WO2018179950A1 - Puce de capteur pour système de détection d'échantillon - Google Patents

Puce de capteur pour système de détection d'échantillon Download PDF

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Publication number
WO2018179950A1
WO2018179950A1 PCT/JP2018/005167 JP2018005167W WO2018179950A1 WO 2018179950 A1 WO2018179950 A1 WO 2018179950A1 JP 2018005167 W JP2018005167 W JP 2018005167W WO 2018179950 A1 WO2018179950 A1 WO 2018179950A1
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WIPO (PCT)
Prior art keywords
flow path
sensor chip
liquid
metal film
excitation light
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PCT/JP2018/005167
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English (en)
Japanese (ja)
Inventor
祐輝 三宅
洋一 青木
野田　哲也
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コニカミノルタ株式会社
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Priority to JP2019508728A priority Critical patent/JP6885458B2/ja
Publication of WO2018179950A1 publication Critical patent/WO2018179950A1/fr

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    • 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
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/15—Preventing contamination of the components of the optical system or obstruction of the light path

Definitions

  • the present invention relates to a sensor chip used in an immunoassay (immunoassay) for measuring the presence or absence of a substance to be measured and its amount, and more specifically, a surface plasmon applying a surface plasmon resonance (SPR) phenomenon.
  • the present invention relates to a sensor chip used in a specimen detection system such as a resonance device or a surface plasmon excitation enhanced fluorescence measuring device based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS: Surface Plasmon-field enhanced Fluorescence Spectroscopy).
  • specimen detection methods when detecting a very small substance, various specimen detection methods have been proposed that can detect such a substance by applying a physical phenomenon of the substance.
  • a specimen detection method for example, by using an antigen-antibody reaction between an antigen, which is a measurement target substance contained in a sample solution, and an antibody or antigen labeled with a labeling substance, the presence or absence of the measurement target substance or its Immunoassay methods (immunoassays) for measuring amounts are known.
  • the immunoassay examples include an enzyme immunoassay (EIA) using an enzyme as a labeling substance, and a fluorescence immunoassay (FIA) using a fluorescent substance as a labeling substance.
  • EIA enzyme immunoassay
  • FIA fluorescence immunoassay
  • a specimen detection device using a fluorescence immunoassay method a phenomenon in which high light output is obtained by resonating electrons and light in a fine region such as a nanometer level (SPR: Surface Plasmon Resonance)
  • SPR apparatus surface plasmon resonance apparatus that detects minute alanite in a living body is used.
  • SPFS device based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) using surface plasmon resonance (SPR) phenomenon, analyte detection can be performed with higher accuracy than SPR equipment.
  • SPFS device The surface plasmon excitation enhanced fluorescence spectrometer (hereinafter also referred to as “SPFS device”) is one of such specimen detection devices.
  • surface plasmon excitation enhanced fluorescence spectroscopy SPFS
  • surface plasmon light is applied to the surface of the metal film under the condition that excitation light such as laser light emitted from a light source attenuates total reflection (ATR) on the surface of the metal film.
  • excitation light such as laser light emitted from a light source attenuates total reflection (ATR) on the surface of the metal film.
  • ATR total reflection
  • a sensor chip including a dielectric member, a metal film adjacent to the upper surface of the dielectric member, and a liquid holding member disposed on the upper surface of the metal film is used.
  • a reaction field having a ligand for capturing an analyte is provided on a metal film.
  • the analyte By supplying a sample solution containing the analyte to the liquid holding member, the analyte is captured by the ligand (primary reaction). In this state, a liquid (labeling liquid) containing a secondary antibody labeled with a fluorescent substance is introduced into the liquid holding member. In the solution holding member, the analyte captured by the ligand is labeled with a fluorescent substance by an antigen-antibody reaction (secondary reaction).
  • the fluorescent material is excited by the surface plasmon light generated on the surface of the metal film, and fluorescence is generated from the fluorescent material. By detecting this fluorescence, the presence or absence of the analyte and the amount thereof can be measured.
  • a well member is used as a liquid holding member, and a sample solution is used in which a sample liquid is stored in the well member, and a sample liquid is used as a liquid holding member.
  • a flow channel chip type hereinafter simply referred to as “flow channel chip” in which a specimen test is performed in a state where the analyte is trapped in a reaction field in the flow channel by flowing the gas through the flow channel.
  • FIG. 7 is a schematic diagram for explaining the structure of a conventional channel chip.
  • the channel chip 200 is formed by bonding a dielectric member 202 and a channel lid 204 with a channel seal 206.
  • the channel lid 204 is provided with an inlet 204a and a reservoir 204b for injecting a sample solution and the like.
  • a space surrounded by the dielectric member 202, the flow path lid 204, and the flow path seal 206 is a flow path 208.
  • a ligand that is an antibody specific to an analyte or a fragment thereof is contained in this flow path 208.
  • a fixed reaction field 210 is provided.
  • the channel 208 has a width of 0.5 mm to 3 mm and a height of 50 ⁇ m to 500 ⁇ m.
  • a circulation type that can improve the reaction efficiency by repeatedly passing the sample solution through the reaction field 210 or A reciprocating liquid feeding method is often used.
  • the liquid L cannot be completely removed from the flow path 208, and the liquid remains near the inlet 204a of the flow path 208 as shown in FIG.
  • the suction of the liquid L is stopped, as shown in FIG. 8C, the liquid L remaining in the vicinity of the injection port 204a may return to the vicinity of the center of the flow path 208 due to capillary action. .
  • the channel lid 204 bends inward, as shown in FIG.
  • bubbles A are often generated.
  • An object of the present invention is to provide a sensor chip for a specimen detection system that can suppress the generation of bubbles in a flow path and prevent a decrease in measurement accuracy in view of the current situation.
  • Sensor chip for A sensor chip having a reaction field inside for capturing an analyte, A flow path having the reaction field; A first through hole formed at one end of the flow path, The channel is provided with a gradient so that the height of the channel near the reaction field is higher than the height of the channel near the first through hole.
  • the flow path structure for keeping the residual liquid in the flow path at a predetermined position suppresses the generation of bubbles due to the residual liquid and prevents the measurement accuracy from being lowered. be able to. Further, since it is possible to suppress the generation of bubbles due to the residual liquid, it is possible to inject and suck the reagent many times in the same flow path, and there is no need to provide an extra flow path in the sensor chip. For this reason, it is possible to reduce the manufacturing cost of the sensor chip and the specimen detection apparatus and contribute to downsizing.
  • FIG. 1 is a schematic diagram for explaining a configuration of a surface plasmon excitation enhanced fluorescence spectrometer (SPFS apparatus) according to an embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing an example of a sensor chip used in the SPFS apparatus of FIG.
  • FIG. 3 is a schematic diagram for explaining the state of the liquid when the liquid is reciprocated into the flow path in the sensor chip of FIG.
  • FIG. 4 is a schematic diagram showing a modification of the sensor chip used in the SPFS device of FIG.
  • FIG. 5 is a schematic diagram showing another modification of the sensor chip used in the SPFS device of FIG.
  • FIG. 6 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus of FIG.
  • FIG. 7 is a schematic diagram for explaining the structure of a conventional channel chip.
  • FIG. 8 is a schematic diagram for explaining the state of the liquid when the liquid is reciprocated into the flow path in the flow path chip of FIG.
  • FIG. 1 is a schematic diagram for explaining a configuration of a surface plasmon excitation enhanced fluorescence spectrometer (SPFS apparatus) according to an embodiment of the present invention
  • FIG. 2 is an example of a sensor chip used in the SPFS apparatus of FIG. It is a schematic diagram which shows.
  • the SPFS device 10 includes an excitation light irradiation unit 20, a fluorescence detection unit 30, a liquid feeding unit 40, a transport unit 50, and a control unit 80.
  • the SPFS device 10 is used in a state where the sensor chip 100 is mounted on the chip holder 54 of the transport unit 50.
  • the sensor chip 100 includes a dielectric member 102 having an incident surface 102a, a film formation surface 102b, and an emission surface 102c, a metal film 104 formed on the film formation surface 102b, and a film formation surface 102b or And a flow path forming member 106 fixed on the metal film 104.
  • the sensor chip 100 is replaced for each specimen test.
  • the sensor chip 100 is preferably a structure having a length of several millimeters to several centimeters on each side, but is a smaller structure or a larger structure that is not included in the category of “chip”. It doesn't matter.
  • the dielectric member 102 can be a prism made of a dielectric that is transparent to the excitation light ⁇ .
  • the incident surface 102 a of the dielectric member 102 is a surface on which the excitation light ⁇ irradiated from the excitation light irradiation unit 20 is incident on the inside of the dielectric member 102.
  • a metal film 104 is formed on the film formation surface 102b.
  • the excitation light ⁇ incident on the inside of the dielectric member 102 is reflected at the interface between the metal film 104 and the film formation surface 102b of the dielectric member 102 (hereinafter referred to as “the back surface of the metal film 104” for convenience), and the emission surface.
  • the excitation light ⁇ is emitted to the outside of the dielectric member 102 through 102c.
  • the shape of the dielectric member 102 is not particularly limited, and the dielectric member 102 shown in FIGS. 1 and 2 is a prism formed of a hexahedron having a substantially trapezoidal vertical cross-sectional shape (a truncated quadrangular pyramid shape). Also, a prism having a vertical cross-sectional shape of a triangle (a so-called triangular prism), a semicircular shape, and a semielliptical shape can be used.
  • the incident surface 102 a is formed so that the excitation light ⁇ does not return to the excitation light irradiation unit 20.
  • the light source of the excitation light ⁇ is, for example, a laser diode (hereinafter also referred to as “LD”)
  • LD laser diode
  • the angle of the incident surface 102a is set so that the excitation light ⁇ does not enter the incident surface 102a perpendicularly in the scanning range centered on the ideal enhancement angle.
  • the resonance angle (and the enhancement angle in the vicinity thereof) is generally determined by the design of the sensor chip 100.
  • the design elements are the refractive index of the dielectric member 102, the refractive index of the metal film 104, the film thickness of the metal film 104, the extinction coefficient of the metal film 104, the wavelength of the excitation light ⁇ , and the like.
  • the resonance angle and the enhancement angle are shifted by the analyte immobilized on the metal film 104, but the amount is less than a few degrees.
  • the dielectric member 102 has a considerable amount of birefringence.
  • the material of the dielectric member 102 includes, for example, various inorganic materials such as glass and ceramics, natural polymers, synthetic polymers, and the like, and is excellent in chemical stability, manufacturing stability, optical transparency, and low birefringence. Is preferred.
  • the material is not particularly limited as described above. In providing, for example, it is preferably formed from a resin material.
  • the method for manufacturing the dielectric member 102 is not particularly limited, but injection molding using a mold is preferable from the viewpoint of manufacturing cost.
  • the dielectric member 102 is formed from a resin material, for example, polyolefins such as polyethylene (PE) and polypropylene (PP), polycyclic olefins such as cyclic olefin copolymer (COC) and cyclic olefin polymer (COP), polystyrene, Polycarbonate (PC), acrylic resin, triacetyl cellulose (TAC), or the like can be used.
  • PE polyethylene
  • PP polypropylene
  • polycyclic olefins such as cyclic olefin copolymer (COC) and cyclic olefin polymer (COP), polystyrene, Polycarbonate (PC), acrylic resin, triacetyl cellulose (TAC), or the like
  • PC polycarbonate
  • TAC triacetyl cellulose
  • the metal film 104 is formed on the film formation surface 102 b of the dielectric member 102.
  • an interaction (surface plasmon resonance) occurs between the photon of the excitation light ⁇ incident on the film formation surface 102b under the total reflection condition and the free electrons in the metal film 104, and is locally on the surface of the metal film 104. In-situ light can be generated.
  • the material of the metal film 104 is not particularly limited as long as it is a metal capable of causing surface plasmon resonance.
  • a metal capable of causing surface plasmon resonance.
  • at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum is used.
  • Such a metal is suitable as the metal film 104 because it is stable against oxidation and has a large electric field enhancement by surface plasmon light.
  • the method for forming the metal film 104 is not particularly limited, and examples thereof include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. It is done.
  • the sputtering method or the vapor deposition method is used because the adjustment of the metal film forming conditions is easy.
  • the thickness of the metal film 104 is not particularly limited, but is preferably in the range of 5 to 500 nm, and more preferably gold, silver, copper, In the case of platinum, it is preferably in the range of 20 to 70 nm, in the case of aluminum, 10 to 50 nm, and in the case of these alloys, it is preferably in the range of 10 to 70 nm.
  • the thickness of the metal film 104 is within the above range, it is preferable that surface plasmon light is easily generated.
  • the size (length ⁇ width) dimensions and shape are not particularly limited.
  • a ligand for capturing the analyte is immobilized on the surface of the metal film 104 that does not face the dielectric member 102 (hereinafter referred to as “the surface of the metal film 104” for convenience). Has been. By immobilizing the ligand, the analyte can be selectively detected.
  • the ligand is uniformly immobilized in a predetermined region (reaction field 116) on the metal film 104.
  • the type of the ligand is not particularly limited as long as the analyte can be captured.
  • the ligand is an analyte specific antibody or fragment thereof.
  • the flow path forming member 106 is disposed on the film formation surface 102 b of the dielectric member 102 or the metal film 104.
  • the flow path forming member 106 is joined to the dielectric member 102 or the metal film 104 by an adhesive sheet (flow path seal 114) in which through holes are formed, and the dielectric member 102, the flow path forming member 106, A space surrounded by the flow path seal 114, that is, a through hole of the flow path seal 114 is used as the flow path 112.
  • the flow path forming member 106 is not limited to this.
  • a flow path groove is formed on the film formation surface 102 b or the surface facing the metal film 104, and the flow path formation member 106 is formed on the metal film 104.
  • the reaction field 116 is disposed so as to cover the space surrounded by the flow path forming member 106 and the dielectric member 102, that is, the flow path groove, for supplying the sample liquid, the labeling liquid, the cleaning liquid, and the like. It can be used as the channel 112.
  • the flow path forming member 106 can be bonded to the dielectric member 102 or the metal film 104 by, for example, adhesion using an adhesive or a transparent adhesive sheet, laser welding, ultrasonic welding, or pressure bonding using a clamp member. it can.
  • the width of the flow path 112 formed in this way in the vicinity of the reaction field 116 is preferably 0.1 mm to 5 mm, and the length is preferably 10 mm to 50 mm.
  • the height of the flow path 112 in the vicinity of the first through hole 110a is preferably 50 ⁇ m to 500 ⁇ m.
  • the flow path forming member 106 has a first through hole 110 a formed at one end of the flow path 112 and a second through hole 110 b formed at the other end of the flow path 112.
  • each of the first through hole 110a and the second through hole 110b has a substantially cylindrical shape.
  • the first through hole 110a and the second through hole 110b serve as an inlet for injecting a sample liquid, a labeling liquid, a cleaning liquid, and the like into the flow path 112, and an outlet for taking out the sample liquid, the labeling liquid, the cleaning liquid, and the like. Function.
  • the material of the flow path forming member 106 is not particularly limited as long as it is formed of a material that is optically transparent to at least the fluorescent ⁇ described later, but the sensor chip 100 that is inexpensive and excellent in handleability is used. In providing, for example, it is preferably formed from a resin material.
  • the manufacturing method of the flow path forming member 106 is not particularly limited, but injection molding using a mold is preferable from the viewpoint of manufacturing cost.
  • the flow path forming member 106 is formed from a resin material, for example, polyesters such as polyethylene terephthalate (PET) and polyethylene naphthalate, polyolefins such as polyethylene (PE) and polypropylene (PP), cyclic olefin copolymer (COC), Polycyclic olefins such as cyclic olefin polymer (COP), vinyl resins such as polyvinyl chloride and polyvinylidene chloride, polystyrene, polyetheretherketone (PEEK), polysulfone (PSF), polyethersulfone (PES), polycarbonate (PC), polyamide, polyimide, acrylic resin, triacetyl cellulose (TAC) and the like can be used.
  • PET polyethylene terephthalate
  • PP polypropylene
  • COC cyclic olefin copolymer
  • COP Polycyclic olefins
  • vinyl resins such as polyvinyl chlor
  • the flow path forming member 106 is provided with a gradient 118 so that the height h2 in the vicinity of the reaction field 116 of the flow path 112 is higher than the height h1 in the vicinity of the first through hole 110a.
  • the height h2 of the flow path 112 in the vicinity of the reaction field 116 is preferably 0.1 mm to 1 mm.
  • the liquid L is injected from the first through hole 110a into the flow path 112 as shown in FIG. 3A, and the flow path from the first through hole 110a as shown in FIG. 3B. Even if the liquid L in the 112 is sucked and the liquid L remains in the vicinity of the first through hole 110a of the flow path 112, even if the suction is stopped as it is, as shown in FIG. It is possible to prevent the liquid L from staying in the vicinity of the first through hole 110a and returning to the vicinity of the reaction field 116.
  • the gradient 118 provided in the flow path forming member 106 only needs to be configured so that the height of the flow path 112 in the vicinity of the reaction field 116 is higher than the height in the vicinity of the first through hole 110a.
  • the gradient 118 is such that the height in the vicinity of the reaction field 116 is the highest in the flow path 112 and the height of the flow path 112 decreases as it approaches the first through hole 110 a and the second through hole 110 b.
  • a gradient 118 may be provided so that the height of the flow path 112 increases from the first through hole 110a toward the second through hole 110b. .
  • the gradient 118 may be a curved surface. Further, as shown in FIG. 4C, the gradient 118 can be a plurality of planes. However, from the viewpoint of preventing liquid pooling when the liquid is sent to the flow path 112, FIG. 2 and FIG. As shown to (a), it is preferable that the surface which forms the flow path 112 is as smooth as possible.
  • the gradient 118 can be provided on the dielectric member 102 as shown in FIG. Furthermore, as shown in FIG. 5B, a gradient 118 can be provided on both the flow path forming member 106 and the dielectric member 102.
  • the sensor chip 100 configured as described above is mounted on the chip holder 54 of the transport unit 50 of the SPFS apparatus 10, and specimen detection is performed by the SPFS apparatus 10.
  • the SPFS apparatus 10 includes the excitation light irradiation unit 20, the fluorescence detection unit 30, the liquid feeding unit 40, the transport unit 50, and the control unit 80.
  • the excitation light irradiation unit 20 irradiates the sensor chip 100 held by the chip holder 54 with the excitation light ⁇ . As will be described later, when measuring the fluorescence ⁇ , the excitation light irradiation unit 20 directs only the P wave with respect to the metal film 104 toward the incident surface 102a so that the incident angle with respect to the metal film 104 is an angle that causes surface plasmon resonance. And exit.
  • the “excitation light” is light that directly or indirectly excites the fluorescent substance.
  • the excitation light ⁇ is irradiated through the dielectric member 102 at an angle at which surface plasmon resonance occurs on the metal film 104, local field light that excites the fluorescent material is generated on the surface of the metal film 104. Light.
  • the excitation light irradiation unit 20 includes a configuration for emitting the excitation light ⁇ toward the dielectric member 102 and a configuration for scanning the incident angle of the excitation light ⁇ with respect to the back surface of the metal film 104.
  • the excitation light irradiation unit 20 includes a light source unit 21, an angle adjustment mechanism 22, and a light source control unit 23.
  • the light source unit 21 irradiates collimated excitation light ⁇ having a constant wavelength and light amount so that the irradiation spot has a substantially circular shape on the back surface of the metal film 104.
  • the light source unit 21 includes, for example, a light source of excitation light ⁇ , a beam shaping optical system, an APC (Automatic Power-Control) mechanism, and a temperature adjustment mechanism (all not shown).
  • the type of light source is not particularly limited, and includes, for example, a laser diode (LD), a light emitting diode, a mercury lamp, and other laser light sources.
  • LD laser diode
  • 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 only a part thereof.
  • the collimator collimates the excitation light ⁇ irradiated from the light source.
  • the band-pass filter turns the excitation light ⁇ irradiated 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 ⁇ irradiated 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 104.
  • 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 104 is a circle having 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 temperature adjustment mechanism is, for example, a heater or a Peltier element.
  • the wavelength and energy of the light emitted from the light source may vary depending on the temperature. For this reason, the wavelength and energy of the light emitted from the light source are controlled to be constant by keeping the temperature of the light source constant by the temperature adjusting mechanism.
  • the angle adjustment mechanism 22 adjusts the incident angle of the excitation light ⁇ to the metal film 104.
  • the angle adjustment mechanism 22 makes the optical axis of the excitation light ⁇ and the chip holder 54 relative to each other. Rotate.
  • the angle adjusting mechanism 22 rotates the light source unit 21 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 104 hardly changes even when the incident angle is scanned.
  • the angle at which the maximum amount of plasmon scattered light can be obtained is the enhancement angle.
  • the basic incident condition of the excitation light ⁇ is determined by the material and shape of the dielectric member 102 of the sensor chip 100, the film thickness of the metal film 104, the refractive index of the sample liquid in the flow path 112, and the like.
  • the optimum incident condition varies slightly depending on the type and amount of the analyte in the flow path 112, the shape error of the dielectric member 102, and the like. For this reason, it is preferable to obtain an optimal enhancement angle for each specimen test.
  • the light source control unit 23 controls various devices included in the light source unit 21 to control the irradiation of the excitation light ⁇ of the light source unit 21.
  • the light source control unit 23 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 fluorescence detection unit 30 detects the fluorescence ⁇ generated from the fluorescent material excited by the irradiation of the excitation light ⁇ to the metal film 104. If necessary, the fluorescence detection unit 30 also detects plasmon scattered light generated by the irradiation of the excitation light ⁇ to the metal film 104.
  • the fluorescence detection unit 30 includes, for example, a light receiving unit 31, a position switching mechanism 37, and a sensor control unit 38.
  • the light receiving unit 31 is disposed in the normal direction of the metal film 104 of the sensor chip 100 (z-axis direction in FIG. 1).
  • the light receiving unit 31 includes a first lens 32, an optical filter 33, a second lens 34, and a light receiving sensor 35.
  • the first lens 32 is, for example, a condensing lens and condenses light generated on the metal film 104.
  • the second lens 34 is, for example, an imaging lens, and forms an image of the light collected by the first lens 32 on the light receiving surface of the light receiving sensor 35.
  • the optical path between both lenses 32 and 34 is a substantially parallel optical path.
  • the optical filter 33 is disposed between the lenses 32 and 34.
  • the optical filter 33 guides only the fluorescent component to the light receiving sensor 35 and removes the excitation light component (plasmon scattered light) in order to detect the fluorescent ⁇ with high S / N.
  • the optical filter 33 includes, for example, an excitation light reflection filter, a short wavelength cut filter, and a band pass filter.
  • the optical filter 33 is, for example, a filter including a multilayer film that reflects a predetermined light component, but may be a colored glass filter that absorbs the predetermined light component.
  • the light receiving sensor 35 detects fluorescence ⁇ .
  • the light receiving sensor 35 is not particularly limited as long as it has a high sensitivity and can detect weak fluorescence ⁇ from a fluorescent substance labeled with a very small amount of analyte.
  • a multiplier tube (PMT), an avalanche photodiode (APD), a low noise photodiode (PD), or the like can be used.
  • the position switching mechanism 37 switches the position of the optical filter 33 on or off the optical path in the light receiving unit 31. Specifically, when the light receiving sensor 35 detects the fluorescence ⁇ , the optical filter 33 is disposed on the optical path of the light receiving unit 31, and when the light receiving sensor 35 detects plasmon scattered light, the optical filter 33 is placed on the light receiving unit 31. Place outside the optical path.
  • the position switching mechanism 37 includes, for example, a rotation driving unit and a known mechanism (such as a turntable or a rack and pinion) that moves the optical filter 33 in the horizontal direction by using a rotational motion.
  • the sensor control unit 38 controls detection of an output value of the light receiving sensor 35, management of sensitivity of the light receiving sensor 35 based on the detected output value, change of sensitivity of the light receiving sensor 35 for obtaining an appropriate output value, and the like.
  • the sensor control unit 38 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 liquid feeding unit 40 supplies a sample liquid, a labeling liquid, a cleaning liquid, and the like into the flow path 112 of the sensor chip 100 mounted on the chip holder 54.
  • the liquid feeding unit 40 includes a syringe pump 41, a pipette nozzle 46, a pipette tip 45, and a liquid feeding pump drive mechanism 44.
  • the liquid feeding unit 40 is used with a pipette tip 45 attached to the tip of the pipette nozzle 46. If the pipette tip 45 is replaceable, the pipette tip 45 need not be washed, and contamination of impurities can be prevented.
  • the syringe pump 41 includes a syringe 42 and a plunger 43 that can reciprocate inside the syringe 42. By the reciprocating motion of the plunger 43, the liquid is sucked and discharged quantitatively.
  • the liquid feed pump driving mechanism 44 includes a driving device for the syringe pump 41 and a moving device for the pipette nozzle 46 to which the pipette tip 45 is attached.
  • the drive device of the syringe pump 41 is a device for reciprocating the plunger 43, and includes, for example, a stepping motor.
  • a drive device including a stepping motor is preferable from the viewpoint of managing the remaining liquid amount of the sensor chip 100 because it can manage the liquid feeding amount and the liquid feeding speed of the syringe pump 41.
  • the moving device of the pipette nozzle 46 freely moves the pipette nozzle 46 in two directions, that is, an axial direction (for example, a vertical direction) of the pipette nozzle 46 and a direction crossing the axial direction (for example, a horizontal direction).
  • the moving device of the pipette nozzle 46 is constituted by, for example, a robot arm, a two-axis stage, or a turntable that can move up and down.
  • the liquid feeding unit 40 is provided at the tip of the pipette tip 45. It is preferable to further have a mechanism for detecting the position.
  • the liquid feeding unit 40 sucks various liquids from a liquid storage unit (not shown) and supplies them into the flow path 112 of the sensor chip 100. At this time, by moving the plunger 43, the liquid reciprocates in the flow path 112 of the sensor chip 100, and the liquid in the flow path 112 is stirred. As a result, it is possible to make the liquid concentration uniform and promote the reaction (for example, antigen-antibody reaction) in the flow path 112.
  • the inlet (first through hole 110a) of the sensor chip 100 is protected by the multilayer film 111 and the first through hole when the pipette chip 45 penetrates the multilayer film.
  • the sensor chip 100 and the pipette chip 45 are preferably configured so that the 110a can be sealed.
  • the lid seal 120 that covers the upper opening of the second through hole 110b is affixed, and serves as a reservoir for temporarily storing the injected liquid through the flow path.
  • the lid seal 120 has a minute hole for air removal.
  • the liquid in the flow path 112 is again sucked by the syringe pump 41 and discharged to a waste liquid portion (not shown).
  • reaction with various liquids, washing, and the like can be performed, and the analyte labeled with the fluorescent substance can be immobilized in the reaction field 116 in the flow path 112.
  • the transport unit 50 transports and fixes the sensor chip 100 mounted on the chip holder 54 to the liquid feeding position or the measurement position by the user.
  • the “liquid feeding position” is a position where the liquid feeding unit 40 supplies the liquid into the flow path 112 of the sensor chip 100 or removes the liquid in the flow path 112.
  • the “measurement position” is a position where the excitation light irradiation unit 20 irradiates the sensor chip 100 with the excitation light ⁇ , and the fluorescence detection unit 30 detects the fluorescence ⁇ generated therewith.
  • the transfer unit 50 includes a transfer stage 52 and a chip holder 54.
  • the chip holder 54 is fixed to the transfer stage 52 and holds the sensor chip 100 in a detachable manner.
  • the shape of the chip holder 54 is not particularly limited as long as it can hold the sensor chip 100 and does not obstruct the optical paths of the excitation light ⁇ and the fluorescence ⁇ .
  • the chip holder 54 is provided with an opening through which excitation light ⁇ and fluorescence ⁇ pass.
  • the transfer stage 52 is configured to be able to move the chip holder 54 in one direction (x-axis direction in FIG. 1) and in the opposite direction.
  • the transport stage 52 is driven by, for example, a stepping motor.
  • FIG. 6 is a flowchart illustrating an example of an operation procedure of the SPFS apparatus 10.
  • the user attaches the sensor chip 100 to the chip holder 54 of the transport unit 50 (S100).
  • the controller 80 operates the transport stage 52 to move the sensor chip 100 mounted on the chip holder 54 to the liquid feeding position (S110).
  • the control unit 80 operates the liquid feeding unit 40 to introduce the cleaning liquid stored in the liquid storage unit (not shown) into the flow path 112, clean the flow path 112, and remove the storage reagent in the flow path 112. It is removed (S120).
  • the cleaning liquid used for cleaning is discharged by the liquid feeding unit 40, and instead, the measurement liquid stored in a liquid storage unit (not shown) is introduced into the flow path 112. If the result of the enhancement angle detection (S140) in the subsequent step is not affected, the preserving reagent cleaning solution and the measurement solution can be used together, and the enhancement angle measurement can be performed without discharging the cleaning solution.
  • control unit 80 operates the transport stage 52 to transport the sensor chip 100 mounted on the chip holder 54 to the measurement position (S130). Then, the control unit 80 operates the excitation light irradiation unit 20 and the fluorescence detection unit 30 to irradiate the sensor chip 100 with the excitation light ⁇ and to detect and enhance the plasmon scattered light having the same wavelength as the excitation light ⁇ . A corner is detected (S140).
  • control unit 80 operates the excitation light irradiation unit 20 to scan the incident angle of the excitation light ⁇ with respect to the metal film 104 and operates the fluorescence detection unit 30 to detect plasmon scattered light. At this time, the control unit 80 operates the position switching mechanism 37 to place the optical filter 33 outside the optical path of the light receiving unit 31. And the control part 80 determines the incident angle of the excitation light (alpha) when the light quantity of plasmon scattered light is the maximum as an enhancement angle.
  • control unit 80 operates the excitation light irradiation unit 20 and the fluorescence detection unit 30 to irradiate the sensor chip 100 arranged at the measurement position with the excitation light ⁇ , and outputs the output value (optical blank value) of the light receiving sensor 35. ) Is recorded (S150).
  • control unit 80 operates the angle adjustment mechanism 22 to set the incident angle of the excitation light ⁇ to the enhancement angle. Further, the control unit 80 operates the position switching mechanism 37 to place the optical filter 33 in the optical path of the light receiving unit 31.
  • control unit 80 operates the transport stage 52 to move the sensor chip 100 to the liquid feeding position (S160). Then, the controller 80 operates the liquid feeding unit 40 to discharge the measurement liquid in the flow path 112 and introduce the sample liquid stored in a liquid storage section (not shown) into the flow path 112 (S170). In the channel 112, the analyte is captured in the reaction field on the metal film 104 by the antigen-antibody reaction (primary reaction).
  • sample liquid used here is a liquid prepared using a specimen, and for example, a specimen and a reagent are mixed to perform a treatment for binding a fluorescent substance to an analyte contained in the specimen. Things. Examples of such specimens include blood, serum, plasma, urine, nasal fluid, saliva, stool, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.).
  • the analyte contained in the sample is, for example, a nucleic acid (DNA that may be single-stranded or double-stranded, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., or nucleoside Nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules thereof, Specific examples thereof include a complex, and may be a carcinoembryonic antigen such as AFP ( ⁇ -fetoprotein), a tumor marker, a signal transduction substance, a hormone, and the like, and is not particularly limited.
  • AFP ⁇ -fetoprotein
  • the control unit 80 operates the liquid feeding unit 40 to introduce the labeled liquid stored in a liquid storage unit (not shown) into the flow path 112 (S190).
  • the analyte captured on the metal film 104 is labeled with a fluorescent substance by an antigen-antibody reaction (secondary reaction).
  • a liquid containing a secondary antibody labeled with a fluorescent substance can be used as the labeling liquid.
  • the labeling liquid in the flow path 112 is removed, the flow path 112 is washed with the cleaning liquid, and after the cleaning liquid is removed, the measurement liquid is introduced into the flow path 112 (S200).
  • control unit 80 operates the transfer stage 52 to move the sensor chip 100 to the measurement position (S210).
  • control unit 80 operates the excitation light irradiation unit 20 and the fluorescence detection unit 30 to irradiate the sensor chip 100 arranged at the measurement position with the excitation light ⁇ and to label the analyte captured by the ligand.
  • the fluorescent ⁇ emitted from the fluorescent material to be detected is detected (S220). Based on the intensity of the detected fluorescence ⁇ , it can be converted into the amount or concentration of the analyte as required.
  • the enhancement angle detection (S140) and the optical blank value measurement (S150) are performed before the primary reaction (S170). However, the enhancement angle detection is performed after the primary reaction (S170). (S140) Optical blank value measurement (S150) may be performed.
  • the detection of the enhancement angle (S140) may be omitted.
  • the secondary reaction (S190) for labeling the analyte with a fluorescent substance is performed after the primary reaction (S170) for reacting the analyte and the ligand (two-step method).
  • the timing for labeling the analyte with a fluorescent substance is not particularly limited.
  • a labeling solution may be added to the sample solution to label the analyte with a fluorescent substance in advance.
  • the analyte labeled with the fluorescent substance is captured by the ligand.
  • the analyte is labeled with a fluorescent substance, and the analyte is captured by the ligand.
  • both the primary reaction and the secondary reaction can be completed by introducing the sample solution into the channel 112 (one-step method).
  • the enhancement angle detection (S140) is performed before the antigen-antibody reaction.
  • the SPFS apparatus has been described in the above embodiment, but the sample detection system according to the present invention has an SPR. It can be applied to a specimen detection system using a fluorescent immunoassay (FIA) such as an apparatus, a specimen detection system using an enzyme immunoassay (EIA), etc. Can be changed.
  • FIA fluorescent immunoassay
  • EIA enzyme immunoassay

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  • General Physics & Mathematics (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

Le problème décrit par la présente invention est de fournir une puce de capteur pour système de détection d'échantillon qui est capable de supprimer l'apparition de bulles dans un trajet d'écoulement et d'empêcher une dégradation de la précision de mesure. A cet effet, le trajet d'écoulement est conçu pour présenter une pente telle à ce que la hauteur du trajet d'écoulement à proximité d'un site de réaction soit supérieure à la hauteur du trajet d'écoulement au voisinage d'un premier trou traversant.
PCT/JP2018/005167 2017-03-30 2018-02-15 Puce de capteur pour système de détection d'échantillon WO2018179950A1 (fr)

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JP2016161546A (ja) * 2015-03-05 2016-09-05 キヤノン株式会社 マイクロ流路チップ
WO2017003079A1 (fr) * 2015-06-30 2017-01-05 한국표준과학연구원 Dispositif de mesure à base de microcanal à immersion, à base de silicium, à incidence de prisme, à incidence oblique et procédé de mesure

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Publication number Priority date Publication date Assignee Title
JP2007071542A (ja) * 2005-09-02 2007-03-22 Fujifilm Holdings Corp 測定セル保持機構、及び、バイオセンサー
JP2007071837A (ja) * 2005-09-09 2007-03-22 Fujifilm Holdings Corp 全反射減衰を利用した測定装置
WO2011062377A2 (fr) * 2009-11-23 2011-05-26 Korea Research Institute Of Standards And Science Appareil et procédé pour quantifier la cinétique de liaison et de dissociation d'interactions moléculaires
JP2011257266A (ja) * 2010-06-09 2011-12-22 Konica Minolta Holdings Inc 光学センサおよび光学センサに用いられるチップ構造体
WO2014007134A1 (fr) * 2012-07-05 2014-01-09 コニカミノルタ株式会社 Capteur sur puce
US20150253243A1 (en) * 2012-10-15 2015-09-10 Korea Research Institute Of Standards And Science Apparatus and method for simultaneously measuring characteristics of molecular junctions and refractive index of buffer solution
WO2016093039A1 (fr) * 2014-12-09 2016-06-16 コニカミノルタ株式会社 Puce de détection et procédé de détection
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WO2017003079A1 (fr) * 2015-06-30 2017-01-05 한국표준과학연구원 Dispositif de mesure à base de microcanal à immersion, à base de silicium, à incidence de prisme, à incidence oblique et procédé de mesure

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020158192A1 (fr) * 2019-01-31 2020-08-06 コニカミノルタ株式会社 Procédé de mesure optique et dispositif de mesure optique

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