WO2019221040A1

WO2019221040A1 - Specimen detecting chip, and specimen detecting device employing same

Info

Publication number: WO2019221040A1
Application number: PCT/JP2019/018863
Authority: WO
Inventors: 高敏彼谷
Original assignee: コニカミノルタ株式会社
Priority date: 2018-05-16
Filing date: 2019-05-13
Publication date: 2019-11-21
Also published as: JPWO2019221040A1

Abstract

[Problem] To provide a specimen detecting chip and a specimen detecting device employing the same, with which it is possible to suppress a deterioration in detection accuracy resulting from a residue of a liquid in a well during a reaction step, with which it is possible to suppress the effects on a detection result of the liquid level of the liquid in the well during a detecting step, and with which the size of the specimen detecting device can be reduced. [Solution] The specimen detecting chip is provided with a side wall member including a reaction field for capturing an analyte, and a pipette tip disposed adjacent to the side wall member, wherein an opening portion is provided in the pipette tip such that the reaction field is in contact with a liquid introduced into the pipette tip, and the pipette tip and a dielectric member are arranged.

Description

Sample detection chip and sample detection apparatus using the same

The present invention relates to a sample detection device for detecting a substance to be measured contained in a sample detection chip (sensor chip) and a sample detection chip used therefor.

Conventionally, 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.
As such 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.

As an immunoassay, a fluorescent immunoassay (FIA) using a fluorescent substance as a labeling substance is known.
For example, as 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) For example, a surface plasmon resonance device (hereinafter, also referred to as “SPR device”) that detects minute analytes in a living body is used.

Further, as disclosed in Patent Document 1, based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS: Surface Plasmon-field enhanced Fluorescence Spectroscopy) applying the surface plasmon resonance (SPR) phenomenon, rather than an SPR device. A surface plasmon excitation enhanced fluorescence spectrometer (hereinafter also referred to as “SPFS device”) that can perform analyte detection with higher accuracy is one of such specimen detection devices.

In the SPFS device, 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. In such a sensor chip, a reaction field having a ligand for capturing an analyte is provided on a metal film.

The analyte is captured by the ligand (primary reaction) by supplying the sample liquid containing the analyte to the liquid holding member. 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 liquid holding member, the analyte captured by the ligand is labeled with a fluorescent substance by an antigen-antibody reaction (secondary reaction).

In this state, when the excitation light is irradiated to the metal film at an angle at which surface plasmon resonance occurs through the dielectric member, 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.

As a liquid holding member of such a sensor chip, as disclosed in Patent Document 2, a well member for temporarily storing a sample liquid or the like, a sample liquid or the like with respect to a reaction field on a metal film A flow path member that can be circulated is used.

JP 2006-208069 A International Publication No. 2014/017433

In the sensor chip (well type sensor chip) using the well member described in Patent Document 2, since a metal film and a reaction field are provided on the bottom surface of the well, when removing the liquid in the well, a pipette or the like is used. There is a possibility that the tip of the liquid feeding means may come into contact with the metal film or the reaction field and break them. For this reason, the tip of the liquid feeding means cannot be pressed against the bottom surface of the well, and it has been difficult to sufficiently remove the liquid in the well. Thus, if the liquid remains in the well, various reactions do not proceed appropriately, and the detection accuracy may be reduced.

Further, in the specimen detection apparatus described in Patent Document 2, the detection unit is disposed above the well and detects fluorescence that has passed through the liquid surface of the liquid in the well. For this reason, when the inner diameter of the well is small, the fluorescence detection result is affected by the meniscus. Even if the inner diameter of the well is large, the fluorescence detection result may be affected by bubbles present on the liquid surface.

Further, in the well type sensor chip, the amount of the necessary sample solution becomes relatively large, and accordingly, the specimen detection apparatus using the well type sensor chip tends to be enlarged.

The present invention can suppress a decrease in detection accuracy due to the remaining liquid in the well during the reaction process, and can suppress the influence on the detection result due to the liquid level of the liquid in the well during the detection process. An object of the present invention is to provide a specimen detection chip that can be used and a specimen detection apparatus using the same.

The present invention also provides a sample detection chip capable of reducing the size of the sample detection device and a sample detection device using the same, while the amount of the necessary sample solution is equivalent to that of the well-type sensor chip. Objective.

The present invention was invented in order to solve the above-described problems in the prior art, and in order to achieve at least one of the above-described objects, a sample detection chip reflecting one aspect of the present invention. Is
An analyte detection chip used for analyte detection,
A sidewall member having a reaction field for capturing the analyte;
A pipette tip disposed adjacent to the side wall member,
The pipette tip is provided with an opening, and the pipette tip and the dielectric member are disposed so that the reaction field contacts the liquid introduced into the pipette tip.

In addition, a specimen detection apparatus reflecting one aspect of the present invention is
A sample detection apparatus that detects a sample using the sensor chip described above,
An excitation light irradiation unit that irradiates the metal film with excitation light via a dielectric member;
A fluorescence detection unit that detects fluorescence generated from the fluorescence-labeled analyte captured in the reaction field based on the excitation light irradiated to the metal film;
The pipette tip of the sensor tip is mounted, and includes a transport unit that moves the pipette tip and sucks and discharges the liquid in the pipette tip.

In addition, a specimen detection apparatus reflecting one aspect of the present invention is
A sample detection apparatus that detects a sample using the sensor chip described above,
An excitation light irradiation unit for irradiating the diffraction grating with excitation light;
A measurement light detection unit that detects fluorescence generated from fluorescently labeled analyte captured in the reaction field based on excitation light irradiated to the diffraction grating, and reflected light of the excitation light reflected by the diffraction grating;
A pipette tip of a sensor tip is attached, and a transport unit that moves the pipette tip and sucks and discharges the liquid in the pipette tip is provided.

According to the present invention, it is possible to suppress a decrease in detection accuracy due to remaining liquid in the well during the reaction process, and to suppress an influence on the detection result due to the liquid level of the liquid in the well during the detection process. be able to.

Further, since the pipette tip and the dielectric member and diffraction grating, which are optical elements, are integrated, the wells that have been used conventionally are no longer necessary, and thus the sample detection apparatus can be downsized.

FIG. 1 is a schematic diagram for explaining the configuration of a prism-coupled surface plasmon excitation enhanced fluorescence spectrometer (PC-SPFS apparatus) according to an embodiment of the present invention. FIG. 2 is a schematic diagram for explaining a configuration of a sensor chip according to an embodiment of the present invention. FIG. 3 is a schematic diagram for explaining a modified example of the sensor chip. FIG. 4 is a flowchart illustrating an example of an operation procedure of the PC-SPFS apparatus. FIG. 5 is a schematic diagram for explaining the configuration of a diffraction grating coupled surface plasmon excitation enhanced fluorescence spectrometer (GC-SPFS apparatus) according to another embodiment of the present invention. FIG. 6 is a schematic diagram for explaining a configuration of a sensor chip according to an embodiment of the present invention. FIG. 7 is a perspective view for explaining an example of the diffraction grating.

Hereinafter, embodiments (examples) of the present invention will be described in more detail based on the drawings.
FIG. 1 is a schematic diagram for explaining the configuration of a prism-coupled surface plasmon excitation enhanced fluorescence spectrometer (PC-SPFS apparatus) according to an embodiment of the present invention, and FIG. 2 shows an embodiment of the present invention. It is a schematic diagram for demonstrating the structure of the sensor chip which concerns, FIG. 2 (A) is sectional drawing in alignment with the height direction of a sensor chip, FIG.2 (B) is sectional drawing in alignment with the horizontal direction of a sensor chip. is there.

As shown in FIG. 1, the PC-SPFS apparatus 10 in this embodiment includes an excitation light irradiation unit 20, a fluorescence detection unit 30, a transport unit 40, and a control unit 50. Note that the PC-SPFS apparatus 10 in the present embodiment is used in a state where the sensor chip 100 is mounted on the transport unit 40.

As shown in FIG. 2, 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 It has a metal film 104 and a pipette tip 106 fixed thereto. In the present embodiment, the dielectric member 102 that is an optical element, the metal film 104, and a reaction field described later form a side wall member. Usually, the sensor chip 100 is replaced for each specimen test.

The dielectric member 102 can be a prism made of a dielectric that is transparent to the excitation light α. The incident surface 102 a of the dielectric member 102 is a surface on which the excitation light α 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 α 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 α 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 α does not return to the excitation light irradiation unit 20. When the light source of the excitation light α is, for example, 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 are changed. It will fluctuate. Therefore, the angle of the incident surface 102a is set so that the excitation light α does not enter the incident surface 102a perpendicularly in the scanning range centered on the ideal enhancement angle.

It should be noted that 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 α, 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.

As long as it is made of a material that is at least optically transparent to the excitation light α and low birefringence, 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.

When 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.

The metal film 104 is formed on the film formation surface 102 b of the dielectric member 102. As a result, an interaction (surface plasmon resonance) occurs between the photon of the excitation light α incident on the film formation surface 102b under total reflection conditions and free electrons in the metal film 104, and is enhanced on the surface of the metal film 104. An electric field 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. For example, at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum is used. Made of gold, more preferably made of gold, and may be made of an alloy of these metals. 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.

Further, 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. Preferably, 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.

If the thickness of the metal film 104 is within the above range, it is preferable that surface plasmon light is easily generated. In addition, as long as the metal film 104 has such a thickness, the size (length × width) dimensions and shape are not particularly limited.

Although not shown in FIGS. 1 and 2, 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.

In this embodiment, the ligand is uniformly immobilized in a predetermined region (reaction field) on the metal film 104. The type of the ligand is not particularly limited as long as the analyte can be captured. In this embodiment, the ligand is an analyte specific antibody or fragment thereof.

As shown in FIG. 2, the pipette tip 106 has a substantially rectangular tube-shaped body portion 108 and a tip portion 110 having a liquid suction / discharge hole 110a. In addition, an opening 106 b is provided in the pipette tip 106 so that the reaction field on the metal film 104 and the liquid in the pipette tip 106 are in contact with each other, and the pipette tip 106 and the dielectric member 102 are disposed. The pipette tip 106 has a mounting hole 106c that can be mounted on a pipette nozzle 46 of the transport unit 40, which will be described later.

In the present embodiment, the body portion 108 of the pipette tip 106 has a substantially rectangular tube shape. However, at least the side wall 108a having the fixing surface 106a with the dielectric member 102 is flat, and the generated fluorescent γ If it is the shape which does not interfere with the fluorescence detection unit 30 detecting, it will not specifically limit. In this specification, “the side wall is a flat surface” means that both the inner surface and the outer surface of the side wall of the pipette tip 106 are flat surfaces.

FIG. 3 is a schematic diagram for explaining a modified example of the sensor chip 100.
As shown in FIG. 3A, only the side wall disposed between the reaction field and the fluorescence detection unit 30, that is, the

side walls

108a and 108b disposed on the optical path of the fluorescence γ is a flat surface, and the other side walls. Can also be curved.

Further, as shown in FIG. 3B, only the side wall 108a having the fixing surface 106a may be a flat surface, and the other side wall may be a curved surface. In addition, as shown in FIGS. 3C to 3E, at least one of the inner surface and the outer surface of the side wall 108b facing the side wall 108a having the fixing surface 106a in the body portion 108 can be a convex curved surface. Thus, by making the inner surface and / or outer surface of the side wall 108b a convex curved surface, the side wall 108b functions as a cylindrical lens and can collect the fluorescent γ generated in the reaction field.

The pipette tip 106 is formed of a material that is transparent to at least light having the wavelength of fluorescence γ, and preferably formed of a material that is transparent to light having the wavelength of excitation light α and light having the wavelength of fluorescence γ. ing. However, a part of the pipette tip 106 may be made of a material that is opaque to light as long as it does not interfere with light extraction in the detection method described later.

The material of the pipette tip 106 includes, for example, various inorganic materials such as glass and ceramics, natural polymers, synthetic polymers, and the like, and materials excellent in chemical stability, manufacturing stability, optical transparency, and low birefringence are used. preferable.

The pipette tip 106 can be joined 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 the like.

The dielectric member 102 is preferably fixed to the body portion 108 of the pipette tip 106. By configuring in this way, the dielectric member 102 can be prevented from coming into contact with the liquid when the liquid is introduced from the liquid reservoir 60 into the pipette tip 106, as will be described later.

Also, the pipette tip 106 may be provided with a filter inside. By providing a filter inside the pipette tip 106, contamination caused by contact of liquid and gas (aerosol by evaporation from the liquid) that is sucked and discharged through the pipette tip 106 to the connection between the pipette nozzle 46 and the pipette tip 106 Can be prevented. As such a filter, for example, a hydrophobic porous filter such as a fluororesin (PTFE) can be used.

The sensor chip 100 configured as described above may be provided with a positioning portion in order to accurately stop at a later-described supply / drainage position or measurement position. The positioning part is, for example, a hole or notch that fits with a positioning pin arranged at the supply / drain liquid position or measurement position, or conversely, a protrusion that fits with a positioning hole arranged at the supply / drain liquid position or measurement position. Or part.

The positioning portion may be provided on the pipette tip 106 or the pipette nozzle 46.
For example, by forming a shape having a characteristic such as a convex portion as a positioning portion in the pipette nozzle 46, it is possible to suppress complication of the shape of the pipette tip 106 and displacement of the positioning portion of the pipette tip 106.

Next, each component of the PC-SPFS apparatus 10 will be described. As described above, the PC-SPFS apparatus 10 in the present embodiment is provided with the excitation light irradiation unit 20, the fluorescence detection unit 30, the transport unit 40, and the control unit 50.

The excitation light irradiation unit 20 irradiates the sensor chip 100 at the measurement position with the excitation light α. As will be described later, when measuring the fluorescence γ, 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.

Here, the “excitation light” is light that directly or indirectly excites the fluorescent substance. For example, when the excitation light α 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 α toward the dielectric member 102 and a configuration for scanning the incident angle of the excitation light α with respect to the back surface of the metal film 104. In the present embodiment, 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 α 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 α, 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. 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. Further, 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.

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 α irradiated from the light source. The band-pass filter turns the excitation light α irradiated 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 irradiated 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 104. 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 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 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.

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 α to the metal film 104. In order to irradiate the excitation light α at a predetermined incident angle toward a predetermined position of the metal film 104 via the dielectric member 102, the angle adjustment mechanism 22 makes the optical axis of the excitation light α and the sensor chip 100 relative to each other. Rotate.

For example, 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 α. At this time, 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. By setting the position of the rotation center 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 formation surface 102b and the incident surface 102a), the irradiation position Can be minimized.

Among the incident angles of the excitation light α to the metal film 104, the angle at which the maximum amount of plasmon scattered light can be obtained is the enhancement angle. By setting the incident angle of the excitation light α to an enhancement angle or an angle in the vicinity thereof, it becomes possible to measure high-intensity fluorescence γ.

Note that the basic incident condition of the excitation light α depends on the material and shape of the dielectric member 102 of the sensor chip 100, the thickness of the metal film 104, the type of metal to be configured, the refractive index of the sample liquid in the pipette chip 106, and the like. However, the optimum incident condition varies slightly depending on the type and amount of the analyte in the pipette tip 106, 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 α 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 γ generated from the fluorescent material excited by the irradiation of the excitation light α 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 α 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 γ 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 γ. The light receiving sensor 35 is not particularly limited as long as it has a high sensitivity and can detect weak fluorescence γ 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 γ, 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 transport unit 40 with the sensor chip 100 attached, sucks and discharges liquids such as sample liquid, labeling liquid, and washing liquid in the pipette chip 106 and moves the pipette chip 106. The transport unit 40 includes a syringe pump 41, a pipette nozzle 46, a pump drive mechanism 44, and a pipette nozzle moving mechanism 45.

The transport unit 40 is used with the pipette tip 106 of the sensor chip 100 attached to the tip of the pipette nozzle 46.

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 pump drive mechanism 44 is a device for reciprocating the plunger 43 in order to drive the syringe pump 41, and includes, for example, a stepping motor. A drive device including a stepping motor is preferable from the viewpoint of managing the amount of liquid remaining in the pipette tip 106 because it can manage the amount and rate of liquid delivery of the syringe pump 41.

The pipette nozzle moving mechanism 45 freely moves the pipette nozzle 46 in two directions, for example, 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 transport unit 40 sucks various liquids from the liquid storage unit 60 by the syringe pump 41 and supplies them into the pipette chip 106 of the sensor chip 100. Thereby, a primary reaction or a secondary reaction can be caused in the pipette tip 106.

Further, the transport unit 40 moves the sensor chip 100 to the supply / drainage position or the measurement position with the sensor chip 100 mounted on the pipette nozzle 46. Here, the “supply / drainage position” is a position where the liquid is supplied to or removed from the pipette tip 106 from the liquid reservoir 60. The “measurement position” is a position where the excitation light irradiation unit 20 irradiates the sensor chip 100 with the excitation light α, and the fluorescence detection unit 30 detects the fluorescence γ generated therewith.

Hereinafter, the flow of specimen detection using the PC-SPFS apparatus 10 will be described. FIG. 4 is a flowchart illustrating an example of an operation procedure of the PC-SPFS apparatus 10.
First, the user attaches the sensor chip 100 to the pipette nozzle 46 of the transport unit 40 (S100).
The control unit 50 operates the pipette nozzle moving mechanism 45 to move the sensor chip 100 attached to the pipette nozzle 46 to the supply / drainage position (S110).

Next, the control unit 50 operates the pipette nozzle moving mechanism 45 and the pump drive mechanism 44 to introduce the cleaning liquid into the pipette tip 106 from the liquid storage unit 60, and cleans the inside of the pipette tip 106 to keep the moisture on the reaction field. The agent is removed (S120). During cleaning, the control unit 50 may agitate the cleaning liquid in the pipette tip 106 by operating the pipette nozzle moving mechanism 45 to vibrate the sensor chip 100. The cleaning liquid used for cleaning can be discharged to the liquid storage section 60 or a waste liquid section (not shown).

Thereafter, the control unit 50 operates the pipette nozzle moving mechanism 45 and the pump drive mechanism 44 to introduce the measurement liquid from the liquid storage unit 60 into the pipette tip 106 (S130). If there is no influence on the result of the enhancement angle detection (S150) in the subsequent step, the enhancement angle measurement can be performed as it is without discharging the cleaning liquid by using both the cleaning liquid and the measurement liquid.

Next, the control unit 50 operates the pipette nozzle moving mechanism 45 to move the sensor chip 100 to the measurement position (S140). Then, the control unit 50 operates the excitation light irradiation unit 20 and the fluorescence detection unit 30 to irradiate the sensor chip 100 with the excitation light α and to detect and enhance the plasmon scattered light having the same wavelength as the excitation light α. A corner is detected (S150).

Specifically, the control unit 50 operates the excitation light irradiation unit 20 to scan the incident angle of the excitation light α 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 50 operates the position switching mechanism 37 to place the optical filter 33 outside the light path of the light receiving unit 31. And the control part 50 determines the incident angle of the excitation light (alpha) when the light quantity of a plasmon scattered light is the maximum as an enhancement angle.

Next, the control unit 50 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 α, and outputs the output value (optical blank value) of the light receiving sensor 35. ) Is recorded (S160).

At this time, the control unit 50 operates the angle adjustment mechanism 22 to set the incident angle of the excitation light α to the enhancement angle. Further, the control unit 50 operates the position switching mechanism 37 to place the optical filter 33 in the optical path of the light receiving unit 31.

Next, the control unit 50 operates the pipette nozzle moving mechanism 45 to move the sensor chip 100 to the supply / drainage position (S170).
Then, the control unit 50 operates the pipette nozzle moving mechanism 45 and the pump drive mechanism 44 to discharge the measurement liquid in the pipette tip 106 and introduce the sample liquid stored in the liquid storage unit 60 into the pipette tip 106. (S180). In the pipette tip 106, the analyte is captured in the reaction field on the metal film 104 by the antigen-antibody reaction (primary reaction). During the primary reaction, the control unit 50 may agitate the sample liquid in the pipette chip 106 by operating the pipette nozzle moving mechanism 45 to vibrate the sensor chip 100.

Note that the 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 (α-fetoprotein), a tumor marker, a signal transduction substance, a hormone, and the like, and is not particularly limited.

Thereafter, the control unit 50 operates the pipette nozzle moving mechanism 45 and the pump drive mechanism 44 to discharge the sample liquid in the pipette tip 106, introduce a cleaning liquid into the pipette tip 106, and wash the inside of the pipette tip 106. (S190).

Next, the control unit 50 operates the pipette nozzle moving mechanism 45 and the pump drive mechanism 44 to introduce the labeling liquid stored in the liquid storage unit 60 into the pipette tip 106 (S200). In the pipette chip 106, the analyte captured on the metal film 104 is labeled with a fluorescent substance by an antigen-antibody reaction (secondary reaction). As the labeling liquid, a liquid containing a secondary antibody labeled with a fluorescent substance can be used. In the secondary reaction, the controller 50 may operate the pipette nozzle moving mechanism 45 to vibrate the sensor chip 100 so that the labeling liquid in the pipette chip 106 is agitated.

Thereafter, the control unit 50 operates the pipette nozzle moving mechanism 45 and the pump drive mechanism 44 to discharge the labeling liquid in the pipette tip 106, introduce a cleaning liquid into the pipette tip 106, and wash the inside of the pipette tip 106. (S210).

Next, the control unit 50 operates the pipette nozzle moving mechanism 45 and the pump drive mechanism 44 to discharge the cleaning liquid in the pipette tip 106 and introduce the measurement liquid into the pipette tip 106 (S220).

Next, the control unit 50 operates the pipette nozzle moving mechanism 45 to move the sensor chip 100 to the measurement position (S230).

Next, the control unit 50 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 α and to label the analyte captured by the ligand. The fluorescent γ emitted from the fluorescent substance to be detected is detected (S240). Based on the intensity of the detected fluorescence γ, it can be converted into the amount or concentration of the analyte as required.

By the above procedure, the presence or amount of the analyte in the sample solution can be detected.
In this embodiment, enhancement angle detection (S150) and optical blank value measurement (S160) are performed before the primary reaction (S180). However, enhancement angle detection (S150) is performed after the primary reaction (S180). ), Optical blank value measurement (S160) may be performed.

Further, when the incident angle of the excitation light α is determined in advance, the detection of the enhancement angle (S150) may be omitted.

In the above description, the secondary reaction (S200) for labeling the analyte with a fluorescent substance is performed after the primary reaction (S180) for reacting the analyte and the ligand (two-step method). However, the timing for labeling the analyte with a fluorescent substance is not particularly limited.

For example, before introducing the sample solution into the pipette tip 106, a labeling solution can be added to the sample solution to label the analyte with a fluorescent substance in advance. Further, by simultaneously injecting the sample solution and the labeling solution into the pipette tip 106, the analyte labeled with the fluorescent substance is captured by the ligand. In this case, the analyte is labeled with a fluorescent substance, and the analyte is captured by the ligand.

In any case, by introducing the sample solution into the pipette tip 106, both the primary reaction and the secondary reaction can be completed (one-step method). Thus, when adopting the one-step method, the enhancement angle detection (S150) is performed before the antigen-antibody reaction.

FIG. 5 is a schematic diagram for explaining a configuration of a diffraction grating coupled surface plasmon excitation enhanced fluorescence spectrometer (GC-SPFS apparatus) according to another embodiment of the present invention, and FIG. 6 is an embodiment of the present invention. FIG. 6A is a schematic view for explaining a configuration of a sensor chip according to the embodiment, FIG. 6A is a cross-sectional view along the height direction of the sensor chip, and FIG. 6B is a cross-section along the horizontal direction of the sensor chip. FIG.

In this embodiment, the same components as those of the PC-SPFS apparatus 10 described with reference to FIGS. 1 to 4 are denoted by the same reference numerals, and detailed description thereof is omitted.

As shown in FIG. 5, the GC-SPFS apparatus 70 in this embodiment includes an excitation light irradiation unit 20, a fluorescence detection unit 30, a transport unit 40, and a control unit 50. Note that the GC-SPFS device 70 in this embodiment is used in a state where the sensor chip 200 is mounted on the transport unit 40.

As shown in FIG. 6, the sensor chip 200 includes a substrate 202 having a film formation surface 202a, a metal film 104 formed on the film formation surface 202a, and a pipette chip 106 fixed to the film formation surface 202a or the metal film 104. And have.

In addition, a diffraction grating 203 as an optical element is formed on the substrate 202 at least at a part corresponding to the reaction field on the metal film 104. In this embodiment, the substrate 202 having a diffraction grating, which is an optical element, the metal film 104, and the reaction field form a side wall member. Usually, the sensor chip 200 is replaced for each specimen test.

The shape of the substrate 202 is not particularly limited. In addition, the substrate 202 can be formed of materials such as various inorganic materials such as glass and ceramics, natural polymers, and synthetic polymers.

As described above, the diffraction grating 203 is formed on the substrate 202. The diffraction grating 203 only needs to be formed on at least a part of the portion corresponding to the reaction field on the metal film 104, and may be formed on the entire surface of the substrate 202 on the pipette tip 106 side. It may be formed only in part.

When the excitation light α is irradiated to the diffraction grating 203 from the excitation light irradiation unit 20, the surface plasmon generated in the metal film 104 and the evanescent light generated by the diffraction grating 203 are combined to generate surface plasmon resonance, and the surface of the metal film 104 is A localized enhanced electric field can be generated.

The diffraction grating 203 generates evanescent light when the excitation light α is irradiated. The shape of the diffraction grating 203 is not particularly limited as long as it can generate evanescent light.

For example, the diffraction grating 203 may be a one-dimensional diffraction grating as shown in FIG. 7A or a two-dimensional diffraction grating as shown in FIG. 7B. In the one-dimensional diffraction grating shown in FIG. 7A, a plurality of ridges parallel to each other are formed on the surface of the substrate 202 at a predetermined interval. In the two-dimensional diffraction grating shown in FIG. 7B, convex portions having a predetermined shape are periodically arranged on the surface of the substrate 202. Examples of the arrangement of the convex portions include a square lattice, a triangular (hexagonal) lattice, and the like. Examples of the cross-sectional shape of the diffraction grating 203 include a rectangular wave shape, a sine wave shape, and a sawtooth shape. The pitch of the diffraction grating 203 is preferably in the range of 100 to 2000 nm from the viewpoint of generating surface plasmon resonance. In this specification, the “diffraction grating pitch” refers to the center-to-center distance Λ of protrusions in the arrangement direction of the protrusions, as shown in FIGS. In this embodiment, the diffraction grating 203 is arranged so that the arrangement direction of the convex portions is along the depth direction of the pipette tip 106.

The formation method of the diffraction grating 203 is not particularly limited. For example, the metal film 104 may be formed on the film formation surface 202 a of the flat substrate 202 and then the uneven shape may be imparted to the metal film 104. Alternatively, the metal film 104 may be formed on the film formation surface 202a of the substrate 202 that has been provided with an uneven shape in advance.

In the present embodiment, the pipette tip 106 is formed of a material that is at least transparent to light having the wavelength of excitation light α and light having a wavelength of fluorescence γ. However, a part of the pipette tip 106 may be made of a material that is opaque to light as long as it does not interfere with light extraction in the detection method described later.

The pipette tip 106 can be bonded to the substrate 202 or the metal film 104 by, for example, adhesion using an adhesive or a transparent adhesive sheet, laser welding, ultrasonic welding, or the like.

The substrate 202 is preferably fixed to the body portion 108 of the pipette tip 106. With this configuration, the substrate 202 can be prevented from coming into contact with the liquid when the liquid is introduced from the liquid storage unit 60 into the pipette tip 106, as will be described later.

Next, each component of the GC-SPFS device 70 will be described. The GC-SPFS apparatus 70 in this embodiment is provided with an excitation light irradiation unit 20, a measurement light detection unit 80, a transport unit 40, and a control unit 50.

The excitation light irradiation unit 20 irradiates the sensor chip 100 at the measurement position with the excitation light α. At this time, the excitation light α is applied to the diffraction grating 203 via the pipette tip 106.

The excitation light irradiation unit 20 applies the excitation light α to the diffraction grating 203 so that the plane including the optical axis of the excitation light α and the optical axis of the reflected light β of the excitation light α is along the arrangement direction of the convex portions of the diffraction grating 203. Irradiate.

The measurement light detection unit 80 includes, for example, a light receiving unit 31, an angle adjustment mechanism 36, a position switching mechanism 37, and a sensor control unit 38.
The angle adjustment mechanism 36 adjusts the angle of the measurement light detection unit 80 in order to detect the reflected light β of the excitation light α reflected by the diffraction grating 203. For example, the angle adjustment mechanism 36 may adjust the angle in conjunction with the angle adjustment mechanism 22 of the excitation light irradiation unit 20.

Among the incident angles of the excitation light α to the diffraction grating 203, the angle at which the amount of the reflected light β is minimized is the enhancement angle. By setting the incident angle of the excitation light α to an enhancement angle or an angle in the vicinity thereof, it becomes possible to measure high-intensity fluorescence γ.

The incident angle of the excitation light α is appropriately selected according to the pitch Λ of the diffraction grating 203, the wavelength of the excitation light α, the thickness of the metal film 104, the type of metal to be configured, the refractive index of the sample liquid in the pipette tip 106, and the like. Is done. For example, the incident angle θ of the excitation light α is set so as to satisfy the following formula (1).

k ₀ : Wave number of excitation light α = 2π / (λ ₀ / n) λ ₀ : Wavelength of excitation light α in vacuum n: Refractive index θ of medium (liquid in pipette tip 106) on diffraction grating 203: Excitation Incident angle m of light α to diffraction grating 203: integer Λ: pitch of diffraction grating 203

Here, ksp is the wave number of the plasmon excited at the interface between the two types of media (the interface between the metal film 104 and the liquid in the pipette tip 106), and is defined as the following equation (2).

ω: angular frequency of excitation light α c: speed of light ε ₁ : dielectric constant of medium (liquid in pipette tip 106) = n2ε ₂ : dielectric constant of medium (metal) constituting metal film 104

However, since the optimum incident angle (enhancement angle) of the excitation light α varies depending on various conditions and the shape error of the diffraction grating 203, it is preferable to obtain the optimum enhancement angle for each specimen examination.

Hereinafter, the flow of specimen detection using the GC-SPFS apparatus 70 will be described. Note that the operation procedure of the GC-SPFS device 70 is basically the same as the operation procedure of the PC-SPFS device 10 shown in FIG. 4, and only the method of the enhancement angle detection (S150) is different. . Therefore, only the procedure for detecting the enhancement angle using the GC-SPFS device 70 will be described.

In this embodiment, in the enhancement angle detection procedure, the controller 50 operates the measurement light detection unit 80 while operating the excitation light irradiation unit 20 to scan the incident angle of the excitation light α with respect to the diffraction grating 203. The reflected light β is detected. At this time, the control unit 50 operates the position switching mechanism 37 to place the optical filter 33 outside the light path of the light receiving unit 31. Then, the control unit 50 determines the incident angle of the excitation light α when the light amount of the reflected light β is minimum as the enhancement angle.

The sensor chip 200 is irradiated with the excitation light α at the enhancement angle determined in this way, and the detected fluorescence is detected by detecting the fluorescence γ emitted from the fluorescent substance that labels the analyte captured by the ligand. Based on the intensity of γ, the amount and concentration of the analyte can be converted as necessary.

The preferred embodiment of the present invention has been described above. However, the present invention is not limited to this. For example, the SPFS apparatus has been described in the above embodiment. Various modifications can be made without departing from the object of the present invention, such as a sample detection apparatus using a fluorescence immunoassay (FIA) such as an SPR apparatus.

DESCRIPTION OF SYMBOLS 10 PC-SPFS apparatus 20 Excitation light irradiation unit 21 Light source unit 22 Angle adjustment mechanism 23 Light source control part 30 Fluorescence detection unit 31 Light reception unit 32 Lens 33 Optical filter 34 Lens 35 Light reception sensor 36 Angle adjustment mechanism 37 Position switching mechanism 38 Sensor control part 40 Transport unit 41 Syringe pump 42 Syringe 43 Plunger 44 Pump drive mechanism 45 Pipette nozzle moving mechanism 46 Pipette nozzle 50 Control unit 60 Liquid storage unit 70 GC-SPFS device 80 Measurement light detection unit 100 Sensor chip 102 Dielectric member

102a Incident surface

102b Deposition surface

102c Emission surface 104 Metal film 106 Pipette tip

106a Fixing surface 106b Opening

106c Mounting hole 108 Body 108a Side wall 108b side 110 tip 110a liquid suction and discharge hole 200 sensor chip 202 substrate 202a deposition surface 203 diffraction grating

Claims

An analyte detection chip used for analyte detection,
A side wall member having a reaction field for capturing the analyte;
A pipette tip disposed adjacent to the side wall member,
An analyte detection chip in which an opening is provided in the pipette chip and the pipette chip and the side wall member are arranged so that the reaction field contacts a liquid introduced into the pipette chip.
The pipette tip has a body portion and a tip portion having a liquid suction / discharge hole,
The specimen detection chip according to claim 1, wherein the side wall member is fixed to the body portion.
3. The sample detection chip according to claim 2, wherein in the pipette chip, a side wall having a fixing surface between the body portion and the side wall member is a flat surface.
4. The sample detection chip according to claim 3, wherein, in the pipette chip, a side wall facing the side wall having the fixing surface is a flat surface.
4. The sample detection chip according to claim 3, wherein in the pipette chip, at least one of an inner surface and an outer surface of the side wall facing the side wall having the fixing surface is a convex curved surface.
The specimen detection chip according to any one of claims 1 to 5, wherein the side wall member includes an optical element.
The specimen detection chip according to claim 6, wherein the optical element is a dielectric member, a member in which fine convex portions or concave portions are periodically arranged, an optical waveguide, or a light reflecting member.
The optical element is a dielectric member including an incident surface for entering light and a reflecting surface for reflecting light incident on the incident surface,
The analyte detection chip according to claim 7, wherein the reaction field is disposed on the reflection surface.
The dielectric member has a metal film disposed on the reflective surface,
The analyte detection chip according to claim 8, wherein the reaction field is disposed on the metal film.
A sample detection apparatus for detecting a sample using the sample detection chip according to claim 9,
An excitation light irradiation unit for irradiating the metal film with excitation light via the dielectric member;
A fluorescence detection unit for detecting fluorescence generated from the fluorescence-labeled analyte captured in the reaction field based on the excitation light irradiated to the metal film;
A specimen detection apparatus comprising: a transport unit that is mounted with the pipette tip of the sensor chip, moves the pipette tip, and sucks and discharges liquid in the pipette tip.
The optical element is a diffraction grating in which fine convex portions or concave portions are periodically arranged, and the surface thereof is coated with a metal,
The diffraction grating is exposed in the pipette tip through the opening,
The analyte detection chip according to claim 6, wherein the reaction field is disposed on the diffraction grating.
A sample detection apparatus for detecting a sample using the sample detection chip according to claim 11,
An excitation light irradiation unit for irradiating the diffraction grating with excitation light;
Measurement light detection for detecting fluorescence generated from the fluorescence-labeled analyte trapped in the reaction field and reflected light of the excitation light reflected by the diffraction grating based on the excitation light irradiated on the diffraction grating Unit,
A specimen detection apparatus comprising: a transport unit that is mounted with the pipette tip of the sensor chip, moves the pipette tip, and sucks and discharges liquid in the pipette tip.