JPWO2019221040A1 - Specimen detection chip and sample detection device using it - Google Patents

Abstract

[Problem] It is possible to suppress a decrease in detection accuracy due to residual liquid in the well during the reaction step, and it is possible to suppress the influence of the liquid level of the liquid in the well on the detection result during the detection step. Further, the present invention provides a sample detection chip capable of downsizing the sample detection device and a sample detection device using the same. SOLUTION: The pipette is provided with a side wall member having a reaction field for capturing the analite and a pipette tip arranged adjacent to the side wall member so that the reaction field is in contact with a liquid introduced into the pipette tip. The tip is provided with an opening, and the pipette tip and the dielectric member are arranged as a sample detection tip.

Description

The present invention relates to a sample detection device that detects a substance to be measured contained in a sample detection chip (sensor chip) and a sample detection chip used therein.

Conventionally, various sample detection methods have been proposed that enable the detection of such a substance by applying the physical phenomenon of the substance in the case of detecting a very small substance.
As such a sample detection method, for example, the presence or absence of the substance to be measured and the presence or absence of the substance to be measured and the presence or absence of the substance to be measured by utilizing the antigen-antibody reaction between the antigen, which is the substance to be measured contained in the sample solution, and the antibody or antigen labeled with the labeling substance. An immunoassay that measures the amount is known.

As an immunoassay, a fluorescent immunoassay (FIA) or the like using a fluorescent substance as a labeling substance is known.
For example, as a sample detection device using a fluorescence immunoassay, a phenomenon in which electrons and light resonate in a minute region such as a nanometer level to obtain a high light output (Surface Plasmon Resonance (SPR)). A surface plasmon resonance apparatus (hereinafter, also referred to as “SPR apparatus”) that applies a phenomenon) to detect extremely minute analysts in a living body can be mentioned.

Further, as disclosed in Patent Document 1, based on the principle of surface plasmon-field enhanced fluorescence spectroscopy (SPFS) to which the surface plasmon resonance (SPR) phenomenon is applied, the SPR apparatus is more than the SPR apparatus. A surface plasmon excitation-enhanced fluorescence spectroscopic measurement device (hereinafter, also referred to as “SPFS device”) capable of performing analyte detection with higher accuracy is one such sample detection device.

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 arranged on the upper surface of the metal film is used. In such a sensor chip, a reaction field having a ligand for capturing the analyte is provided on the metal membrane.

By supplying the sample liquid containing the analite to the liquid holding member, the analyte is captured by the ligand (primary reaction). In this state, a liquid containing a secondary antibody labeled with a fluorescent substance (labeled liquid) is introduced into the liquid holding member. In the liquid holding member, the antigen-antibody reaction (secondary reaction) causes the analyte supplemented by the ligand to be labeled with a fluorescent substance.

In this state, when the metal film is irradiated with excitation light at an angle at which surface plasmon resonance occurs via the dielectric member, the fluorescent substance is excited by the surface plasmon light generated on the surface of the metal film, and fluorescence is generated from the fluorescent substance. By detecting this fluorescence, it is possible to measure the presence or absence of and the amount of the announcer.

As the 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 or a sample liquid or the like is placed on a reaction field on a metal film. A flow path member or the like that can be circulated is used.

Japanese Unexamined Patent Publication No. 2006-208069 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 was a risk that the tip of the liquid feeding means would come into contact with the metal film or the reaction field and damage them. Therefore, the tip of the liquid feeding means could not be pressed against the bottom surface of the well, and it was difficult to sufficiently remove the liquid in the well. If the liquid remains in the well in this way, various reactions may not proceed properly and the detection accuracy may decrease.

Further, in the sample detection device described in Patent Document 2, the detection unit is arranged above the well and detects the fluorescence that has passed through the liquid surface of the liquid in the well. Therefore, when the inner diameter of the well is small, the fluorescence detection result is affected by the meniscus. Further, even when the inner diameter of the well is large, the fluorescence detection result may be affected by the bubbles existing on the liquid surface.

Further, in the well type sensor chip, the amount of the required sample liquid becomes relatively large, and the sample detection device using the well type sensor chip tends to become large in size accordingly.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to suppress a decrease in detection accuracy due to residual liquid in the well during the reaction step, and it is possible to suppress the influence of the liquid level of the liquid in the well on the detection result during the detection step. It is an object of the present invention to provide a sample detection chip capable of the present invention and a sample detection device using the same.

Further, the present invention provides a sample detection chip capable of downsizing the sample detection device while the required amount of sample solution is the same as that of the well type sensor chip, and a sample detection device using the sample detection chip. The purpose.

The present invention has been invented to solve the above-mentioned problems in the prior art, and is a sample detection chip that reflects one aspect of the present invention in order to realize at least one of the above-mentioned objects. Is
A sample detection chip used to detect analysts.
A side wall member with a reaction field that captures the analite,
With a pipette tip located adjacent to the side wall member,
An opening is provided in the pipette tip and the pipette tip and the dielectric member are arranged so that the reaction field comes into contact with the liquid introduced into the pipette tip.

In addition, the sample detection device that reflects one aspect of the present invention is
A sample detection device that detects a sample using the sensor chip described above.
An excitation light irradiation unit that irradiates a metal film with excitation light via a dielectric member,
A fluorescence detection unit that detects the fluorescence generated from the fluorescently labeled analyzer captured in the reaction field based on the excitation light applied to the metal film.
The pipette tip of the sensor tip is mounted, and the pipette tip is provided with a transfer unit for moving the pipette tip and sucking and discharging the liquid in the pipette tip.

In addition, the sample detection device that reflects one aspect of the present invention is
A sample detection device that detects a sample using the sensor chip described above.
An excitation light irradiation unit that irradiates the diffraction grating with excitation light,
Based on the excitation light emitted to the diffraction grating, the measurement light detection unit that detects the fluorescence generated from the fluorescently labeled analyzer captured in the reaction field and the reflected light of the excitation light reflected by the diffraction grating, and
A pipette tip of a sensor tip is attached, and the pipette tip is provided with a transfer unit for moving the pipette tip and sucking and discharging the liquid in the pipette tip.

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

Further, by integrating the pipette tip with the dielectric member and the diffraction grating that are optical elements, the wells that have been conventionally used are no longer required, so that the sample detection device can be miniaturized.

FIG. 1 is a schematic diagram for explaining the configuration of a prism-coupled surface plasmon excitation-enhanced fluorescence spectroscopy measurement device (PC-SPFS device) 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 showing an example of the operation procedure of the PC-SPFS device. FIG. 5 is a schematic diagram for explaining the configuration of a diffraction grating-coupled surface plasmon excitation-enhanced fluorescence spectroscopy measurement device (GC-SPFS device) according to another embodiment of the present invention. FIG. 6 is a schematic diagram for explaining the configuration of the sensor chip according to the embodiment of the present invention. FIG. 7 is a perspective view for explaining an example of a diffraction grating.

Hereinafter, embodiments (examples) of the present invention will be described in more detail with reference to the drawings.
FIG. 1 is a schematic diagram for explaining the configuration of a prism-coupled surface plasmon excitation-enhanced fluorescence spectroscopy measurement device (PC-SPFS device) according to an embodiment of the present invention, and FIG. 2 is an embodiment of the present invention. FIG. 2A is a schematic view for explaining the configuration of the sensor chip, FIG. 2A is a cross-sectional view taken along the height direction of the sensor chip, and FIG. 2B is a cross-sectional view taken along the horizontal direction of the sensor chip. is there.

As shown in FIG. 1, the PC-SPFS device 10 in the present embodiment includes an excitation light irradiation unit 20, a fluorescence detection unit 30, a transfer unit 40, and a control unit 50. The PC-SPFS device 10 in this embodiment is used with the sensor chip 100 attached to 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 forming surface 102b, and an emitting surface 102c, a metal film 104 formed on the film forming surface 102b, and a film forming surface 102b or It has a metal film 104 and a fixed pipette tip 106. In the present embodiment, the dielectric member 102, which is an optical element, the metal film 104, and the reaction field described later form a side wall member. Normally, the sensor chip 100 is replaced every time a sample test is performed.

The dielectric member 102 can be a prism made of a dielectric material that is transparent to the excitation light α. The incident surface 102a of the dielectric member 102 is a surface on which the excitation light α emitted from the excitation light irradiation unit 20 is incident inside the dielectric member 102. Further, a metal film 104 is formed on the film-forming 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-forming surface 102b of the dielectric member 102 (hereinafter, referred to as “the back surface of the metal film 104” for convenience) and is emitted from the exit surface. The excitation light α is emitted to the outside of the dielectric member 102 via the 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 (ellipse quadrangular pyramid shape) having a substantially trapezoidal vertical cross-sectional shape. , The vertical cross-sectional shape may be a triangular (so-called triangular prism), semicircular, or semi-elliptical prism.

The incident surface 102a 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 excited state of the LD is disturbed, and the wavelength and output of the excitation light α are changed. It fluctuates. Therefore, the angle of the incident surface 102a is set so that the excitation light α is not incident perpendicularly to the incident surface 102a in the scanning range centered on the ideal enhancement angle.

The resonance angle (and the augmentation angle in the immediate vicinity thereof) is roughly determined by the design of the sensor chip 100. The design elements include 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 and augmentation angles are shifted by the analysts immobilized on the metal film 104, but the amount is less than a few degrees.

The dielectric member 102 has not a little birefringence characteristic. The material of the dielectric member 102 includes, for example, various inorganic substances such as glass and ceramics, natural polymers, synthetic polymers, etc., and is excellent in chemical stability, production stability, optical transparency, and low birefringence. Is preferable.

At least, as long as it is formed of a material that is optically transparent to the excitation light α and has low birefringence, the material is not particularly limited as described above, but the sensor chip 100 is inexpensive and has excellent handleability. 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, etc. Polycarbonate (PC), acrylic resin, triacetyl cellulose (TAC) and the like can be used.

The metal film 104 is formed on the film-forming surface 102b of the dielectric member 102. As a result, an interaction (surface plasmon resonance) occurs between the photons of the excitation light α incident on the film formation surface 102b under total reflection conditions and the free electrons in the metal film 104, and the photons are 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, and for example, at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum. It is made of, more preferably made of gold, and may be further composed of an alloy of these metals. Such a metal is suitable as a metal film 104 because it is stable against oxidation and the electric field enhancement by surface plasmon light is large.

The method for forming the metal film 104 is not particularly limited, and examples thereof include a sputtering method, a vapor deposition method (resistive heating vapor deposition method, electron beam vapor deposition method, etc.), electrolytic plating, electroless plating, and the like. Be done. It is preferable to use a sputtering method or a thin-film deposition method because the metal film forming conditions can be easily adjusted.

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, from the viewpoint of the electric field enhancing effect. It is preferably in the range of 20 to 70 nm in the case of platinum, 10 to 50 nm in the case of aluminum, and 10 to 70 nm in the case of these alloys.

When the thickness of the metal film 104 is within the above range, surface plasmon light is likely to be generated, which is suitable. Further, the size (length x width) of the metal film 104 having such a thickness is not particularly limited.

Further, 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 done. Immobilization of the ligand makes it possible to selectively detect analysts.

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

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

In this embodiment, the body portion 108 of the pipette tip 106 has a substantially square tubular shape, but at least the side wall 108a having the fixing surface 106a with the dielectric member 102 is flat and the generated fluorescence γ is generated. The shape is not particularly limited as long as it does not interfere with the detection by the fluorescence detection unit 30. In addition, in this specification, "the side wall is flat" means that both the inner surface and the outer surface of the side wall of the pipette tip 106 are flat.

FIG. 3 is a schematic diagram for explaining a modified example of the sensor chip 100.
As shown in FIG. 3A, only the side walls arranged between the reaction field and the fluorescence detection unit 30, that is, the side walls 108a and 108b arranged on the optical path of the fluorescence γ are flat, and the other side walls are flat. Can also be a curved surface.

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 walls may be a curved surface. Further, 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. By making the inner surface and / or the outer surface of the side wall 108b a convex curved surface in this way, the side wall 108b functions as a cylindrical lens and can collect the fluorescent γ generated in the reaction field.

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

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

The pipette tip 106 can be bonded to the dielectric member 102 or the metal film 104 by, for example, bonding with an adhesive or a transparent pressure-sensitive adhesive sheet, laser welding, ultrasonic welding, or the like.

The dielectric member 102 is preferably fixed to the body 108 of the pipette tip 106. With this configuration, as will be described later, it is possible to prevent the dielectric member 102 from coming into contact with the liquid when the liquid is introduced from the liquid storage unit 60 into the pipette tip 106.

Further, the pipette tip 106 may be provided with a filter inside. By providing a filter inside the pipette tip 106, contamination by contact between a liquid and a gas (aerosol due to evaporation from the liquid) moving by suction and discharge via the pipette tip 106 at the connection portion 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 fluororesin (PTFE) can be used.

The sensor chip 100 configured in this way may be provided with a positioning portion in order to accurately stop the sensor chip 100 at a liquid supply / drainage position or a measurement position, which will be described later. The positioning portion may be, for example, a hole or notch for fitting with a positioning pin arranged at a liquid supply / drainage position or a measurement position, or conversely, a protrusion for fitting with a positioning hole arranged at a liquid supply / drainage position or a measurement position. It can be a part.

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

Next, each component of the PC-SPFS device 10 will be described. As described above, the PC-SPFS device 10 in the present embodiment is provided with the excitation light irradiation unit 20, the fluorescence detection unit 30, the transfer 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 to the metal film 104 toward the incident surface 102a so that the angle of incidence on the metal film 104 is an angle that causes surface plasmon resonance. And emit.

Here, the "excitation light" is light that directly or indirectly excites a fluorescent substance. For example, the excitation light α generates localized field light on the surface of the metal film 104 that excites a fluorescent substance when the metal film 104 is irradiated through the dielectric member 102 at an angle at which surface plasmon resonance occurs. It is 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 adjusting mechanism 22, and a light source control unit 23.

The light source unit 21 irradiates the back surface of the metal film 104 with excitation light α that is collimated and has a constant wavelength and amount of light so that the shape of the irradiation spot is substantially circular. The light source unit 21 includes, for example, a light source for excitation light α, a beam shaping optical system, an APC (Automatic Power-Control) mechanism, and a temperature adjusting 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 bandpass filter, a linear polarizing filter, a half-wave plate, a slit, a zoom means, and the like. The beam shaping optical system may include all of these, or may include only a part of them.

The collimator collimates the excitation light α emitted from the light source. The bandpass filter converts the excitation light α emitted from the light source into narrow-band light having only a central wavelength. This is because the excitation light α from the light source has a slight wavelength distribution width.

The linearly polarized light filter converts the excitation light α emitted from the light source into completely linearly polarized light. The half-wave plate adjusts the polarization direction of the excitation light α so that the P wave component is incident on the metal film 104. The slit and zoom means adjust the beam diameter and contour shape 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 of a predetermined size.

The APC mechanism controls the light source so that the output of the light source is constant. More specifically, the APC mechanism detects the amount of light branched from the excitation light α with a photodiode (not shown) or the like. Then, the APC mechanism controls the output of the light source to be constant by controlling the input energy with the regression circuit.

The temperature adjusting mechanism is, for example, a heater or a Perche element. The wavelength and energy of the emitted light from the light source may vary with temperature. Therefore, by keeping the temperature of the light source constant by the temperature adjusting mechanism, the wavelength and energy of the emitted light of the light source are controlled to be constant.

The angle adjusting mechanism 22 adjusts the angle of incidence of the excitation light α on the metal film 104. The angle adjusting mechanism 22 makes the optical axis of the excitation light α and the sensor chip 100 relative to each other 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. Rotate.

For example, the angle adjusting mechanism 22 rotates the light source unit 21 around an axis orthogonal to the optical axis of the excitation light α (an axis perpendicular to the paper surface of FIG. 1). 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 if the incident angle is scanned. By setting the position of the center of rotation near the intersection of the optical axes of the two excitation lights α at both ends of the scanning range of the incident angle (between the irradiation position on the film forming surface 102b and the incident surface 102a), the irradiation position The deviation can be minimized.

Of the angles of incidence of the excitation light α on 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 at the enhancement angle or an angle in the vicinity thereof, it is possible to measure the high-intensity fluorescence γ.

The basic incident conditions of the excitation light α depend on the material and shape of the dielectric member 102 of the sensor chip 100, the film thickness of the metal film 104, the type of metal constituting the sensor chip 100, the refractive index of the sample liquid in the pipette chip 106, and the like. However, the optimum incident conditions vary slightly depending on the type and amount of the analyze in the pipette tip 106, the shape error of the dielectric member 102, and the like. Therefore, it is preferable to obtain the optimum enhancement angle for each sample 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 is composed of, for example, a known computer or microcomputer including an arithmetic unit, 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 substance excited by irradiating the metal film 104 with the excitation light α. If necessary, the fluorescence detection unit 30 also detects the plasmon scattered light generated by irradiating the metal film 104 with the excitation light α. 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 arranged in the normal direction (z-axis direction in FIG. 1) of the metal film 104 of the sensor chip 100. 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, which condenses light generated on the metal film 104. The second lens 34 is, for example, an imaging lens, and the light collected by the first lens 32 is imaged on the light receiving surface of the light receiving sensor 35. The optical paths between the lenses 32 and 34 are substantially parallel optical paths. The optical filter 33 is arranged between the lenses 32 and 34.

The optical filter 33 guides only the fluorescence component to the light receiving sensor 35, and removes the excitation light component (plasmon scattered light) in order to detect the fluorescence γ with a high S / N. The optical filter 33 includes, for example, an excitation light reflection filter, a short wavelength cut filter, and a bandpass 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 a predetermined light component.

The light receiving sensor 35 detects the fluorescence γ. The light receiving sensor 35 is not particularly limited as long as it has a high sensitivity capable of detecting a weak fluorescent γ from a fluorescent substance labeled with a minute amount of analyst, but is not particularly limited, for example, a photomultiplier tube. A photomultiplier tube (PMT), an avalanche photodiode (APD), a low noise follow diode (PD), or the like can be used.

The position switching mechanism 37 switches the position of the optical filter 33 on or out of the optical path of the light receiving unit 31. Specifically, when the light receiving sensor 35 detects fluorescence γ, the optical filter 33 is arranged 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 it outside the optical path. The position switching mechanism 37 is composed of, for example, a rotation drive 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 movement.

The sensor control unit 38 controls detection of the output value of the light receiving sensor 35, management of the sensitivity of the light receiving sensor 35 based on the detected output value, change of the sensitivity of the light receiving sensor 35 in order to obtain an appropriate output value, and the like. The sensor control unit 38 is composed of, for example, a known computer or microcomputer including an arithmetic unit, a control device, a storage device, an input device, and an output device.

The transport unit 40 sucks and discharges liquids such as a sample liquid, a labeling liquid, and a cleaning liquid into the pipette tip 106 with the sensor tip 100 attached, and moves the pipette tip 106. The transfer unit 40 includes a syringe pump 41, a pipette nozzle 46, a pump drive mechanism 44, and a pipette nozzle moving mechanism 45.

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

The syringe pump 41 is composed of a syringe 42 and a plunger 43 capable of reciprocating in the syringe 42. The reciprocating motion of the plunger 43 quantitatively sucks and discharges the liquid.

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 controlling the residual liquid amount in the pipette tip 106 because the liquid feeding amount and the liquid feeding speed of the syringe pump 41 can be controlled.

The pipette nozzle moving mechanism 45, for example, freely moves the pipette nozzle 46 in two directions, 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 composed of, for example, a robot arm, a two-axis stage, or a vertically movable turntable.

The transfer unit 40 sucks various liquids from the liquid storage unit 60 by the syringe pump 41 and supplies them into the pipette tip 106 of the sensor tip 100. As a result, a primary reaction or a secondary reaction can occur in the pipette tip 106.

Further, the transport unit 40 moves the sensor tip 100 to the liquid supply / drainage position or the measurement position with the sensor tip 100 attached to the pipette nozzle 46. Here, the “liquid supply / drainage position” is a position where the liquid is supplied or removed from the liquid storage unit 60 into the pipette tip 106. 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 accordingly.

Hereinafter, the flow of sample detection using the PC-SPFS device 10 will be described. FIG. 4 is a flowchart showing an example of the operation procedure of the PC-SPFS device 10.
First, the user attaches the sensor tip 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 tip 100 mounted on the pipette nozzle 46 to the liquid 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, clean the inside of the pipette tip 106, and moisturize the reaction field. Remove the agent (S120). At the time of cleaning, the control unit 50 may operate the pipette nozzle moving mechanism 45 to vibrate the sensor tip 100 to agitate the cleaning liquid in the pipette tip 106. The cleaning liquid used for cleaning can be discharged to a liquid storage unit 60 or a waste liquid unit (not shown).

After that, the control unit 50 operates the pipette nozzle moving mechanism 45 and the pump drive mechanism 44 to introduce the measuring liquid from the liquid storage unit 60 into the pipette tip 106 (S130). If the result of the augmented angle detection (S150) in the subsequent step is not affected, the cleaning liquid and the measuring liquid can be used in combination, and the augmented angle measurement can be performed as it is without discharging the cleaning 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 detects and enhances the plasmon scattered light having the same wavelength as the excitation light α. Detect the corner (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 arrange the optical filter 33 outside the optical path of the light receiving unit 31. Then, the control unit 50 determines the incident angle of the excitation light α when the amount of the plasmon scattered light is maximum as the 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 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 adjusting 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 arrange 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 tip 100 to the liquid 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 antigen-antibody reaction (primary reaction) captures the analytes in the reaction field on the metal membrane 104. At the time of the primary reaction, the control unit 50 may operate the pipette nozzle moving mechanism 45 to vibrate the sensor tip 100 to agitate the sample liquid in the pipette tip 106.

The sample liquid used here is a liquid prepared by using the sample. For example, the sample and the reagent are mixed and treated to bind the fluorescent substance to the analyst contained in the sample. Things can be mentioned. Examples of such a sample include blood, serum, plasma, urine, nasal fluid, saliva, stool, and body cavity fluid (cerebrospinal fluid, ascites, pleural effusion, etc.).

The analysts contained in the sample include, for example, nucleic acids (DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., which may be single-stranded or double-stranded, or nucleosides. , Nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), sugars (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules thereof, Examples thereof include complexes, and specific examples thereof may be cancer fetal antigens such as AFP (α-fet protein), tumor markers, signaling substances, hormones, and the like, and are not particularly limited.

After that, 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 the 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 tip 106, the analite 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. Further, at the time of the secondary reaction, the control unit 50 may operate the pipette nozzle moving mechanism 45 to vibrate the sensor tip 100 to agitate the labeling liquid in the pipette tip 106.

After that, 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 the 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 measuring 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 label the analysts captured by the ligand. Fluorescent γ emitted from the fluorescent substance is detected (S240). Based on the detected intensity of the fluorescent γ, it can be converted into the amount and concentration of the analyte, if necessary.

By the above procedure, the presence or amount of Analite in the sample liquid can be detected.
In the present embodiment, the enhanced angle detection (S150) and the optical blank value measurement (S160) are performed before the primary reaction (S180), but the enhanced 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 predetermined, the detection of the augmentation angle (S150) may be omitted.

Further, in the above description, after the primary reaction (S180) in which the analyte is reacted with the ligand, the secondary reaction (S200) in which the analyte is labeled with a fluorescent substance is carried out (two-step method). However, the timing of 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 may be added to the sample solution to pre-label the analyte with a fluorescent substance. Further, by simultaneously injecting the sample solution and the labeling solution into the pipette tip 106, the analysis 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 either case, both the primary reaction and the secondary reaction can be completed by introducing the sample solution into the pipette tip 106 (one-step method). When the one-step method is adopted as described above, the enhanced angle detection (S150) is performed before the antigen-antibody reaction.

FIG. 5 is a schematic diagram for explaining the configuration of a diffraction grating-coupled surface plasmon excitation-enhanced fluorescence spectroscopy measurement device (GC-SPFS device) 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 the configuration of the 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-sectional view along the horizontal direction of the sensor chip. It is a figure.

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

As shown in FIG. 5, the GC-SPFS device 70 in the present embodiment includes an excitation light irradiation unit 20, a fluorescence detection unit 30, a transfer unit 40, and a control unit 50. The GC-SPFS device 70 in this embodiment is used with the sensor chip 200 attached to the transport unit 40.

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

Further, on the substrate 202, a diffraction grating 203 as an optical element is formed on at least a part of a portion corresponding to the reaction field on the metal film 104. In the present 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. Normally, the sensor chip 200 is replaced every time a sample test is performed.

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

As described above, the diffraction grating 203 is formed on the substrate 202. The diffraction grating 203 may 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 partially.

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

The diffraction grating 203 produces evanescent light when irradiated with the excitation light α. 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. 7 (A) or a two-dimensional diffraction grating as shown in FIG. 7 (B). In the one-dimensional diffraction grating shown in FIG. 7A, a plurality of ridges parallel to each other are formed at predetermined intervals on the surface of the substrate 202. 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 sinusoidal 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 the present specification, the “pitch of the diffraction grating” refers to the distance Λ between the centers of the convex portions in the arrangement direction of the convex portions, as shown in FIGS. 7 (A) and 7 (B). 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 method of forming the diffraction grating 203 is not particularly limited. For example, after forming the metal film 104 on the film-forming surface 202a of the flat plate-shaped substrate 202, the metal film 104 may be provided with an uneven shape. Further, the metal film 104 may be formed on the film-forming surface 202a of the substrate 202 to which the concave-convex shape is previously provided.

In this embodiment, the pipette tip 106 is made of a material that is transparent to at least light having a 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 opaque to light as long as it does not interfere with the extraction of light 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, bonding with an adhesive or a transparent pressure-sensitive adhesive sheet, laser welding, ultrasonic welding, or the like.

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

Next, each component of the GC-SPFS device 70 will be described. The GC-SPFS device 70 in the present embodiment is provided with an excitation light irradiation unit 20, a measurement light detection unit 80, a transfer 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 transmits 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 adjusting mechanism 36, a position switching mechanism 37, and a sensor control unit 38.
The angle adjusting 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. The angle adjusting mechanism 36 may, for example, adjust the angle in conjunction with the angle adjusting mechanism 22 of the excitation light irradiation unit 20.

Of the angles of incidence of the excitation light α on the diffraction grating 203, the angle at which the amount of reflected light β is minimized is the enhancement angle. By setting the incident angle of the excitation light α at the enhancement angle or an angle in the vicinity thereof, it is possible to measure the 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 film thickness of the metal film 104, the type of metal constituting the diffraction grating 203, the refractive index of the sample liquid in the pipette tip 106, and the like. Will be done. For example, the incident angle θ of the excitation light α is set so as to satisfy the following equation (1).

ｋ₀：励起光αの波数＝２π／（λ₀／ｎ）λ₀：真空中の励起光αの波長ｎ：回折格子２０３上の媒質（ピペットチップ１０６内の液体）の屈折率θ：励起光αの回折格子２０３に対する入射角ｍ：整数Λ：回折格子２０３のピッチ

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

Here, ksp is the wave number of plasmons excited at the interface between 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：励起光αの角周波数ｃ：光速度ε₁：回折格子２０３上の媒質（ピペットチップ１０６内の液体）の誘電率＝ｎ２ε₂：金属膜１０４を構成する媒質（金属）の誘電率

ω: Angular frequency of excitation light α: Speed of light ε ₁ : Permittivity of medium (liquid in pipette tip 106) on diffraction grating 203 = n2ε ₂ : Permittivity 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, the shape error of the diffraction grating 203, and the like, it is preferable to obtain the optimum enhancement angle for each sample test.

Hereinafter, the flow of sample detection using the GC-SPFS device 70 will be described. 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, except for the method of augmentation angle detection (S150). .. Therefore, only the procedure for detecting the augmented angle using the GC-SPFS device 70 will be described.

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

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

Although the preferred embodiment of the present invention has been described above, the present invention is not limited to this. For example, the SPFS device has been described in the above examples, but the sample detection device according to the present invention is, for example. , A sample detection device using a fluorescence immunoassay (FIA) such as an SPR device can also be applied, and various changes can be made without departing from the object of the present invention.

10 PC-SPFS device 20 Excitation light irradiation unit 21 Light source unit 22 Angle adjustment mechanism 23 Light source control unit 30 Fluorescence detection unit 31 Light receiving unit 32 Lens 33 Optical filter 34 Lens 35 Light receiving sensor 36 Angle adjustment mechanism 37 Position switching mechanism 38 Sensor control unit 40 Transfer unit 41 Syringe pump 42 Syringe 43 Plunger 44 Pump drive mechanism 45 Pipet nozzle movement mechanism 46 Pipet 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 Formation surface 102c Exit surface 104 Metal film 106 Pipet tip 106a Fixing surface 106b Opening 106c Mounting hole 108 Body 108a Side wall 108b Side wall 110 Tip 110a Liquid suction / discharge hole 200 Sensor chip 202 Substrate 202a Formation surface 203 Diffraction lattice

Claims (12)

A sample detection chip used to detect analysts.
A side wall member having a reaction field that captures the analite, and
A pipette tip arranged adjacent to the side wall member is provided.
A sample detection chip in which an opening is provided in the pipette tip and the pipette tip and the side wall member are arranged so that the reaction field comes into contact with a liquid introduced into the pipette tip. The pipette tip has a body portion and a tip portion having a liquid suction / drainage hole.
The sample detection chip according to claim 1, wherein the side wall member is fixed to the body portion. The sample detection tip according to claim 2, wherein in the pipette tip, the side wall having the fixing surface between the body portion and the side wall member is a flat surface. The sample detection tip according to claim 3, wherein in the pipette tip, the side wall facing the side wall having the fixing surface is flat. The sample detection tip according to claim 3, wherein in the pipette tip, at least one of the inner surface and the outer surface of the side wall facing the side wall having the fixing surface is a convex curved surface. The sample detection chip according to any one of claims 1 to 5, wherein the side wall member includes an optical element. The sample detection chip according to claim 6, wherein the optical element is a dielectric member, a member in which fine protrusions or recesses are periodically arranged, an optical waveguide, or a light reflection member. The optical element is a dielectric member including an incident surface for incident light and a reflecting surface for reflecting light incident on the incident surface.
The sample detection chip according to claim 7, wherein the reaction field is arranged on the reflective surface. The dielectric member has a metal film arranged on the reflective surface and has a metal film.
The sample detection chip according to claim 8, wherein the reaction field is arranged on the metal film. A sample detection device that detects a sample using the sample detection chip according to claim 9.
An excitation light irradiation unit that irradiates the metal film with excitation light via the dielectric member,
A fluorescence detection unit that detects fluorescence generated from the fluorescently labeled analyzer captured in the reaction field based on the excitation light applied to the metal film.
A sample detection device including a transport unit to which the pipette tip of the sensor tip is attached, the pipette tip is moved, and the liquid in the pipette tip is sucked and discharged. The optical element is a diffraction grating in which fine protrusions or recesses are periodically arranged and the surface thereof is coated with metal.
The diffraction grating is exposed into the pipette tip through the opening.
The sample detection chip according to claim 6, wherein the reaction field is arranged on the diffraction grating. A sample detection device that detects a sample using the sample detection chip according to claim 11.
An excitation light irradiation unit that irradiates the diffraction grating with excitation light,
Measurement light detection that detects the fluorescence generated from the fluorescently labeled analyzer captured in the reaction field based on the excitation light irradiated on the diffraction grating and the reflected light of the excitation light reflected by the diffraction grating. With the unit
A sample detection device including a transport unit to which the pipette tip of the sensor tip is attached, the pipette tip is moved, and the liquid in the pipette tip is sucked and discharged.

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