JP6885458B2 - Sensor chip for sample detection system - Google Patents

Description

The present invention relates to a sensor chip used in an immunoassay (immunoassay) for measuring the presence or absence of a substance to be measured and its amount, and more specifically, a surface plasmon to which a surface plasmon resonance (SPR) phenomenon is applied. The present invention relates to a sensor chip used in a resonance device and a sample detection system such as a surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) principle based on a surface plasmon excitation enhanced fluorescence spectroscopy.

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.

Immunoassays include an enzyme immunoassay (EIA) that uses an enzyme as a labeling substance, and a fluorescence immunoassay (FIA) that uses a fluorescent substance as a labeling substance.
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 a very small amount of alanite in a living body can be mentioned.

In addition, based on the principle of Surface Plasmon-field enhanced Fluorescence Spectroscopy (SPFS), which applies the surface plasmon resonance (SPR) phenomenon, it is possible to perform analysis detection with higher accuracy than the SPR device. The surface plasmon excitation enhanced fluorescence spectroscopic measurement device (hereinafter, also referred to as “SPFS device”) is one such sample detection device.

In this surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), surface plasmon light is applied to the surface of the metal film under the condition that the excitation light such as laser light emitted from the light source is totally reflected and attenuated (ATR) on the surface of the metal film. By generating (dense wave), the amount of photons contained in the excitation light emitted from the light source is increased several tens to several hundred times, and the electric field enhancement effect of the surface plasmon light is obtained.

In such an 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 (first-order 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 solution 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 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.

For such a sensor chip, a well chip type is used as a liquid holding member to perform a sample inspection while the sample liquid is stored in the well member, and a flow path member is used as a liquid holding member to prepare a sample liquid. There is a flow path chip type (hereinafter, simply referred to as "flow path chip") in which a sample is inspected in a state where the analyzer is captured in the reaction field in the flow path by flowing the sample through the flow path.

FIG. 7 is a schematic diagram for explaining the structure of the conventional flow path chip.
As shown in FIG. 7, the flow path chip 200 is formed by bonding a dielectric member 202 and a flow path lid 204 by a flow path seal 206. The flow path lid 204 is provided with an injection port 204a and a storage portion 204b for injecting a sample solution or the like.

Here, the space surrounded by the dielectric member 202, the flow path lid 204, and the flow path seal 206 is the flow path 208, and the flow path 208 contains a ligand that is an antibody or a fragment thereof specific to the analyst. A immobilized reaction field 210 is provided.

In such a flow path chip 200, the width of the flow path 208 is 0.5 mm to 3 mm, and the height is 50 μm to 500 μm.

In the sample inspection using such a flow path chip 200, for example, as disclosed in Patent Document 1, when the sample liquid is flowed through the flow path 208 of the flow path chip 200, it passes through the reaction field 210 only once. A one-pass type is used, a circulation type in which the sample solution is circulated and passed through the reaction field 210 repeatedly, and a reciprocating type in which the sample solution is reciprocated and passed through the reaction field 210 repeatedly is used.

In particular, in the case of a small amount of sample solution or a sample solution containing rare analysts, the reaction efficiency can be improved by passing the sample solution through the reaction field 210 repeatedly. A reciprocating liquid feeding method is often used.

Japanese Unexamined Patent Publication No. 2012-018159

By the way, in the sample inspection using the flow path chip 200, it is necessary to sequentially inject a liquid such as a measuring liquid or a cleaning liquid in addition to the sample liquid. Therefore, as shown in FIG. 8A, the liquid L injected into the flow path 208 is sequentially sucked from the injection port 204a to remove the liquid from the flow path 208.

However, the liquid L may not be completely removed from the flow path 208, and the liquid remains in the vicinity of the injection port 204a of the flow path 208 as shown in FIG. 8 (b).
Then, when the suction of the liquid L is stopped, as shown in FIG. 8C, the liquid L remaining in the vicinity of the injection port 204a may return to the vicinity of the central portion of the flow path 208 due to the capillary phenomenon. ..

When the next liquid L is injected from the injection port 204a in this state, the injected liquid L is attracted to the residual liquid in the flow path 208 as shown in FIG. 8D, and bubbles A are generated. It may end up. Further, due to the pressure applied in the flow path 208 at the time of injecting the liquid L, the residual liquids on both sides of the flow path 208 may move and be connected to block the flow path 208 in the lateral direction. In this case, an air layer is formed between the liquid L that has been injected and has entered the flow path 208 and the closed residual liquid, and this may be generated as bubbles A.

In particular, in the manufacturing process of the flow path chip 200, for example, when the dielectric member 202 and the flow path lid 204 are bonded to each other via the flow path seal 206, the flow path lid 204 bends inward, as shown in FIG. In addition, when the height near the central portion of the flow path 208 becomes low, bubbles A are often generated.

If the bubbles A are generated in this way and the bubbles A gather in the reaction field 210 in the flow path 208, the measurement accuracy of the analyzer is lowered.
In view of such a current situation, it is an object of the present invention to provide a sensor chip for a sample detection system capable of suppressing the generation of bubbles in a flow path and preventing a decrease in measurement accuracy.

The present invention has been invented to solve the above-mentioned problems in the prior art, and is a sample detection system that reflects one aspect of the present invention in order to realize at least one of the above-mentioned objects. Sensor chip for
A sensor chip that has a reaction field inside that captures Anna Reed.
The flow path having the reaction field and
It has a first through hole formed at one end of the flow path.
The flow path is provided with a gradient so that the height of the flow path in the vicinity of the reaction field is higher than the height of the flow path in the vicinity of the first through hole.
The height of the flow path in the vicinity of the reaction field is 0.1 mm to 1 mm .

According to the present invention, by adopting a flow path structure for keeping the residual liquid in the flow path at a predetermined position, it is possible to suppress the generation of bubbles due to the residual liquid and prevent a decrease in measurement accuracy. be able to.
Further, since it is possible to suppress the generation of air bubbles caused by the residual liquid, it is possible to inject and suck the reagent many times in the same flow path, and it is not necessary to provide an extra flow path in the sensor chip. Therefore, it is possible to reduce the manufacturing cost of the sensor chip and the sample detection device and contribute to the miniaturization.

FIG. 1 is a schematic view for explaining the configuration of a surface plasmon excitation enhanced fluorescence spectroscopic measurement device (SPFS device) according to an embodiment of the present invention. FIG. 2 is a schematic view showing an example of a sensor chip used in the SPFS device of FIG. FIG. 3 is a schematic diagram for explaining the state of the liquid when the liquid is reciprocally sent to the flow path in the sensor chip of FIG. FIG. 4 is a schematic view showing a modified example of the sensor chip used in the SPFS device of FIG. FIG. 5 is a schematic view showing another modification of the sensor chip used in the SPFS apparatus of FIG. FIG. 6 is a flowchart showing an example of the operation procedure of the SPFS apparatus of FIG. FIG. 7 is a schematic diagram for explaining the structure of the conventional flow path chip. FIG. 8 is a schematic diagram for explaining a state of the liquid when the liquid is reciprocally sent to the flow path in the flow path chip of FIG. 7.

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 surface plasmon excitation enhanced fluorescence spectroscopic measurement device (SPFS device) according to an embodiment of the present invention, and FIG. 2 is an example of a sensor chip used in the SPFS device of FIG. It is a schematic diagram which shows.

As shown in FIG. 1, the SPFS device 10 includes an excitation light irradiation unit 20, a fluorescence detection unit 30, a liquid feeding unit 40, a transport unit 50, and a control unit 80. The SPFS device 10 is used with the sensor chip 100 mounted on the chip holder 54 of the transport unit 50.

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 flow path forming member 106 fixed on the metal film 104. Normally, the sensor chip 100 is replaced every time a sample test is performed.

The sensor chip 100 is preferably a structure having a length of several mm to several cm on each side, but is a smaller structure or a larger structure that is not included in the category of “chip”. It doesn't matter.

The dielectric member 102 can be a prism made of a dielectric 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 having a substantially trapezoidal vertical cross-sectional shape (a quadrangular pyramid shape), for example. , The vertical cross-sectional shape may be a triangle (so-called triangular prism), a semicircular shape, or a semi-elliptical shape.

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-forming surface 102b under total reflection conditions and the free electrons in the metal film 104, and the station is stationed on the surface of the metal film 104. It can generate presence light.

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 116) 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.

The flow path forming member 106 is arranged on the film forming surface 102b of the dielectric member 102 or the metal film 104. In the present embodiment, the flow path forming member 106 is joined to the dielectric member 102 or the metal film 104 by an adhesive sheet (flow path seal 114) having through holes formed therein, and the dielectric member 102, the flow path forming member 106, The space surrounded by the flow path seal 114, that is, the through hole of the flow path seal 114 is used as the flow path 112.

The flow path forming member 106 is not limited to this, and for example, a flow path groove is formed on the film forming surface 102b or the surface facing the metal film 104, and the flow path forming member 106 is formed on the metal film 104. By arranging so as to cover the reaction field 116 of the above, the space surrounded by the flow path forming member 106 and the dielectric member 102, that is, the flow path groove, is used to send a sample solution, a labeling solution, a cleaning solution, or the like. It can be used as a flow path 112.

In this case, the flow path forming member 106 may be joined to the dielectric member 102 or the metal film 104 by, for example, bonding with an adhesive or a transparent adhesive sheet, laser welding, ultrasonic welding, crimping using a clamp member, or the like. it can.

The width of the flow path 112 thus formed in the vicinity of the reaction field 116 is preferably 0.1 mm to 5 mm, and the length is preferably 10 mm to 50 mm. Further, the height of the flow path 112 in the vicinity of the first through hole 110a is preferably 50 μm to 500 μm.

Further, the flow path forming member 106 has a first through hole 110a formed at one end of the flow path 112 and a second through hole 110b formed at the other end of the flow path 112. In the present embodiment, the first through hole 110a and the second through hole 110b each have a substantially cylindrical shape. Further, the first through hole 110a and the second through hole 110b serve as an injection port for injecting the sample liquid, the labeling liquid, the cleaning liquid, etc. into the flow path 112, and an outlet for taking out the sample liquid, the labeling liquid, the cleaning liquid, etc. Function.

The material of the flow path forming member 106 is not particularly limited as long as it is formed of at least a material that is optically transparent to fluorescence γ, which will be described later, 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 flow path forming member 106 is not particularly limited, but injection molding using a mold is preferable from the viewpoint of manufacturing cost.

When the flow path forming member 106 is formed from a resin material, for example, polyesters such as polyethylene terephthalate (PET) and polyethylene naphthalate, polyolefins such as polyethylene (PE) and polypropylene (PP), cyclic olefin copolymer (COC), and the like. Polycyclic olefins such as cyclic olefin polymer (COP), vinyl resins such as polyvinyl chloride and polyvinylidene chloride, polystyrene, polyetheretherketone (PEEK), polysalphon (PSF), polyethersalphon (PES), polycarbonate (PC), polypropylene, polyimide, acrylic resin, triacetyl cellulose (TAC) and the like can be used, and it is particularly preferable to use acrylic resin because it is chemically stable and has excellent moldability. As described above, by using the acrylic resin, the gradient 118 as described later can be easily formed.

In the present embodiment, the flow path forming member 106 is provided with a gradient 118 so that the height h2 of the flow path 112 in the vicinity of the reaction field 116 is higher than the height h1 in the vicinity of the first through hole 110a. .. The height h2 of the flow path 112 in the vicinity of the reaction field 116 is preferably 0.1 mm to 1 mm.

With this configuration, the liquid L is injected into the flow path 112 from the first through hole 110a as shown in FIG. 3A, and the flow path is injected from the first through hole 110a as shown in FIG. 3B. Even if the liquid L in the 112 is sucked and the liquid L remains in the vicinity of the first through hole 110a of the flow path 112, or if the suction is stopped as it is, the liquid is the first as shown in FIG. 3C. It is possible to prevent the liquid L from returning to the vicinity of the reaction field 116 by staying in the vicinity of the through hole 110a.

The gradient 118 provided in the flow path forming member 106 may be configured so that the height of the flow path 112 in the vicinity of the reaction field 116 is higher than the height in the vicinity of the first through hole 110a. In the example shown in 2, the gradient 118 has the highest height in the vicinity of the reaction field 116 in the flow path 112, and the height of the flow path 112 decreases as it approaches the first through hole 110a and the second through hole 110b. Although it is provided, for example, as shown in FIG. 4A, a gradient 118 may be provided so that the height of the flow path 112 increases from the first through hole 110a toward the second through hole 110b. ..

Further, as shown in FIG. 4B, the gradient 118 may be a curved surface. Further, as shown in FIG. 4C, the gradient 118 may be formed into a plurality of planes, but from the viewpoint of preventing liquid accumulation when the liquid is sent to the flow path 112, FIGS. 2 and 4 As shown in (a), the surface forming the flow path 112 is preferably as smooth as possible.

Further, the gradient 118 may not be provided on the flow path forming member 106, but may be provided on the dielectric member 102 as shown in FIG. 5 (a). Further, as shown in FIG. 5B, the gradient 118 can be provided on both the flow path forming member 106 and the dielectric member 102.

As shown in FIG. 1, the sensor chip 100 configured in this way is attached to the chip holder 54 of the transport unit 50 of the SPFS device 10, and the sample is detected by the SPFS device 10.

Next, each component of the SPFS device 10 will be described. As described above, the SPFS device 10 in the present embodiment is provided with the excitation light irradiation unit 20, the fluorescence detection unit 30, the liquid feeding unit 40, the transport unit 50, and the control unit 80.

The excitation light irradiation unit 20 irradiates the sensor chip 100 held in the chip holder 54 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 the 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 Peltier 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 chip holder 54 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 in 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 are determined by the material and shape of the dielectric member 102 of the sensor chip 100, the film thickness of the metal film 104, the refractive index of the sample liquid in the flow path 112, and the like. The optimum incident conditions vary slightly depending on the type and amount of the analyte in the flow path 112, the shape error of the dielectric member 102, and the like. 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 liquid feeding unit 40 supplies a sample liquid, a labeling liquid, a cleaning liquid, and the like into the flow path 112 of the sensor chip 100 mounted on the chip holder 54. The liquid feed unit 40 includes a syringe pump 41, a pipette nozzle 46, a pipette tip 45, and a liquid feed pump drive mechanism 44.

The liquid feeding unit 40 is used with the pipette tip 45 attached to the tip of the pipette nozzle 46. If the pipette tip 45 is replaceable, it is not necessary to clean the pipette tip 45, and it is possible to prevent impurities from being mixed.

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 liquid feed pump drive mechanism 44 includes a drive device for the syringe pump 41 and a moving device for the pipette nozzle 46 to which the pipette tip 45 is mounted. The driving device of the syringe pump 41 is a device for reciprocating the plunger 43, and includes, for example, a stepping motor. A drive device including a stepping motor is preferable from the viewpoint of controlling the residual liquid amount of the sensor chip 100 because the liquid feeding amount and the liquid feeding speed of the syringe pump 41 can be controlled. The moving device of the pipette nozzle 46, 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.

From the viewpoint of adjusting the relative height of the pipette tip 45 and the sensor tip 100 to be constant and controlling the amount of residual liquid in the sensor tip 100 to be constant, the liquid feeding unit 40 is the tip of the pipette tip 45. It is preferable to further have a mechanism for detecting the position.

The liquid feeding unit 40 sucks various liquids from a liquid storage unit (not shown) and supplies them into the flow path 112 of the sensor chip 100. At this time, by moving the plunger 43, the liquid reciprocates in the flow path 112 of the sensor chip 100, and the liquid in the flow path 112 is agitated. As a result, it is possible to make the concentration of the liquid uniform and promote the reaction (for example, the antigen-antibody reaction) in the flow path 112.

Since such an operation is performed, the injection port (first through hole 110a) of the sensor chip 100 is protected by the multilayer film 111 or the like, and the first through hole is formed when the pipette tip 45 penetrates the multilayer film. It is preferable that the sensor tip 100 and the pipette tip 45 are configured so that the 110a can be sealed.

On the other hand, the second through hole 110b has a lid seal 120 that covers the upper opening thereof, and serves as a storage portion in which the injected liquid passes through the flow path and is temporarily stored. The lid seal 120 is provided with a minute hole for venting air.

The liquid in the flow path 112 is sucked again by the syringe pump 41 and discharged to a waste liquid portion (not shown). By repeating these operations, it is possible to carry out reactions with various liquids, washing and the like, and to immobilize the analysis labeled with the fluorescent substance in the reaction field 116 in the flow path 112.

The transport unit 50 transports and fixes the sensor chip 100 mounted on the chip holder 54 by the user to the liquid feeding position or the measuring position. Here, the "liquid feeding position" is a position where the liquid feeding unit 40 supplies the liquid into the flow path 112 of the sensor chip 100 or removes the liquid in the flow path 112. The "measurement position" is a position where the excitation light irradiation unit 20 irradiates the sensor chip 100 with the excitation light α and the fluorescence detection unit 30 detects the fluorescence γ generated accordingly.

The transport unit 50 includes a transport stage 52 and a tip holder 54. The chip holder 54 is fixed to the transport stage 52 and holds the sensor chip 100 in a detachable manner. The shape of the chip holder 54 is not particularly limited as long as it can hold the sensor chip 100 and does not obstruct the optical path of the excitation light α and the fluorescence γ. For example, the chip holder 54 is provided with an opening through which the excitation light α and the fluorescence γ pass.

The transfer stage 52 is configured so that the chip holder 54 can be moved in one direction (x-axis direction in FIG. 1) and vice versa. The transport stage 52 is driven by, for example, a stepping motor or the like.

Hereinafter, the flow of sample detection using the SPFS device 10 will be described. FIG. 6 is a flowchart showing an example of the operation procedure of the SPFS device 10.
First, the user attaches the sensor chip 100 to the chip holder 54 of the transport unit 50 (S100).

The control unit 80 operates the transfer stage 52 to move the sensor chip 100 mounted on the chip holder 54 to the liquid feeding position (S110).
Next, the control unit 80 operates the liquid feeding unit 40 to introduce the cleaning liquid stored in the liquid storage unit (not shown) into the flow path 112, wash the flow path 112, and use the storage reagent in the flow path 112. Remove (S120). The cleaning liquid used for cleaning is discharged by the liquid feeding unit 40, and instead, a measuring liquid stored in a liquid storage unit (not shown) is introduced into the flow path 112. If the result of the enhancement angle detection (S140) in the subsequent step is not affected, the storage reagent cleaning solution and the measurement solution can be used in combination, and the enhancement angle measurement can be performed as it is without discharging the cleaning solution.

Next, the control unit 80 operates the transport stage 52 to transport the sensor chip 100 mounted on the chip holder 54 to the measurement position (S130). Then, the control unit 80 operates the excitation light irradiation unit 20 and the fluorescence detection unit 30 to irradiate the sensor chip 100 with the excitation light α, and detects and enhances the plasmon scattered light having the same wavelength as the excitation light α. Detect the corner (S140).

Specifically, the control unit 80 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 80 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 80 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 80 operates the excitation light irradiation unit 20 and the fluorescence detection unit 30 to irradiate the sensor chip 100 arranged at the measurement position with the excitation light α, and the output value (optical blank value) of the light receiving sensor 35. ) Is recorded (S150).

At this time, the control unit 80 operates the angle adjusting mechanism 22 to set the incident angle of the excitation light α to the enhancement angle. Further, the control unit 80 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 80 operates the transport stage 52 to move the sensor chip 100 to the liquid feeding position (S160).
Then, the control unit 80 operates the liquid feeding unit 40 to discharge the measurement liquid in the flow path 112, and introduces the sample liquid stored in the liquid storage unit (not shown) into the flow path 112 (S170). In the flow path 112, an antigen-antibody reaction (primary reaction) traps the analyst in the reaction field on the metal membrane 104.

The sample liquid used here is a liquid prepared 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 sample liquid in the flow path 112 is removed, and the inside of the flow path 112 is washed with a cleaning liquid (S180).

Next, the control unit 80 operates the liquid feeding unit 40 to introduce the labeling liquid stored in the liquid storage unit (not shown) into the flow path 112 (S190). In the flow path 112, 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. After that, the labeling liquid in the flow path 112 is removed, the inside of the flow path 112 is washed with the cleaning liquid, and after the cleaning liquid is removed, the measuring liquid is introduced into the flow path 112 (S200).

Next, the control unit 80 operates the transport stage 52 to move the sensor chip 100 to the measurement position (S210).

Next, the control unit 80 operates the excitation light irradiation unit 20 and the fluorescence detection unit 30 to irradiate the sensor chip 100 arranged at the measurement position with the excitation light α and label the analysts captured by the ligand. Fluorescent γ emitted from the fluorescent substance is detected (S220). 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 solution can be detected.
In the present embodiment, the enhancement angle detection (S140) and the optical blank value measurement (S150) are performed before the primary reaction (S170), but the enhancement angle detection is performed after the primary reaction (S170). (S140), the optical blank value measurement (S150) may be performed.

Further, when the incident angle of the excitation light α is predetermined, the detection of the augmentation angle (S140) may be omitted.

Further, in the above description, after the primary reaction (S170) in which the analyte is reacted with the ligand, the secondary reaction (S190) 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 flow path 112, a labeling solution may be added to the sample solution to label the analyst with a fluorescent substance in advance. Further, by injecting the sample solution and the labeling solution into the flow path 112 at the same time, 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 trapped by the ligand.

In either case, both the primary reaction and the secondary reaction can be completed by introducing the sample solution into the flow path 112 (one-step method). When the one-step method is adopted as described above, the enhanced angle detection (S140) is performed before the antigen-antibody reaction.

Although the preferred embodiment of the present invention has been described above, the present invention is not limited to this. For example, although the SPFS apparatus has been described in the above examples, the sample detection system according to the present invention is SPR. It can also be applied to a sample detection system using a fluorescence immunoassay (FIA) such as an apparatus, a sample detection system using an enzyme immunoassay (EIA), and the like, as long as it does not deviate from the object of the present invention. Can be changed.

10 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 First lens 33 Optical filter 34 Second lens 35 Light receiving sensor 37 Position switching mechanism 38 Sensor control unit 40 Sending Liquid unit 41 Syringe pump 42 Syringe 43 Plunger 44 Liquid feed pump drive mechanism 45 Pipette tip 46 Pipette nozzle 50 Transfer unit 52 Transfer stage 54 Chip holder 80 Control unit 100 Sensor chip 102 Dielectric member 102a Incident surface 102b Film formation surface 102c Emission Surface 104 Metal film 106 Flow path forming member 110a First through hole 110b Second through hole 111 Multilayer film 112 Flow path 114 Flow path seal 116 Reaction field 118 Gradient 120 Lid seal 200 Flow path chip 202 Dielectric member 204 Flow path lid 204a Injection 206 Flow path seal 208 Flow path 210 Reaction field A Bubble L Liquid

Claims (10)

A sensor chip that has a reaction field inside that captures Anna Reed.
The flow path having the reaction field and
It has a first through hole formed at one end of the flow path.
The flow path is provided with a gradient so that the height of the flow path in the vicinity of the reaction field is higher than the height of the flow path in the vicinity of the first through hole.
A sensor chip having a height of the flow path in the vicinity of the reaction field of 0.1 mm to 1 mm. The sensor chip according to claim 1, wherein the height of the flow path in the vicinity of the reaction field is the highest in the flow path. The width of the flow path in the vicinity of the reaction field is 0.1 mm to 5 mm.
The sensor chip according to claim 1 or 2 , wherein the length of the flow path is 10 mm to 50 mm. The dielectric member and the flow path forming member are joined by a flow path seal in which a through hole is formed.
The sensor chip according to any one of claims 1 to 3 , wherein the flow path is a space surrounded by the dielectric member, the flow path forming member, and the flow path seal. The dielectric member and the flow path forming member in which the flow path groove is formed are joined to each other.
The sensor chip according to any one of claims 1 to 3 , wherein the flow path is a space surrounded by the dielectric member and the flow path forming member. The sensor chip according to claim 4 or 5 , wherein the flow path forming member is made of a material that is optically highly transparent, chemically stable, and has excellent moldability. The sensor chip according to any one of claims 4 to 6 , wherein the gradient is provided on the flow path forming member. The sensor chip according to any one of claims 4 to 7 , wherein the gradient is provided on the dielectric member. The sensor chip according to any one of claims 4 to 8 , wherein a metal film is formed on the surface of the dielectric member of the flow path. The sensor chip according to claim 9 and
An excitation light irradiation unit that irradiates the metal film with excitation light via the dielectric member,
A sample detection system including a fluorescence detection unit that detects fluorescence generated from the fluorescence-labeled analyzer captured in the reaction field based on the excitation light applied to the metal film.

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