置、制御装置、記憶装置、入力装置および出力装置を含む公知のコンピュータやマイコンなどによって構成される。 The light source control unit 143 controls various devices included in the light source unit 141 to control the emission of the excitation light α from the light source unit 141. The light source control unit 143 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
　光検出部１５０は、光照射部１４０が検出チップ１０の金属膜３０に励起光αを照射したときに、金属膜３０の流路６０側の面の近傍から放出される蛍光δの光量を検出する。また、必要に応じて、光検出部１５０は、金属膜３０への励起光αの照射によって生じたプラズモン散乱光γも検出する。光検出部１５０は、受光ユニット１５１、位置切り替え機構１５２およびセンサー制御部１５３を含む。 The light detection unit 150 detects the amount of fluorescence δ emitted from the vicinity of the surface of the metal film 30 on the flow path 60 side when the light irradiation unit 140 irradiates the metal film 30 of the detection chip 10 with the excitation light α. To do. Further, as necessary, the light detection unit 150 also detects plasmon scattered light γ generated by irradiation of the excitation light α to the metal film 30. The light detection unit 150 includes a light receiving unit 151, a position switching mechanism 152, and a sensor control unit 153.
　受光ユニット１５１は、検出チップ１０の金属膜３０の表面に対する法線方向に配置される。受光ユニット１５１は、第１レンズ１５４、光学フィルター１５５、第２レンズ１５６および受光センサー１５７を含む。 The light receiving unit 151 is disposed in the normal direction to the surface of the metal film 30 of the detection chip 10. The light receiving unit 151 includes a first lens 154, an optical filter 155, a second lens 156, and a light receiving sensor 157.
　第１レンズ１５４は、例えば、集光レンズであり、金属膜３０上から出射される光を集光する。第２レンズ１５６は、例えば、結像レンズであり、第１レンズ１５４で集光された光を受光センサー１５７の受光面に結像させる。第１レンズ１５４および第２レンズ１５６の間の光路は、略平行になっている。 The first lens 154 is, for example, a condensing lens, and condenses light emitted from the metal film 30. The second lens 156 is an imaging lens, for example, and forms an image of the light collected by the first lens 154 on the light receiving surface of the light receiving sensor 157. The optical path between the first lens 154 and the second lens 156 is substantially parallel.
　光学フィルター１５５は、第１レンズ１５４および第２レンズ１５６の間に配置されている。光学フィルター１５５は、蛍光成分のみを受光センサー１５７に導き、高いＳ／Ｎ比で蛍光δを検出するために、励起光成分（プラズモン散乱光γ）を除去する。光学フィルター１５５の例には、励起光反射フィルター、短波長カットフィルターおよびバンドパスフィルターが含まれる。光学フィルター１５５は、例えば、所定の光成分を反射する多層膜を含むフィルター、または所定の光成分を吸収する色ガラスフィルターである。 The optical filter 155 is disposed between the first lens 154 and the second lens 156. The optical filter 155 guides only the fluorescence component to the light receiving sensor 157 and removes the excitation light component (plasmon scattered light γ) in order to detect the fluorescence δ with a high S / N ratio. Examples of the optical filter 155 include an excitation light reflection filter, a short wavelength cut filter, and a band pass filter. The optical filter 155 is, for example, a filter including a multilayer film that reflects a predetermined light component, or a color glass filter that absorbs a predetermined light component.
　受光センサー１５７は、蛍光δおよびプラズモン散乱光γを検出する。受光センサー１５７は、微量の被検出物質からの微弱な蛍光δを検出することが可能な高い感度を有する。受光センサー１５７は、例えば、光電子増倍管（ＰＭＴ）やアバランシェフォトダイオード（ＡＰＤ）などである。 The light receiving sensor 157 detects fluorescence δ and plasmon scattered light γ. The light receiving sensor 157 has a high sensitivity capable of detecting weak fluorescence δ from a very small amount of a substance to be detected. The light receiving sensor 157 is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD).
　位置切り替え機構１５２は、光学フィルター１５５の位置を、受光ユニット１５１における光路上または光路外に切り替える。具体的には、受光センサー１５７が蛍光δを検出する時には、光学フィルター１５５を受光ユニット１５１の光路上に配置し、受光センサー１５７がプラズモン散乱光γを検出する時には、光学フィルター１５５を受光ユニット１５１の光路外に配置する。 The position switching mechanism 152 switches the position of the optical filter 155 on or off the optical path in the light receiving unit 151. Specifically, when the light receiving sensor 157 detects the fluorescence δ, the optical filter 155 is disposed on the optical path of the light receiving unit 151, and when the light receiving sensor 157 detects the plasmon scattered light γ, the optical filter 155 is placed on the light receiving unit 151. Placed outside the optical path.
　センサー制御部１５３は、受光センサー１５７の出力値の検出や、検出した出力値による受光センサー１５７の感度の管理、適切な出力値を得るための受光センサー１５７の感度の変更、などを制御する。センサー制御部１５３は、例えば、演算装置、制御装置、記憶装置、入力装置および出力装置を含む公知のコンピュータやマイコンなどによって構成される。 The sensor control unit 153 controls detection of an output value of the light receiving sensor 157, management of sensitivity of the light receiving sensor 157 based on the detected output value, change of sensitivity of the light receiving sensor 157 for obtaining an appropriate output value, and the like. The sensor control unit 153 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
　制御部１６０は、送液ポンプ駆動機構１１３、搬送ステージ１２２、角度調整機構１４２、光源制御部１４３、位置切り替え機構１５２、およびセンサー制御部１５３を制御する。制御部１６０は、例えば、演算装置、制御装置、記憶装置、入力装置および出力装置を含む公知のコンピュータやマイコンなどによって構成される。 The controller 160 controls the liquid feed pump drive mechanism 113, the transport stage 122, the angle adjustment mechanism 142, the light source controller 143, the position switching mechanism 152, and the sensor controller 153. The control unit 160 includes, for example, a known computer or microcomputer including an arithmetic device, a control device, a storage device, an input device, and an output device.
　（検出装置の検出動作）
　次に、実施の形態１に係る反応方法を含むＳＰＦＳ装置１００の検出動作について説明する。図４は、ＳＰＦＳ装置１００の動作手順の一例を示すフローチャートである。図５は、ピペットチップ１７０の先端の位置情報を取得する工程（図４における工程Ｓ１２０）の内容を示すフローチャートである。図６Ａは、注入部７０を塞いでいるシール５０を突き破る工程を説明するための図であり、図６Ｂは、凹部４６を塞いでいるシール５０を突き破る工程を説明するための図であり、図６Ｃは、凹部４６から液体を吸引する工程を説明するための図である。この例では、捕捉体として１次抗体が金属膜３０上に固定化されている。また、蛍光標識に使用する捕捉体として、蛍光物質で標識された２次抗体を使用している。また、注入部７０、貯留部８０、凹部４６は、それぞれシール５０によって塞がれている。さらに、基準部１８０は、流路６０の底面とした。 (Detection operation of the detection device)
Next, the detection operation of the SPFS device 100 including the reaction method according to Embodiment 1 will be described. FIG. 4 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 100. FIG. 5 is a flowchart showing the contents of the step (step S120 in FIG. 4) of acquiring the position information of the tip of the pipette tip 170. 6A is a diagram for explaining a process of breaking through the seal 50 blocking the injection portion 70, and FIG. 6B is a diagram for explaining a process of breaking through the seal 50 closing the recess 46. 6C is a diagram for explaining a step of sucking the liquid from the recess 46. FIG. In this example, the primary antibody is immobilized on the metal film 30 as a capturing body. Further, a secondary antibody labeled with a fluorescent substance is used as a capturing body used for fluorescent labeling. In addition, the injection part 70, the storage part 80, and the concave part 46 are each closed by a seal 50. Further, the reference portion 180 is the bottom surface of the flow path 60.
　まず、測定の準備をする（工程Ｓ１１０）。具体的には、検出チップ１０を準備して、検出チップ１０のセット位置においてチップホルダー１２１に検出チップ１０を設置する。また、ピペットノズル１１６の先端部にピペットチップ１７０を装着する。 First, preparation for measurement is performed (step S110). Specifically, the detection chip 10 is prepared, and the detection chip 10 is installed in the chip holder 121 at the set position of the detection chip 10. A pipette tip 170 is attached to the tip of the pipette nozzle 116.
　次いで、ピペットチップ１７０の先端の位置情報を取得する（工程Ｓ１２０）。まず、ピペットチップ１７０が突き破るべき複数のシール５０のうち、突き破るために必要な力が最大であるシール５０をピペットチップ１７０で突き破る（工程Ｓ１２１）。具体的には、制御部１６０は、搬送ステージ１２２を操作して、検出チップ１０を送液位置に移動させる。そして、制御部１６０は、ピペット移動部１１２を駆動して、ピペットチップ１７０が突き破るべき複数のシール５０のうち、突き破るために必要な力が最大であるシール５０をピペットチップ１７０で突き破る。なお、本実施の形態では、ピペットチップ１７０が突き破るべき複数のシール５０のうち、突き破るために必要な力が最大であるシール５０は、注入部７０の開口を塞いでいるシール５０である。前述したように、シール５０の裏面と流路６０の底面との間の距離ｄ１は、予め記憶されている（図６Ａ）。したがって、ピペット移動部１１２は、ピペットチップ１７０の先端が流路６０の底面に接することがないように制御する。 Next, the position information of the tip of the pipette tip 170 is acquired (step S120). First, among the plurality of seals 50 to be pierced by the pipette tip 170, the seal 50 having the maximum force required to pierce is pierced by the pipette tip 170 (step S121). Specifically, the control unit 160 operates the transport stage 122 to move the detection chip 10 to the liquid feeding position. Then, the control unit 160 drives the pipette moving unit 112 to break through the pipette tip 170 with the seal 50 having the maximum force required to pierce among the plurality of seals 50 to be pierced by the pipette tip 170. In the present embodiment, among the plurality of seals 50 to be pierced by the pipette tip 170, the seal 50 having the maximum force necessary for piercing is the seal 50 closing the opening of the injection portion 70. As described above, the distance d1 between the back surface of the seal 50 and the bottom surface of the flow path 60 is stored in advance (FIG. 6A). Therefore, the pipette moving unit 112 controls so that the tip of the pipette tip 170 does not contact the bottom surface of the flow path 60.
　また、前述したように、シール５０と凹部４６の底面との間の距離（図６Ｂ；ｄ３参照）、凹部４６に貯留された液体の深さ（図６Ｂ；ｄ４参照）は、予め記憶されている。よって、ピペットチップ１７０が突き破るべき複数のシール５０のうち、突き破るために必要な力が最大であるシール５０が凹部４６の開口を塞いでいるシール５０である場合、ピペット移動部１１２は、ピペットチップ１７０の先端が凹部４６に貯留された液体に接しないように制御する。 As described above, the distance between the seal 50 and the bottom surface of the recess 46 (see FIG. 6B; d3) and the depth of the liquid stored in the recess 46 (see FIG. 6B; d4) are stored in advance. Yes. Therefore, when the seal 50 having the maximum force required to pierce among the plurality of seals 50 to be pierced by the pipette tip 170 is the seal 50 closing the opening of the recess 46, the pipette moving unit 112 has the pipette tip 112. Control is performed so that the tip of 170 does not come into contact with the liquid stored in the recess 46.
　次いで、ピペットチップ１７０内の空気圧を測定する（工程Ｓ１２２）。具体的には、制御部１６０は、ピペット移動部１１２を駆動して、ピペットチップ１７０の先端を流路６０の底面（基準部１８０）に向かって移動させる。このとき、制御部１６０は、送液ポンプ駆動機構１１３を駆動して、プランジャー１１５をシリンジ１１４に対して進行させる。すなわち、制御部１６０は、ピペットチップ１７０の先端から空気を連続してはき出しながら、ピペットチップ１７０の先端を基準部１８０に近づける。ピペットチップ１７０の先端を基準部１８０に近づけると、ピペットチップ１７０の先端から空気がはき出されにくくなるため、空気圧センサー１３１の検出値が上昇する。これにより、制御部１６０は、空気圧センサー１３１が空気圧を検出することで、基準部１８０に対するピペットチップ１７０の先端の位置情報を取得する。 Next, the air pressure in the pipette tip 170 is measured (step S122). Specifically, the control unit 160 drives the pipette moving unit 112 to move the tip of the pipette tip 170 toward the bottom surface (reference unit 180) of the flow channel 60. At this time, the controller 160 drives the liquid feed pump drive mechanism 113 to advance the plunger 115 relative to the syringe 114. That is, the control unit 160 brings the tip of the pipette tip 170 closer to the reference unit 180 while continuously ejecting air from the tip of the pipette tip 170. When the tip of the pipette tip 170 is brought close to the reference portion 180, the air is less likely to be expelled from the tip of the pipette tip 170, so that the detection value of the air pressure sensor 131 increases. Accordingly, the control unit 160 acquires position information of the tip of the pipette tip 170 with respect to the reference unit 180 when the air pressure sensor 131 detects the air pressure.
　なお、ピペットチップ１７０の先端から空気を間欠的にはき出しながら、ピペットチップ１７０の先端を基準部１８０に近づけてもよい。また、ピペットチップ１７０の先端から空気を連続的にまたは間欠的に吸引しながら、ピペットチップ１７０の先端を基準部１８０に近づけてもよい。これらの場合であっても基準部１８０に対するピペットチップ１７０の先端の位置情報を高精度に取得できる。 Note that the tip of the pipette tip 170 may be brought close to the reference portion 180 while air is intermittently ejected from the tip of the pipette tip 170. Further, the tip of the pipette tip 170 may be brought close to the reference portion 180 while sucking air from the tip of the pipette tip 170 continuously or intermittently. Even in these cases, the position information of the tip of the pipette tip 170 with respect to the reference portion 180 can be obtained with high accuracy.
　また、シール５０を突き破る工程（工程Ｓ１２１）は、ＳＰＦＳ装置１００の検出動作（反応方法）に含まれる全工程においてピペットチップ１７０の位置精度が最も求められる操作を行う前までに行われることが好ましい。この場合は、ピペットチップ１７０の位置精度が最も求められる操作を行う前までにピペットチップ１７０が突き破るべき複数のシール５０のうち、突き破るために必要な力が最大であるシール５０をピペットチップ１７０で突き破るようにしてもよい。本実施の形態では、ピペットチップ１７０の位置精度が最も求められる操作は、１次反応において、流路６０内から検体を除去する工程であり、当該操作までに突き破るべきシール５０のうち、突き破るために必要な力が最大であるシール５０は、注入部７０の開口を塞いでいるシール５０である。したがって、本実施の形態では、１次反応（工程Ｓ１４０）の前にピペットチップの先端の位置情報を取得する工程（工程Ｓ１２０）を行っている。 Further, the step of breaking through the seal 50 (step S121) is preferably performed before performing an operation in which the position accuracy of the pipette tip 170 is most required in all steps included in the detection operation (reaction method) of the SPFS device 100. . In this case, of the plurality of seals 50 to be pierced by the pipette tip 170 before performing the operation for which the positional accuracy of the pipette tip 170 is most required, the pipette tip 170 has the seal 50 having the maximum force required for piercing. You may make it break through. In the present embodiment, the operation for which the position accuracy of the pipette tip 170 is most required is a step of removing the specimen from the flow path 60 in the primary reaction. The seal 50 that requires the maximum force is the seal 50 that closes the opening of the injection portion 70. Therefore, in the present embodiment, the step (step S120) of acquiring the position information of the tip of the pipette tip is performed before the primary reaction (step S140).
　なお、検出チップ１０の金属膜３０上に保湿剤が存在する場合は、１次抗体が適切に被検出物質を捕捉できるように、金属膜３０上を洗浄して保湿剤を除去する必要がある。この場合は、金属膜３０を洗浄した後に金属膜３０上から洗浄液を除去する操作においてもピペットチップ１７０の位置精度が求められる。したがって、ピペットチップ１７０の先端の位置情報を取得する工程（工程Ｓ１２０）は、洗浄液を金属膜３０上から除去する前までに行ってもよい。 When a humectant is present on the metal film 30 of the detection chip 10, it is necessary to clean the metal film 30 and remove the humectant so that the primary antibody can appropriately capture the substance to be detected. . In this case, the positional accuracy of the pipette tip 170 is also required in the operation of removing the cleaning liquid from the metal film 30 after cleaning the metal film 30. Therefore, the step of acquiring the position information of the tip of the pipette tip 170 (step S120) may be performed before the cleaning liquid is removed from the metal film 30.
　また、工程Ｓ１２１では、複数のシール５０を突き破ってもよい。このとき、突き破るために必要な力が最大であるシール５０を突き破った後に、その他のシール５０を突き破ってもよいし、その他のシール５０を突き破った後に、突き破るために必要な力が最大であるシール５０を突き破ってもよい。このように、シール５０を１回または２回以上突き破った後に、ピペットチップ１７０の先端の位置情報を取得する。 In step S121, a plurality of seals 50 may be broken. At this time, after piercing through the seal 50 that requires the maximum force to pierce, the other seal 50 may be pierced, or after piercing through the other seal 50, the force required to pierce is the maximum. The seal 50 may be pierced. As described above, the position information of the tip of the pipette tip 170 is acquired after breaking the seal 50 once or twice or more.
　次いで、励起光αの入射角を決定する（工程Ｓ１３０）。具体的には、制御部１６０は、搬送ステージ１２２を操作して、検出チップ１０を検出位置に移動させる。そして、制御部１６０は、角度調整機構１４２を駆動して励起光αの入射角を走査しながら、センサー制御部１５３を駆動して受光センサー１５７によりプラズモン散乱光γを検出する。そして、プラズモン散乱光γの光量が最大となる角度を励起光αの入射角（増強角）とする。 Next, the incident angle of the excitation light α is determined (step S130). Specifically, the control unit 160 operates the transfer stage 122 to move the detection chip 10 to the detection position. The controller 160 drives the sensor controller 153 to detect the plasmon scattered light γ by the light receiving sensor 157 while driving the angle adjusting mechanism 142 to scan the incident angle of the excitation light α. The angle at which the amount of plasmon scattered light γ is maximized is defined as the incident angle (enhancement angle) of the excitation light α.
　なお、ピペットチップ１７０の先端の位置情報を取得する工程（工程Ｓ１２０）と励起光αの入射角を決定する工程（工程Ｓ１３０）との順番は、これに限定されない。励起光αの入射角を決定する工程（工程Ｓ１３０）を行った後に、ピペットチップ１７０の先端の位置情報を取得する工程（工程Ｓ１２０）を行ってもよい。 In addition, the order of the process (process S120) which acquires the positional information on the front-end | tip of the pipette tip 170 and the process (process S130) which determines the incident angle of excitation light (alpha) is not limited to this. After performing the step of determining the incident angle of the excitation light α (step S130), the step of acquiring the position information of the tip of the pipette tip 170 (step S120) may be performed.
　次いで、検体中の被検出物質と１次抗体とを反応させる（１次反応；工程Ｓ１４０）。制御部１６０は、搬送ステージ１２２を操作して、検体が貯留されている容器をピペットチップ１７０の直下に移動させる。そして、ピペットチップ１７０の先端を検体が貯留されている容器に向かって移動させて、ピペットチップ１７０内に検体を吸入させる。制御部１６０は、搬送ステージ１２２を操作して、検出チップ１０を送液位置に移動させる。そして、制御部１６０は、ピペット移動部１１２を駆動して、ピペットチップ１７０の先端を注入部７０内に移動させて、流路６０内に検体を注入する。このとき、ピペットチップ１７０の位置が高精度に特定されているため、ピペットチップ１７０の先端と流路６０の底面との間の距離（図６Ａ；ｄ２参照）を高精度に制御できる。したがって、ピペットチップ１７０の先端と流路６０の底面との間の距離を離しすぎて、検体が注入部７０の側壁に付着することも、ピペットチップ１７０の先端と流路６０の底面との間の距離を近づけすぎて、流路６０の底面（金属膜３０）に傷を付けてしまうこともない。検体中に被検出物質が存在する場合は、被検出物質の少なくとも一部は１次抗体に結合する。１次反応後、検体を流路６０内から除去する。この場合、ピペットチップ１７０の先端の位置情報に基づいて、ピペットチップ１７０の先端を流路６０の底面に近接させる。そして、ピペットチップ１７０内に検体を吸入することで流路６０から検体を除去する。 Next, the detection target substance in the specimen is reacted with the primary antibody (primary reaction; step S140). The control unit 160 operates the transport stage 122 to move the container in which the specimen is stored directly below the pipette tip 170. Then, the tip of the pipette tip 170 is moved toward the container in which the specimen is stored, and the specimen is inhaled into the pipette tip 170. The controller 160 operates the transfer stage 122 to move the detection chip 10 to the liquid feeding position. Then, the control unit 160 drives the pipette moving unit 112 to move the tip of the pipette tip 170 into the injection unit 70 and injects the sample into the flow channel 60. At this time, since the position of the pipette tip 170 is specified with high accuracy, the distance (see FIG. 6A; d2) between the tip of the pipette tip 170 and the bottom surface of the flow path 60 can be controlled with high accuracy. Therefore, the distance between the tip of the pipette tip 170 and the bottom surface of the flow path 60 is too large, and the specimen may adhere to the side wall of the injection unit 70. Therefore, the bottom surface (metal film 30) of the flow path 60 is not damaged. When a substance to be detected exists in the sample, at least a part of the substance to be detected binds to the primary antibody. After the primary reaction, the specimen is removed from the flow path 60. In this case, the tip of the pipette tip 170 is brought close to the bottom surface of the channel 60 based on the position information of the tip of the pipette tip 170. Then, the sample is removed from the flow path 60 by inhaling the sample into the pipette tip 170.
　なお、検体が貯留されている容器は、検出チップ１０に配置してもよい。この場合、検出チップ１０の流路蓋４０には、当該容器を収容するための収容穴が形成される。 Note that the container in which the specimen is stored may be disposed on the detection chip 10. In this case, an accommodation hole for accommodating the container is formed in the flow path lid 40 of the detection chip 10.
　なお、検体および被検出物質の種類は、特に限定されない。検体の例には、血液や血清、血漿、尿、鼻孔液、唾液、精液などの体液およびその希釈液が含まれる。また、被検出物質の例には、核酸（ＤＮＡやＲＮＡなど）、タンパク質（ポリペプチドやオリゴペプチドなど）、アミノ酸、糖質、脂質およびこれらの修飾分子が含まれる。 Note that the types of the specimen and the substance to be detected are not particularly limited. Examples of the specimen include body fluids such as blood, serum, plasma, urine, nasal fluid, saliva, semen, and diluted solutions thereof. Examples of substances to be detected include nucleic acids (such as DNA and RNA), proteins (such as polypeptides and oligopeptides), amino acids, carbohydrates, lipids, and modified molecules thereof.
　また、１次反応（工程Ｓ１４０）において、検体を流路６０内で往復動作させてもよい。この場合、流路６０内への検体を注入する工程と同様に、ピペットチップ１７０の先端の位置情報に基づいて、ピペットチップ１７０の先端を流路６０の底面に近接させる。そして、ピペットチップ１７０の先端の位置を固定した状態で、プランジャー１１５を往復動作させる。これにより、ピペットチップ１７０で検体の吸入および排出を繰り返すことで、流路６０内で検体を往復させることができる。流路６０内で検体を往復させた後、ピペットチップ１７０内に検体を吸入することで流路６０から検体を除去する。 In the primary reaction (step S140), the specimen may be reciprocated in the flow path 60. In this case, the tip of the pipette tip 170 is brought close to the bottom surface of the channel 60 based on the positional information of the tip of the pipette tip 170 as in the step of injecting the sample into the channel 60. Then, the plunger 115 is reciprocated while the position of the tip of the pipette tip 170 is fixed. Accordingly, the sample can be reciprocated in the flow path 60 by repeatedly inhaling and discharging the sample with the pipette tip 170. After reciprocating the sample in the flow channel 60, the sample is removed from the flow channel 60 by inhaling the sample into the pipette tip 170.
　そして、金属膜３０上を緩衝液などの洗浄液で洗浄する。制御部１６０は、ピペットチップ１７０の先端を凹部４６内の洗浄液に向かって移動させて、ピペットチップ１７０内に洗浄液を吸入させる。このとき、ピペットチップ１７０の位置情報が高精度に特定されているため、ピペットチップ１７０の先端と洗浄液の表面との間の距離（図６Ｃ；ｄ５参照）およびピペットチップ１７０の先端と凹部４６の底面との間の距離（図６Ｃ；ｄ６参照）も高精度に制御できる。したがって、ピペットチップ１７０内に洗浄液を適切に吸入できる。そして、制御部１６０は、ピペット移動部１１２を駆動して、ピペットチップ１７０の先端を注入部７０内に移動させて、流路６０内に洗浄液を注入する。 Then, the metal film 30 is cleaned with a cleaning solution such as a buffer solution. The controller 160 moves the tip of the pipette tip 170 toward the cleaning liquid in the recess 46 and causes the cleaning liquid to be sucked into the pipette tip 170. At this time, since the position information of the pipette tip 170 is specified with high accuracy, the distance between the tip of the pipette tip 170 and the surface of the cleaning liquid (see FIG. 6C; d5) and the tip of the pipette tip 170 and the recess 46 The distance from the bottom surface (see FIG. 6C; d6) can also be controlled with high accuracy. Therefore, the cleaning liquid can be appropriately sucked into the pipette tip 170. Then, the control unit 160 drives the pipette moving unit 112 to move the tip of the pipette tip 170 into the injection unit 70 and injects the cleaning liquid into the flow channel 60.
　次いで、流路６０内から１次抗体に結合しなかった物質を含む洗浄液を除去する。具体的には、制御部１６０は、ピペット移動部１１２を駆動して、ピペットチップ１７０の先端を注入部７０に移動させる。そして、前述したピペットチップ１７０の先端の位置情報に基づいて、ピペットチップ１７０の先端を流路６０の底面に近づけて、流路６０から洗浄液を除去する。このとき、ピペットチップ１７０の先端を前述したピペットチップ１７０の先端の位置情報に基づいて、プリズム２０（金属膜３０）に近づけているため、流路６０内に残留する液量を最小限とすることができる。洗浄液を除去するときのピペットチップ１７０の先端の位置は、流路６０内から検体を除去する工程におけるピペットチップ１７０の先端の位置と同じであることが好ましい。これにより、流路６０内に残留する液量を一定にできる。 Next, the washing liquid containing the substance that has not bound to the primary antibody is removed from the flow path 60. Specifically, the control unit 160 drives the pipette moving unit 112 to move the tip of the pipette tip 170 to the injection unit 70. Then, based on the position information on the tip of the pipette tip 170 described above, the tip of the pipette tip 170 is brought close to the bottom surface of the channel 60 to remove the cleaning liquid from the channel 60. At this time, since the tip of the pipette tip 170 is brought close to the prism 20 (metal film 30) based on the position information of the tip of the pipette tip 170 described above, the amount of liquid remaining in the flow path 60 is minimized. be able to. The position of the tip of the pipette tip 170 when removing the cleaning liquid is preferably the same as the position of the tip of the pipette tip 170 in the step of removing the specimen from the flow path 60. Thereby, the amount of liquid remaining in the flow path 60 can be made constant.
　次いで、金属膜３０上に捕捉されている被検出物質を蛍光物質で標識する（２次反応；工程Ｓ１５０）。具体的には、制御部１６０は、ピペットチップ１７０の先端を蛍光物質で標識された捕捉体を含む液体（標識液）が貯留された凹部４６に向かって移動させて、ピペットチップ１７０内に標識液を吸入させる。このとき、ピペットチップ１７０の位置情報が高精度に特定されているため、ピペットチップ１７０の先端と標識液の表面との間の距離（図６Ｃのｄ５参照）およびピペットチップ１７０の先端と凹部４６の底面との間の距離（図６Ｃのｄ６参照）も高精度に制御できる。したがって、ピペットチップ１７０内に標識液を適切に吸入できる。そして、制御部１６０は、ピペット移動部１１２を駆動して、ピペットチップ１７０の先端を注入部７０内に移動させて、流路６０内に標識液を注入する。流路６０内では、抗原抗体反応によって、金属膜３０上に捕捉されている被検出物質が蛍光物質で標識される。この後、流路６０内の標識液は除去され、流路６０内は洗浄液で洗浄される。流路６０内の標識液を除去するときのピペットチップ１７０の先端の位置は、前述したピペットチップ１７０の先端の位置情報に基づいて位置決めされる。これにより、流路６０内に残留する液量を最小に、かつ一定にすることができる。 Next, the target substance captured on the metal film 30 is labeled with a fluorescent substance (secondary reaction; step S150). Specifically, the control unit 160 moves the tip of the pipette tip 170 toward the concave portion 46 in which a liquid (labeling liquid) containing a capturing body labeled with a fluorescent substance is stored, and labels the pipette tip 170 in the pipette tip 170. Inhale fluid. At this time, since the position information of the pipette tip 170 is specified with high accuracy, the distance between the tip of the pipette tip 170 and the surface of the labeling liquid (see d5 in FIG. 6C), the tip of the pipette tip 170, and the recess 46 The distance (see d6 in FIG. 6C) to the bottom surface of the can also be controlled with high accuracy. Therefore, the labeling solution can be appropriately inhaled into the pipette tip 170. Then, the control unit 160 drives the pipette moving unit 112 to move the tip of the pipette tip 170 into the injection unit 70 and injects the labeling solution into the flow channel 60. In the flow channel 60, the detection target substance captured on the metal film 30 is labeled with a fluorescent substance by an antigen-antibody reaction. Thereafter, the labeling liquid in the flow path 60 is removed, and the inside of the flow path 60 is cleaned with a cleaning liquid. The position of the tip of the pipette tip 170 when removing the labeling liquid in the flow channel 60 is determined based on the position information of the tip of the pipette tip 170 described above. As a result, the amount of liquid remaining in the flow path 60 can be kept to a minimum and constant.
　なお、１次反応（工程Ｓ１４０）と２次反応（工程Ｓ１５０）との順番は、これに限定されない。たとえば、第１被検出物質を第１の２次抗体に結合させるとともに、第２被検出物質を第２の２次抗体に結合させた後に、これらの複合体を含む液体を金属膜３０上に提供してもよい。また、金属膜３０上に検体と標識液を同時に提供してもよい。このように、検出チップ１０内において２以上の物質を反応させるためにピペットチップ１７０の先端の位置情報に基づいて１回または２回以上ピペット１１１を移動させる。 Note that the order of the primary reaction (step S140) and the secondary reaction (step S150) is not limited to this. For example, after the first substance to be detected is bound to the first secondary antibody and the second substance to be detected is bound to the second secondary antibody, the liquid containing these complexes is deposited on the metal film 30. May be provided. Further, the specimen and the labeling solution may be provided on the metal film 30 at the same time. Thus, in order to react two or more substances in the detection chip 10, the pipette 111 is moved once or twice or more based on the position information of the tip of the pipette chip 170.
　次いで、被検出物質を検出する（工程Ｓ１６０）。具体的には、制御部１６０は、搬送ステージ１２２を操作して、検出チップ１０を検出位置に移動させる。そして、光源制御部１４３を駆動して励起光αを工程Ｓ１３０で決定した入射角（増強角）で金属膜３０の所定の位置に照射させながら、センサー制御部１５３を駆動して金属膜３０（金属膜３０表面およびその近傍）上から放出される蛍光δの強度を検出するように受光センサー１５７を制御する。 Next, a substance to be detected is detected (step S160). Specifically, the control unit 160 operates the transfer stage 122 to move the detection chip 10 to the detection position. The sensor control unit 153 is driven to drive the metal film 30 (while the light source control unit 143 is driven to irradiate the excitation light α to a predetermined position of the metal film 30 at the incident angle (enhancement angle) determined in step S130. The light receiving sensor 157 is controlled so as to detect the intensity of the fluorescence δ emitted from the surface of the metal film 30 and the vicinity thereof.
　なお、制御部１６０は、２次反応（工程Ｓ１５０）の前にブランク値を測定してもよい。この場合、増強角で励起光αを金属膜３０に照射し、受光センサー１５７の検出値をブランク値とする。そして、被検出物質を検出する工程（工程Ｓ１６０）では、蛍光δの検出値からブランク値を引くことで、検体中の被検出物質の量を示す蛍光δの量を算出する。 In addition, the control part 160 may measure a blank value before a secondary reaction (process S150). In this case, the excitation light α is irradiated onto the metal film 30 at an enhancement angle, and the detection value of the light receiving sensor 157 is set as a blank value. In the step of detecting the substance to be detected (step S160), the amount of fluorescence δ indicating the amount of the substance to be detected in the sample is calculated by subtracting the blank value from the detected value of fluorescence δ.
　（効果）
　以上のように、本実施の形態に係るＳＰＦＳ装置１００は、ピペットチップ１７０でシール５０を突き破った後に、ピペットチップ１７０の先端の位置情報を取得しているため、ピペットチップ１７０の先端の位置情報を取得した後にピペットチップ１７０の先端の位置がずれることなく、高精度にピペットチップ１７０の先端の位置を制御することができる。また、これにより、流路６０内の残液量を一定にすることで、検出結果の精度を向上させることができる。 (effect)
As described above, since the SPFS device 100 according to the present embodiment acquires the position information of the tip of the pipette tip 170 after breaking through the seal 50 with the pipette tip 170, the position information of the tip of the pipette tip 170 is obtained. The position of the tip end of the pipette tip 170 can be controlled with high accuracy without the position of the tip end of the pipette tip 170 being shifted after obtaining the above. Moreover, by this, the precision of a detection result can be improved by making constant the residual liquid amount in the flow path 60. FIG.
　［実施の形態２］
　実施の形態２に係るＳＰＦＳ装置は、位置情報取得部２３０の構成が異なる点において、実施の形態１に係るＳＰＦＳ装置１００と異なる。そこで、実施の形態１に係るＳＰＦＳ装置１００と同様の構成については同一の符号を付してその説明を省略し、異なる部分を中心に説明する。 [Embodiment 2]
The SPFS device according to the second embodiment differs from the SPFS device 100 according to the first embodiment in that the configuration of the position information acquisition unit 230 is different. Therefore, the same components as those of the SPFS device 100 according to the first embodiment are denoted by the same reference numerals, description thereof is omitted, and different portions are mainly described.
　（ＳＰＦＳ装置の構成）
　図７Ａは、実施の形態２に係るＳＰＦＳ装置の一部の構成を示す図である。 (Configuration of SPFS device)
FIG. 7A is a diagram illustrating a partial configuration of the SPFS apparatus according to the second embodiment.
　図７Ａに示されるように、実施の形態２に係る位置情報取得部２３０は、歪みセンサー２３１を有している。歪みセンサー２３１は、ピペットノズル１１６に配置されている。ピペットチップ１７０の先端の位置情報の取得は、ピペットチップ１７０の先端と、基準部１８０とが接触したときのピペットノズル１１６の歪みを歪みセンサー２３１により測定することで行う。ピペットチップ１７０の先端と基準部１８０とが接触すると、ピペットチップ１７０およびピペットチップ１７０が装着されているピペットノズル１１６が歪む。このピペットノズル１１６の歪みを歪みセンサー２３１で検出することで、ピペットチップ１７０の先端と基準部１８０との接触を検出することができる。よって、ピペットチップ１７０の先端の位置情報を取得できる。 7A, the position information acquisition unit 230 according to Embodiment 2 includes a strain sensor 231. The strain sensor 231 is disposed on the pipette nozzle 116. The position information of the tip of the pipette tip 170 is acquired by measuring the strain of the pipette nozzle 116 when the tip of the pipette tip 170 and the reference unit 180 come into contact with each other by the strain sensor 231. When the tip of the pipette tip 170 comes into contact with the reference portion 180, the pipette tip 170 and the pipette nozzle 116 to which the pipette tip 170 is attached are distorted. By detecting the distortion of the pipette nozzle 116 with the distortion sensor 231, the contact between the tip of the pipette tip 170 and the reference portion 180 can be detected. Therefore, the position information of the tip of the pipette tip 170 can be acquired.
　（検出装置の検出動作）
　次に、実施の形態２に係る反応方法を含むＳＰＦＳ装置の検出動作について、実施の形態１に係るＳＰＦＳ装置１００の検出動作と異なる工程を中心に説明する。 (Detection operation of the detection device)
Next, the detection operation of the SPFS device including the reaction method according to the second embodiment will be described focusing on the steps different from the detection operation of the SPFS device 100 according to the first embodiment.
　図７Ｂは、ピペットチップ１７０の先端の位置情報を取得する工程を示すフローチャートである。 FIG. 7B is a flowchart showing a process of acquiring the position information of the tip of the pipette tip 170.
　図７Ｂに示されるように、実施の形態２におけるピペットチップ１７０の先端の位置情報を取得する工程では、まず、ピペットチップ１７０が突き破るべき複数のシール５０のうち、突き破るために必要な力が最大であるシール５０をピペットチップ１７０で突き破る（工程Ｓ１２１）。 As shown in FIG. 7B, in the step of acquiring the position information of the tip of the pipette tip 170 according to the second embodiment, first, the force required to break through the plurality of seals 50 that the pipette tip 170 should break through is maximized. The seal 50 is broken with the pipette tip 170 (step S121).
　次いで、ピペットノズル１１６の歪みを測定する（工程Ｓ２２２）。具体的には、制御部１６０は、ピペット移動部１１２を操作して、ピペットチップ１７０の先端を流路６０の底面（基準部１８０）に向かって移動させる。ピペットチップ１７０の先端と基準部１８０とが接触すると、ピペットチップ１７０およびピペットチップ１７０が装着されているピペットノズル１１６が歪む。これにより、ピペットノズル１１６に配置されている歪みセンサー２３１によって、ピペットチップ１７０の先端と基準部１８０との接触を検出することで、ピペットチップ１７０の先端の位置情報を取得する。 Next, the distortion of the pipette nozzle 116 is measured (step S222). Specifically, the control unit 160 operates the pipette moving unit 112 to move the tip of the pipette tip 170 toward the bottom surface (reference unit 180) of the channel 60. When the tip of the pipette tip 170 comes into contact with the reference portion 180, the pipette tip 170 and the pipette nozzle 116 to which the pipette tip 170 is attached are distorted. Thereby, the position information of the tip of the pipette tip 170 is acquired by detecting the contact between the tip of the pipette tip 170 and the reference portion 180 by the strain sensor 231 disposed in the pipette nozzle 116.
　（効果）
　以上のように、実施の形態２に係るＳＰＦＳ装置は、実施の形態１に係るＳＰＦＳ装置１００と同様の効果を有する。 (effect)
As described above, the SPFS device according to the second embodiment has the same effects as the SPFS device 100 according to the first embodiment.
　［実施の形態３］
　実施の形態３に係るＳＰＦＳ装置１００は、位置情報取得部３３０の構成が異なる点において、実施の形態１に係るＳＰＦＳ装置１００と異なる。そこで、実施の形態１に係るＳＰＦＳ装置１００と同様の構成については同一の符号を付してその説明を省略する。 [Embodiment 3]
The SPFS apparatus 100 according to the third embodiment is different from the SPFS apparatus 100 according to the first embodiment in that the configuration of the position information acquisition unit 330 is different. Therefore, the same components as those of the SPFS device 100 according to Embodiment 1 are denoted by the same reference numerals, and the description thereof is omitted.
　（ＳＰＦＳ装置の構成）
　図８Ａは、実施の形態３に係るＳＰＦＳ装置の一部の構成を示す図である。 (Configuration of SPFS device)
FIG. 8A is a diagram illustrating a partial configuration of the SPFS apparatus according to the third embodiment.
　図８Ａに示されるように、実施の形態３に係る位置情報取得部３３０は、静電容量センサー３３１を有している。静電容量センサー３３１が配置される位置は、特に限定されない。本実施の形態では、静電容量センサー３３１は、搬送ステージ１２２に配置されている。ピペットチップ１７０の先端の位置情報の取得は、ピペットチップ１７０の先端と、基準部１８０（静電容量センサー３３１の検出面）とを近づけたときの、ピペットチップ１７０の先端と基準部１８０との間の静電容量の変化を静電容量センサー３３１により測定することで行う。 As shown in FIG. 8A, the position information acquisition unit 330 according to Embodiment 3 has a capacitance sensor 331. The position where the capacitance sensor 331 is disposed is not particularly limited. In the present embodiment, the capacitance sensor 331 is disposed on the transfer stage 122. Acquisition of the position information of the tip of the pipette tip 170 is performed between the tip of the pipette tip 170 and the reference portion 180 when the tip of the pipette tip 170 is brought close to the reference portion 180 (detection surface of the capacitance sensor 331). The change in the electrostatic capacity is measured by the electrostatic capacity sensor 331.
　（検出装置の検出動作）
　次に、実施の形態３に係る反応方法を含むＳＰＦＳ装置の検出動作について、実施の形態１に係るＳＰＦＳ装置１００の検出動作と異なる工程を中心に説明する。 (Detection operation of the detection device)
Next, the detection operation of the SPFS device including the reaction method according to the third embodiment will be described focusing on steps different from the detection operation of the SPFS device 100 according to the first embodiment.
　図８Ｂは、ピペットチップ１７０の先端の位置情報を取得する工程を示すフローチャートである。 FIG. 8B is a flowchart showing a process of acquiring position information of the tip of the pipette tip 170.
　図８Ｂに示されるように、実施の形態３におけるピペットチップ１７０の先端の位置情報を取得する工程では、まず、ピペットチップ１７０が突き破るべき複数のシール５０のうち、突き破るために必要な力が最大であるシール５０をピペットチップ１７０で突き破る（工程Ｓ１２１）。 As shown in FIG. 8B, in the step of acquiring the position information of the tip of the pipette tip 170 in the third embodiment, first, the force required to pierce out of the plurality of seals 50 to be pierced by the pipette tip 170 is maximized. The seal 50 is broken with the pipette tip 170 (step S121).
　次いで、ピペットチップ１７０の先端と基準部との間の静電容量の変化を測定する（工程Ｓ３２２）。具体的には、制御部１６０は、ピペット移動部１１２を操作して、ピペットチップ１７０の先端を静電容量センサー３３１の検出面（基準部１８０）に向かって移動させる。ピペットチップ１７０の先端と基準部１８０とを近づけると、ピペットチップ１７０の先端と基準部１８０との間の静電容量が変化する。このときの静電容量の変化を静電容量センサー３３１により測定することでピペットチップ１７０の先端の位置情報を取得する。 Next, the change in capacitance between the tip of the pipette tip 170 and the reference portion is measured (step S322). Specifically, the control unit 160 operates the pipette moving unit 112 to move the tip of the pipette tip 170 toward the detection surface (reference unit 180) of the capacitance sensor 331. When the tip of the pipette tip 170 and the reference portion 180 are brought close to each other, the capacitance between the tip of the pipette tip 170 and the reference portion 180 changes. By measuring the change in capacitance at this time by the capacitance sensor 331, position information of the tip of the pipette tip 170 is acquired.
　（効果）
　以上のように、実施の形態３に係るＳＰＦＳ装置は、実施の形態１に係るＳＰＦＳ装置１００と同様の効果を有する。 (effect)
As described above, the SPFS device according to the third embodiment has the same effects as the SPFS device 100 according to the first embodiment.
　［実施の形態４］
　実施の形態４に係るＳＰＦＳ装置は、位置情報取得部４３０の構成が異なる点において、実施の形態１に係るＳＰＦＳ装置１００と異なる。そこで、実施の形態１に係るＳＰＦＳ装置１００と同様の構成については同一の符号を付してその説明を省略し、異なる部分を中心に説明する。 [Embodiment 4]
The SPFS apparatus according to the fourth embodiment differs from the SPFS apparatus 100 according to the first embodiment in that the configuration of the position information acquisition unit 430 is different. Therefore, the same components as those of the SPFS device 100 according to the first embodiment are denoted by the same reference numerals, description thereof is omitted, and different portions are mainly described.
　（ＳＰＦＳ装置の構成）
　図９Ａは、実施の形態４に係るＳＰＦＳ装置の一部の構成を示す図である。 (Configuration of SPFS device)
FIG. 9A is a diagram illustrating a partial configuration of the SPFS apparatus according to the fourth embodiment.
　図９Ａに示されるように、実施の形態４に係る位置情報取得部４３０は、電源４３１および電流計４３２を有している。電源４３１および電流計４３２は、ピペットチップ１７０の先端と基準部１８０とにそれぞれ接続されている。この場合、ピペットチップ１７０は、カーボンなどが含有されている電導性のピペットチップを使用する。ピペットチップ１７０の先端の位置情報の取得は、電源４３１によりピペットチップ１７０の先端と基準部１８０（金属膜３０）との間に電圧を印加して、電流計４３２によりピペットチップ１７０の先端と基準部１８０（金属膜３０）との間のインピーダンス特性を測定することで行う。 As shown in FIG. 9A, the position information acquisition unit 430 according to the fourth embodiment includes a power source 431 and an ammeter 432. The power source 431 and the ammeter 432 are connected to the tip of the pipette tip 170 and the reference unit 180, respectively. In this case, the pipette tip 170 uses a conductive pipette tip containing carbon or the like. The position information of the tip of the pipette tip 170 is acquired by applying a voltage between the tip of the pipette tip 170 and the reference portion 180 (metal film 30) by the power source 431, and using the ammeter 432 for the tip of the pipette tip 170 and the reference. This is done by measuring the impedance characteristic between the portion 180 (metal film 30).
　（検出装置の検出動作）
　次に、実施の形態４に係る反応方法を含むＳＰＦＳ装置の検出動作について、実施の形態１に係るＳＰＦＳ装置１００の検出動作と異なる工程を中心に説明する。 (Detection operation of the detection device)
Next, the detection operation of the SPFS device including the reaction method according to the fourth embodiment will be described focusing on steps different from the detection operation of the SPFS device 100 according to the first embodiment.
　図９Ｂは、ピペットチップ１７０の先端の位置情報を取得する工程を示すフローチャートである。 FIG. 9B is a flowchart showing a process of acquiring position information of the tip of the pipette tip 170.
　図９Ｂに示されるように、実施の形態４におけるピペットチップ１７０の先端の位置情報を取得する工程では、まず、ピペットチップ１７０が突き破るべき複数のシール５０のうち、突き破るために必要な力が最大であるシール５０をピペットチップ１７０で突き破る（工程Ｓ１２１）。 As shown in FIG. 9B, in the step of acquiring the position information of the tip of the pipette tip 170 according to the fourth embodiment, first, the force required to break through the plurality of seals 50 that the pipette tip 170 should break through is maximized. The seal 50 is broken with the pipette tip 170 (step S121).
　次いで、ピペットチップ１７０の先端と基準部１８０との間のインピーダンスを測定する（工程Ｓ４２２）。具体的には、制御部１６０は、ピペット移動部１１２を操作して、ピペットチップ１７０の先端を流路６０の底面（基準部１８０）に向かって移動させる。ピペットチップ１７０の先端と基準部１８０とを近づけた状態で、電源４３１によりピペットチップ１７０の先端と基準部１８０（金属膜３０）との間に電圧を印加して、電流計４３２によりピペットチップ１７０の先端と基準部１８０（金属膜３０）との間のインピーダンス特性を測定することでピペットチップ１７０の先端の位置情報を取得する。 Next, the impedance between the tip of the pipette tip 170 and the reference portion 180 is measured (step S422). Specifically, the control unit 160 operates the pipette moving unit 112 to move the tip of the pipette tip 170 toward the bottom surface (reference unit 180) of the channel 60. In a state where the tip of the pipette tip 170 and the reference portion 180 are brought close to each other, a voltage is applied between the tip of the pipette tip 170 and the reference portion 180 (metal film 30) by the power source 431, and the pipette tip 170 is drawn by the ammeter 432. The position information of the tip of the pipette tip 170 is obtained by measuring the impedance characteristic between the tip of the pipe and the reference portion 180 (metal film 30).
　（効果）
　以上のように、実施の形態４に係るＳＰＦＳ装置は、実施の形態１に係るＳＰＦＳ装置１００と同様の効果を有する。 (effect)
As described above, the SPFS device according to the fourth embodiment has the same effects as the SPFS device 100 according to the first embodiment.
　［実施の形態５］
　実施の形態５に係るＳＰＦＳ装置は、位置情報取得部５３０の構成が異なる点において、実施の形態１に係るＳＰＦＳ装置１００と異なる。そこで、実施の形態１に係るＳＰＦＳ装置１００と同様の構成については同一の符号を付してその説明を省略し、異なる部分を中心に説明する。 [Embodiment 5]
The SPFS device according to the fifth embodiment is different from the SPFS device 100 according to the first embodiment in that the configuration of the position information acquisition unit 530 is different. Therefore, the same components as those of the SPFS device 100 according to the first embodiment are denoted by the same reference numerals, description thereof is omitted, and different portions are mainly described.
　（ＳＰＦＳ装置の構成）
　図１０Ａは、実施の形態５に係るＳＰＦＳ装置の一部の構成を示す図である。 (Configuration of SPFS device)
FIG. 10A is a diagram illustrating a partial configuration of the SPFS apparatus according to the fifth embodiment.
　図１０Ａに示されるように、実施の形態５に係る位置情報取得部５３０は、感圧センサー５３１を有している。感圧センサー５３１が配置される位置は、特に限定されない。本実施の形態では、感圧センサー５３１は、搬送ステージ１２２に配置されている。ピペットチップ１７０の先端の位置情報の取得は、ピペットチップ１７０の先端と、基準部１８０（感圧センサー５３１の検出面）とが接触したときの圧力を感圧センサー５３１により測定することで行う。ピペットチップ１７０の先端と基準部１８０とが接触すると、感圧センサー５３１が所定の圧力を検出する。これにより、ピペットチップ１７０の先端と基準部１８０との接触を検出することができる。 As shown in FIG. 10A, the position information acquisition unit 530 according to Embodiment 5 includes a pressure-sensitive sensor 531. The position where the pressure-sensitive sensor 531 is disposed is not particularly limited. In the present embodiment, the pressure-sensitive sensor 531 is disposed on the transfer stage 122. The position information of the tip of the pipette tip 170 is acquired by measuring the pressure when the tip of the pipette tip 170 comes into contact with the reference portion 180 (detection surface of the pressure sensor 531) by the pressure sensor 531. When the tip of the pipette tip 170 comes into contact with the reference portion 180, the pressure-sensitive sensor 531 detects a predetermined pressure. Thereby, the contact between the tip of the pipette tip 170 and the reference portion 180 can be detected.
　（検出装置の検出動作）
　次に、実施の形態５に係る反応方法を含むＳＰＦＳ装置の検出動作について、実施の形態１に係るＳＰＦＳ装置１００の検出動作と異なる工程を中心に説明する。 (Detection operation of the detection device)
Next, the detection operation of the SPFS device including the reaction method according to the fifth embodiment will be described focusing on the steps different from the detection operation of the SPFS device 100 according to the first embodiment.
　図１０Ｂは、ピペットチップ１７０の先端の位置情報を取得する工程を示すフローチャートである。 FIG. 10B is a flowchart showing a process of acquiring the position information of the tip of the pipette tip 170.
　図１０Ｂに示されるように、実施の形態２におけるピペットチップ１７０の先端の位置情報を取得する工程では、まず、ピペットチップ１７０が突き破るべき複数のシール５０のうち、突き破るために必要な力が最大であるシール５０をピペットチップ１７０で突き破る（工程Ｓ１２１）。 As shown in FIG. 10B, in the step of acquiring the position information of the tip of the pipette tip 170 according to the second embodiment, first, the force required to break through the plurality of seals 50 that the pipette tip 170 should break through is maximized. The seal 50 is broken with the pipette tip 170 (step S121).
　次いで、ピペットチップ１７０の先端と基準部１８０との接触圧を測定する（工程Ｓ５２２）。具体的には、制御部１６０は、ピペット移動部１１２を操作して、ピペットチップ１７０の先端を流路６０の底面（基準部１８０）に向かって移動させる。ピペットチップ１７０の先端と基準部１８０とが接触すると、感圧センサー５３１がそのときの圧力を検出する。このとき、感圧センサー５３１が所定の圧力を検出することでピペットチップ１７０の先端の位置情報を取得する。 Next, the contact pressure between the tip of the pipette tip 170 and the reference portion 180 is measured (step S522). Specifically, the control unit 160 operates the pipette moving unit 112 to move the tip of the pipette tip 170 toward the bottom surface (reference unit 180) of the channel 60. When the tip of the pipette tip 170 comes into contact with the reference portion 180, the pressure-sensitive sensor 531 detects the pressure at that time. At this time, the pressure sensor 531 detects a predetermined pressure to acquire position information of the tip of the pipette tip 170.
　（効果）
　以上のように、実施の形態５に係るＳＰＦＳ装置は、実施の形態１に係るＳＰＦＳ装置１００と同様の効果を有する。 (effect)
As described above, the SPFS device according to the fifth embodiment has the same effect as the SPFS device 100 according to the first embodiment.
　［実施の形態６］
　実施の形態６に係るＳＰＦＳ装置は、位置情報取得部６３０の構成が異なる点において、実施の形態１に係るＳＰＦＳ装置１００と異なる。そこで、実施の形態１に係るＳＰＦＳ装置１００と同様の構成については同一の符号を付してその説明を省略し、異なる部分を中心に説明する。 [Embodiment 6]
The SPFS device according to the sixth embodiment differs from the SPFS device 100 according to the first embodiment in that the configuration of the position information acquisition unit 630 is different. Therefore, the same components as those of the SPFS device 100 according to the first embodiment are denoted by the same reference numerals, description thereof is omitted, and different portions are mainly described.
　（ＳＰＦＳ装置の構成）
　図１１Ａは、実施の形態６に係るＳＰＦＳ装置の一部の構成を示す図である。 (Configuration of SPFS device)
FIG. 11A is a diagram illustrating a partial configuration of the SPFS apparatus according to the sixth embodiment.
　図１１Ａに示されるように、実施の形態６に係る位置情報取得部６３０は、光センサー６３１を有している。光センサー６３１は、光学的にピペットチップ１７０の先端の位置情報を取得することができれば特に限定されない。光センサー６３１の例には、発光素子と受光素子との間のレーザー光の受け渡しの有無で検出する方式であってもよいし、ＣＣＤカメラで撮像して検出する方式であってもよい。本実施の形態では、光センサー６３１は、ＣＣＤカメラで撮像して検出する方式である。光センサー６３１が配置される位置は、特に限定されない。本実施の形態では、光センサー６３１は、支持部材を介して搬送ステージ１２２に固定されている。ピペットチップ１７０の先端の位置情報の取得は、ピペットチップ１７０の先端が光センサー６３１の撮像領域に進入した場合に撮像することで行う。ピペットチップ１７０の先端が光センサー６３１の撮像領域に進入すると、光センサー６３１は、ピペットチップ１７０の先端を検知して撮像する。これにより、ピペットチップ１７０の先端の位置情報を取得できる。 As shown in FIG. 11A, the position information acquisition unit 630 according to the sixth embodiment includes an optical sensor 631. The optical sensor 631 is not particularly limited as long as it can optically acquire the position information of the tip of the pipette tip 170. An example of the optical sensor 631 may be a method of detecting by the presence / absence of laser light passing between the light emitting element and the light receiving element, or a method of detecting by imaging with a CCD camera. In the present embodiment, the optical sensor 631 is a method of detecting by imaging with a CCD camera. The position where the optical sensor 631 is arranged is not particularly limited. In the present embodiment, the optical sensor 631 is fixed to the transfer stage 122 via a support member. Acquisition of the position information of the tip of the pipette tip 170 is performed by imaging when the tip of the pipette tip 170 enters the imaging region of the optical sensor 631. When the tip of the pipette tip 170 enters the imaging area of the optical sensor 631, the optical sensor 631 detects and images the tip of the pipette tip 170. Thereby, the position information of the tip of the pipette tip 170 can be acquired.
　（検出装置の検出動作）
　次に、実施の形態６に係る反応方法を含むＳＰＦＳ装置の検出動作について、実施の形態１に係るＳＰＦＳ装置１００の検出動作と異なる工程を中心に説明する。 (Detection operation of the detection device)
Next, the detection operation of the SPFS device including the reaction method according to the sixth embodiment will be described focusing on steps different from the detection operation of the SPFS device 100 according to the first embodiment.
　図１１Ｂは、ピペットチップ１７０の先端の位置情報を取得する工程を示すフローチャートである。 FIG. 11B is a flowchart showing a process of acquiring the position information of the tip of the pipette tip 170.
　図１１Ｂに示されるように、実施の形態６におけるピペットチップ１７０の先端の位置情報を取得する工程では、まず、ピペットチップ１７０が突き破るべき複数のシール５０のうち、突き破るために必要な力が最大であるシール５０をピペットチップ１７０で突き破る（工程Ｓ１２１）。 As shown in FIG. 11B, in the step of acquiring the position information of the tip of the pipette tip 170 in the sixth embodiment, first, the force required for breaking through the plurality of seals 50 that the pipette tip 170 should break through is maximized. The seal 50 is broken with the pipette tip 170 (step S121).
　次いで、ピペットチップ１７０の先端位置を検出する（工程Ｓ６２２）。具体的には、制御部１６０は、ピペット移動部１１２を操作して、ピペットチップ１７０の先端を光センサー６３１の撮像領域に向かって移動させる。光センサー６３１は、光センサー６３１の撮像領域にピペットチップ１７０の先端が浸入するのを検知して、ピペットチップ１７０の先端を撮像してピペットチップ１７０の先端の位置情報を取得する。 Next, the tip position of the pipette tip 170 is detected (step S622). Specifically, the control unit 160 operates the pipette moving unit 112 to move the tip of the pipette tip 170 toward the imaging area of the optical sensor 631. The optical sensor 631 detects that the tip of the pipette tip 170 has entered the imaging area of the optical sensor 631, captures the tip of the pipette tip 170, and acquires positional information of the tip of the pipette tip 170.
　（効果）
　以上のように、実施の形態６に係るＳＰＦＳ装置は、実施の形態１に係るＳＰＦＳ装置１００と同様の効果を有する。 (effect)
As described above, the SPFS device according to the sixth embodiment has the same effect as the SPFS device 100 according to the first embodiment.
　なお、実施の形態１～６では、ＳＰＦＳ装置の検出動作において、ピペットチップ１７０の先端の位置情報を取得した後に、標識液や洗浄液をピペットチップ１７０に吸入したが、実施の形態２～４および６においては、標識液や洗浄液をピペットチップ１７０に吸入した状態でピペットチップ１７０の先端の位置情報を取得してもよい。また、実施の形態２および４においては、ピペットチップ１７０の先端の位置情報の取得と、流路６０内への標識液や洗浄液の注入操作とを同時に行うことができ、検出時間を短縮できる。さらに、実施の形態１～４および６では、ＳＰＦＳ装置を汚染することがない。 In the first to sixth embodiments, in the detection operation of the SPFS device, after acquiring the position information of the tip of the pipette tip 170, the labeling solution and the cleaning solution are sucked into the pipette tip 170. 6, the position information of the tip of the pipette tip 170 may be acquired in a state where the labeling solution or the cleaning solution is sucked into the pipette tip 170. In the second and fourth embodiments, the acquisition of the position information of the tip of the pipette tip 170 and the injection operation of the labeling liquid and the cleaning liquid into the flow channel 60 can be performed simultaneously, and the detection time can be shortened. Further, in Embodiments 1 to 4 and 6, the SPFS apparatus is not contaminated.
　また、実施の形態１～６では、本発明に係る反応装置を含む検出装置について説明したが、被検出物質を検出するための構成（例えば、搬送部１２０、光照射部１４０、光検出部１５０）などを含んでいなくてもよい。また、検出方法においても、検出工程（工程Ｓ１６０）を含んでいなくてもよい。 In the first to sixth embodiments, the detection apparatus including the reaction apparatus according to the present invention has been described. However, a configuration for detecting a substance to be detected (for example, the conveyance unit 120, the light irradiation unit 140, and the light detection unit 150). ) Etc. may not be included. Further, the detection method may not include the detection step (step S160).
　なお、実施の形態１～６においては、反応装置を含む検出装置としてＳＰＲを利用したＳＰＦＳ装置１００に適用した例を示したが、ＳＰＲを利用したＳＰＲ装置（反射光βを検出）に適用してもよい。この場合も実施の形態１～６と同様の効果を得られる。また、金属膜３０を配置していない分析チップを用いる検出系に適用してもよい。この場合は、ＳＰＲを利用せずに全反射測定（ＡＴＲ）法などにより測定する検出装置となるが、実施の形態１～６と同様の効果を得られる。 In the first to sixth embodiments, an example is shown in which the present invention is applied to the SPFS device 100 using SPR as a detection device including a reaction device. However, the present invention is applied to an SPR device (detecting reflected light β) using SPR. May be. In this case, the same effect as in the first to sixth embodiments can be obtained. Moreover, you may apply to the detection system using the analysis chip | tip which has not arrange | positioned the metal film 30. FIG. In this case, the detection apparatus performs measurement by the total reflection measurement (ATR) method or the like without using SPR, but the same effects as in the first to sixth embodiments can be obtained.
　本出願は、２０１５年２月２０日出願の特願２０１５－０３１６８９に基づく優先権を主張する。当該出願明細書および図面に記載された内容は、すべて本願明細書に援用される。 This application claims priority based on Japanese Patent Application No. 2015-031689 filed on Feb. 20, 2015. The contents described in the application specification and the drawings are all incorporated herein.
　本発明に係る反応方法および反応装置は、被検出物質を高い信頼性で測定することができる。よって、非常に簡易な定量免疫測定システムの開発、普及および発展に寄与することも期待される。 The reaction method and reaction apparatus according to the present invention can measure a substance to be detected with high reliability. Therefore, it is expected to contribute to the development, spread and development of a very simple quantitative immunoassay system.
　１０、１０’　検出チップ
　２０　プリズム
　２１　入射面
　２２　成膜面
　２３　出射面
　３０　金属膜
　４０　流路蓋
　４１　反応領域
　４２　試薬貯留領域
　４３　流路溝
　４４　第１貫通孔
　４５　第２貫通孔
　４６　凹部
　５０　シール
　６０　流路
　６０’　ウェル
　７０　注入部
　８０　貯留部
　１００　ＳＰＦＳ装置
　１１０　送液部
　１１１　ピペット
　１１２　ピペット移動部
　１１３　送液ポンプ移動機構
　１１４　シリンジ
　１１５　プランジャー
　１１６　ピペットノズル
　１２０　搬送部
　１２１　チップホルダー
　１２２　搬送ステージ
　１３０、２３０、３３０、４３０、５３０、６３０　位置情報取得部
　１３１　空気圧センサー
　１４０　光照射部
　１４１　光源ユニット
　１４２　角度調整機構
　１４３　光源制御部
　１５０　光検出部
　１５１　受光ユニット
　１５２　位置切り替え機構
　１５３　センサー制御部
　１５４　第１レンズ
　１５５　光学フィルター
　１５６　第２レンズ
　１５７　受光センサー
　１６０　制御部
　１７０　ピペットチップ
　１８０　基準部
　２３１　歪みセンサー
　３３１　静電容量センサー
　４３１　電源
　４３２　電流計
　５３１　感圧センサー
　６３１　光センサー
　α　励起光
　β　反射光
　γ　プラズモン散乱光
　δ　蛍光 DESCRIPTION OF SYMBOLS 10, 10 'Detection chip | tip 20 Prism 21 Incident surface 22 Film-forming surface 23 Output surface 30 Metal film 40 Channel cover 41 Reaction region 42 Reagent storage region 43 Channel groove 44 1st through-hole 45 2nd through-hole 46 Recess 50 Seal 60 flow path 60 'well 70 injection part 80 storage part 100 SPFS device 110 liquid feeding part 111 pipette 112 pipette moving part 113 liquid feeding pump moving mechanism 114 syringe 115 plunger 116 pipette nozzle 120 conveying part 121 chip holder 122 conveying stage 130, 230, 330, 430, 530, 630 Position information acquisition unit 131 Air pressure sensor 140 Light irradiation unit 141 Light source unit 142 Angle adjustment mechanism 143 Light source control unit 150 Light detection unit 151 Light reception unit 152 Position switching mechanism 153 Sensor control unit 154 First lens 155 Optical filter 156 Second lens 157 Light receiving sensor 160 Control unit 170 Pipette tip 180 Reference unit 231 Strain sensor 331 Capacitance sensor 431 Power supply 432 Ammeter 531 Pressure sensor 631 Optical sensor α Excitation light β Reflected light γ Plasmon scattered light δ fluorescence

Claims (20)

  1. 　ピペットノズルに装着されたピペットチップと、凹部および前記凹部の開口を封止しているシールを有する反応チップとを使用して、前記反応チップ内において２以上の物質を反応させる反応方法であって、
    　前記反応チップの前記シールを前記ピペットチップで突き破る第１工程と、
    　前記第１工程の後、前記ピペットチップの先端の位置情報を取得する第２工程と、
    　前記第２工程の後、前記ピペットチップの先端の位置情報に基づいて前記ピペットチップを操作して、前記反応チップ内において２以上の物質を反応させる第３工程と、を有する、
    　反応方法。     A reaction method in which two or more substances are reacted in a reaction chip using a pipette chip attached to a pipette nozzle and a reaction chip having a recess and a seal that seals the opening of the recess. ,
    A first step of breaking through the seal of the reaction tip with the pipette tip;
    After the first step, a second step of acquiring position information of the tip of the pipette tip;
    After the second step, the third step of operating the pipette tip based on the position information of the tip of the pipette tip to react two or more substances in the reaction tip,
    Reaction method.
  2. 　前記反応チップは、前記シールにより封止されている複数の前記凹部を有し、
    　前記第１工程では、前記ピペットチップが突き破るべき複数の前記シールのうち、突き破るために必要な力が最大である前記シールを前記ピペットチップで突き破る、
    　請求項１に記載の反応方法。     The reaction chip has a plurality of the recesses sealed by the seal,
    In the first step, among the plurality of seals to be pierced by the pipette tip, the pipette tip pierces the seal that requires the maximum force to pierce,
    The reaction method according to claim 1.
  3. 　前記反応チップは、前記シールにより封止されている複数の前記凹部を有し、
    　前記第２工程は、前記反応方法に含まれる全工程において前記ピペットチップの位置精度が最も求められる操作を行う前までに行われ、
    　前記第１工程では、前記ピペットチップの位置精度が最も求められる操作までに前記ピペットチップが突き破るべき複数の前記シールのうち、突き破るために必要な力が最大である前記シールを突き破る、
    　請求項１に記載の反応方法。     The reaction chip has a plurality of the recesses sealed by the seal,
    The second step is performed before performing the operation in which the position accuracy of the pipette tip is most required in all steps included in the reaction method,
    In the first step, out of a plurality of the seals that the pipette tip should break through before the operation where the positional accuracy of the pipette tip is most required, break through the seal that requires the maximum force to break through,
    The reaction method according to claim 1.
  4. 　前記ピペットノズルには、前記ピペットノズルの歪みを検出する歪みセンサーが配置されており、
    　前記第２工程では、前記ピペットチップの先端と、前記ピペットチップの先端の基準位置となる基準部とが接触したときの前記ピペットノズルの歪みを前記歪みセンサーにより測定することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１～３のいずれか一項に記載の反応方法。     The pipette nozzle is provided with a strain sensor for detecting the strain of the pipette nozzle,
    In the second step, the strain of the pipette tip is measured by the strain sensor when the tip of the pipette tip comes into contact with a reference portion serving as a reference position of the tip of the pipette tip. Get the position information of the tip,
    The reaction method according to any one of claims 1 to 3.
  5. 　前記ピペットノズルには、前記ピペットチップ内の空気圧を測定する空気圧センサーが接続されており、
    　前記第２工程では、前記ピペットチップの先端と前記ピペットチップの先端の基準位置となる基準部との間隔を変えつつ、前記ピペットチップの先端から気体を吸引または排出したときの前記ピペットチップ内の空気圧の変化を前記空気圧センサーにより測定することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１～３のいずれか一項に記載の反応方法。     The pipette nozzle is connected to an air pressure sensor that measures the air pressure in the pipette tip,
    In the second step, the inside of the pipette tip when the gas is sucked or discharged from the tip of the pipette tip while changing the distance between the tip of the pipette tip and the reference portion serving as a reference position of the tip of the pipette tip. By measuring the change in air pressure with the air pressure sensor, the position information of the tip of the pipette tip is obtained.
    The reaction method according to any one of claims 1 to 3.
  6. 　前記ピペットチップの先端の基準位置となる基準部には、静電容量センサーが配置されており、
    　前記第２工程では、前記ピペットチップの先端と前記基準部とを近づけたときの、前記ピペットチップの先端と前記基準部との間の静電容量の変化を前記静電容量センサーにより測定することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１～３のいずれか一項に記載の反応方法。     A capacitance sensor is arranged at a reference portion that is a reference position of the tip of the pipette tip,
    In the second step, the capacitance sensor measures a change in capacitance between the tip of the pipette tip and the reference portion when the tip of the pipette tip and the reference portion are brought close to each other. To obtain the position information of the tip of the pipette tip,
    The reaction method according to any one of claims 1 to 3.
  7. 　前記ピペットチップの先端と前記ピペットチップの先端の基準位置となる基準部とには、電源および電流計がそれぞれ接続されており、
    　前記第２工程では、前記ピペットチップの先端と前記基準部との間に前記電源により電圧を印加した状態で、前記ピペットチップの先端と前記基準部とを近づけたときの、前記ピペットチップの先端と前記基準部との間のインピーダンスを前記電流計により測定することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１～３のいずれか一項に記載の反応方法。     A power source and an ammeter are respectively connected to the tip of the pipette tip and the reference portion serving as the reference position of the tip of the pipette tip.
    In the second step, the tip of the pipette tip when the tip of the pipette tip and the reference portion are brought close to each other with a voltage applied by the power source between the tip of the pipette tip and the reference portion. By measuring the impedance between the reference portion and the ammeter, the position information of the tip of the pipette tip is obtained.
    The reaction method according to any one of claims 1 to 3.
  8. 　前記ピペットチップの先端の基準位置となる基準部には、感圧センサーが配置されており、
    　前記第２工程では、前記ピペットチップの先端と前記感圧センサーとの接触を前記感圧センサーにより検出することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１～３のいずれか一項に記載の反応方法。     A pressure-sensitive sensor is arranged at the reference portion that is the reference position of the tip of the pipette tip,
    In the second step, the position information of the tip of the pipette tip is obtained by detecting contact between the tip of the pipette tip and the pressure sensitive sensor with the pressure sensitive sensor.
    The reaction method according to any one of claims 1 to 3.
  9. 　前記第２工程では、前記ピペットチップの先端を光センサーにより検出することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１～３のいずれか一項に記載の反応方法。     In the second step, the position information of the tip of the pipette tip is obtained by detecting the tip of the pipette tip with an optical sensor.
    The reaction method according to any one of claims 1 to 3.
  10. 　前記反応チップは、使い捨てであり、
    　前記基準部は、前記反応チップに含まれる、
    　請求項４～７のいずれか一項に記載の反応方法。     The reaction chip is disposable,
    The reference part is included in the reaction chip.
    The reaction method according to any one of claims 4 to 7.
  11. 　凹部および前記凹部の開口を封止しているシールを有する反応チップを使用し、ピペットチップで前記シールを突き破った後に前記反応チップ内において２以上の物質を反応させる反応装置であって、
    　前記反応チップを保持するチップホルダーと、
    　前記ピペットチップを着脱可能なピペットノズルを有するピペットと、
    　前記ピペットを移動させるピペット移動部と、
    　前記ピペットチップの先端の位置情報を取得する位置情報取得部と、を有し、
    　前記位置情報取得部は、前記ピペット移動部により移動させられた前記ピペットチップが前記シールを１回または２回以上突き破った後に、前記ピペットチップの先端の位置情報を取得し、
    　前記ピペット移動部は、前記位置情報取得部が前記ピペットチップの先端の位置情報を取得した後に、前記反応チップ内において２以上の物質を反応させるために前記ピペットチップの先端の位置情報に基づいて１回または２回以上前記ピペットを移動させる、
    　反応装置。     Using a reaction chip having a recess and a seal sealing the opening of the recess, and a reaction apparatus for reacting two or more substances in the reaction chip after breaking through the seal with a pipette chip,
    A chip holder for holding the reaction chip;
    A pipette having a pipette nozzle to which the pipette tip can be attached and detached;
    A pipette moving section for moving the pipette;
    A position information acquisition unit that acquires position information of the tip of the pipette tip,
    The position information acquisition unit acquires the position information of the tip of the pipette tip after the pipette tip moved by the pipette moving unit breaks the seal once or twice or more,
    The pipette moving unit is configured to allow the two or more substances to react in the reaction chip after the position information acquisition unit acquires the position information of the tip of the pipette tip based on the position information of the tip of the pipette tip. Move the pipette one or more times,
    Reactor.
  12. 　前記反応チップは、前記シールにより封止されている複数の前記凹部を有し、
    　前記位置情報取得部は、前記ピペットチップが突き破るべき複数の前記シールのうち、突き破るために必要な力が最大である前記シールを前記ピペットチップが突き破った後に、前記ピペットチップの先端の位置情報を取得する、
    　請求項１１に記載の反応装置。     The reaction chip has a plurality of the recesses sealed by the seal,
    The position information acquisition unit obtains position information of the tip of the pipette tip after the pipette tip has pierced the seal that requires the maximum force among the plurality of seals to be pierced by the pipette tip. get,
    The reaction apparatus according to claim 11.
  13. 　前記反応チップは、前記シールにより封止されている複数の前記凹部を有し、
    　前記位置情報取得部は、前記ピペット移動部が前記ピペットを移動させる全操作において前記ピペットチップの位置精度が最も求められる操作を行う前であって、かつ当該操作までに前記ピペットチップが突き破るべき複数の前記シールのうち、突き破るために必要な力が最大である前記シールを前記ピペットチップが突き破った後に、前記ピペットチップの先端の位置情報を取得する、
    　請求項１１に記載の反応装置。     The reaction chip has a plurality of the recesses sealed by the seal,
    The position information acquisition unit includes a plurality of pipette tips that must be broken by the operation before the operation in which the pipette tip is required to have the highest position accuracy in all the operations in which the pipette moving unit moves the pipette. After the pipette tip breaks through the seal that has the maximum force required to break through the seal, the position information of the tip of the pipette tip is obtained.
    The reaction apparatus according to claim 11.
  14. 　前記位置情報取得部は、前記ピペットノズルの歪みを検出する歪みセンサーを含み、
    　前記位置情報取得部は、前記ピペット移動部が、前記ピペットチップの先端と、前記ピペットチップの先端の基準位置となる基準部とを接触させたときの前記ピペットノズルの歪みを前記歪みセンサーが測定することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１１～１３のいずれか一項に記載の反応装置。     The position information acquisition unit includes a distortion sensor that detects distortion of the pipette nozzle,
    In the position information acquisition unit, the strain sensor measures the distortion of the pipette nozzle when the pipette moving unit contacts the tip of the pipette tip and the reference portion that is the reference position of the tip of the pipette tip. To obtain the position information of the tip of the pipette tip,
    The reaction apparatus according to any one of claims 11 to 13.
  15. 　前記位置情報取得部は、前記ピペットチップ内の空気圧を測定する空気圧センサーを含み、
    　前記位置情報取得部は、前記ピペット移動部が、前記ピペットチップの先端と、前記ピペットチップの先端の基準位置となる基準部との間隔を変えつつ、前記ピペットが前記ピペットチップの先端から気体を吸引または排出したときの前記ピペットチップ内の空気圧の変化を前記空気圧センサーが測定することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１１～１３のいずれか一項に記載の反応装置。     The position information acquisition unit includes an air pressure sensor that measures air pressure in the pipette tip,
    In the position information acquisition unit, the pipette moving unit changes the distance between the tip of the pipette tip and a reference portion that is a reference position of the tip of the pipette tip, and the pipette releases gas from the tip of the pipette tip. The air pressure sensor measures the change in air pressure in the pipette tip when aspirating or discharging, thereby obtaining positional information of the tip of the pipette tip.
    The reaction apparatus according to any one of claims 11 to 13.
  16. 　前記位置情報取得部は、前記ピペットチップの先端の基準位置となる基準部に配置された静電容量センサーを含み、
    　前記位置情報取得部は、前記ピペット移動部が前記ピペットチップの先端と前記基準部とを近づけたときの前記ピペットチップの先端と前記基準部との間の静電容量の変化を前記静電容量センサーが測定することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１１～１３のいずれか一項に記載の反応装置。     The position information acquisition unit includes a capacitance sensor arranged in a reference unit serving as a reference position of the tip of the pipette tip,
    The position information acquisition unit is configured to detect a change in capacitance between the tip of the pipette tip and the reference unit when the pipette moving unit brings the tip of the pipette tip close to the reference unit. The sensor measures to obtain the position information of the tip of the pipette tip.
    The reaction apparatus according to any one of claims 11 to 13.
  17. 　前記位置情報取得部は、前記ピペットチップの先端と前記ピペットチップの先端の基準位置となる基準部とにそれぞれ接続された電源および電流計を含み、
    　前記位置情報取得部は、前記電源が前記ピペットチップの先端と前記基準部との間に電圧を印加した状態で、前記ピペット移動部が前記ピペットチップの先端と前記基準部とを近づけたときの、前記ピペットチップの先端と前記基準部との間のインピーダンスを前記電流計が測定することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１１～１３のいずれか一項に記載の反応装置。     The position information acquisition unit includes a power source and an ammeter respectively connected to a tip portion of the pipette tip and a reference portion serving as a reference position of the tip of the pipette tip.
    The position information acquisition unit is configured such that when the power source applies a voltage between the tip of the pipette tip and the reference unit, the pipette moving unit brings the tip of the pipette tip and the reference unit close to each other. The ammeter measures the impedance between the tip of the pipette tip and the reference portion, thereby obtaining positional information of the tip of the pipette tip.
    The reaction apparatus according to any one of claims 11 to 13.
  18. 　前記位置情報取得部は、前記ピペットチップの先端の基準位置となる基準部に配置された感圧センサーを含み、
    　前記位置情報取得部は、前記ピペット移動部が前記ピペットチップの先端と前記基準部とを接触させたときの前記ピペットチップと前記感圧センサーとの接触を前記感圧センサーが検出することで、前記ピペットチップの先端の位置情報を取得する、
    　請求項１１～１３のいずれか一項に記載の反応装置。     The position information acquisition unit includes a pressure-sensitive sensor disposed in a reference unit serving as a reference position of the tip of the pipette tip.
    The position information acquisition unit is configured so that the pressure sensor detects contact between the pipette tip and the pressure sensor when the pipette moving unit contacts the tip of the pipette tip and the reference unit. Obtaining position information of the tip of the pipette tip;
    The reaction apparatus according to any one of claims 11 to 13.
  19. 　前記位置情報取得部は、前記ピペットチップの先端の位置を検出できる光センサーを含む、請求項１１～１３のいずれか一項に記載の反応装置。 The reaction apparatus according to any one of claims 11 to 13, wherein the position information acquisition unit includes an optical sensor capable of detecting a position of a tip of the pipette tip.
  20. 　前記反応チップは、使い捨てであり、
    　前記基準部は、前記反応チップに含まれる、
    　請求項１４～１７のいずれか一項に記載の反応装置。     The reaction chip is disposable,
    The reference part is included in the reaction chip.
    The reaction apparatus according to any one of claims 14 to 17.
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