Detection chip and detection method

The detection chip according to the present invention has an accommodating part, a metal film, a first reaction field, and a second reaction field. The accommodating part accommodates a liquid. The metal film is arranged in a bottom part of the accommodating part so that one face thereof faces into the accommodating part. The first reaction field and the second reaction field are arranged in mutually different regions on one face of the metal film. A capture body is immobilized in the first reaction field and the second reaction field. When the liquid is accommodated in the accommodating part, the depth of the liquid on the first reaction field differs from the depth of the liquid on the second reaction field.

TECHNICAL FIELD

The present invention relates to a detection chip for use in detecting an analyte utilizing surface plasmon resonance, and a detection method using the detection chip.

BACKGROUND ART

In a clinical test or the like, highly sensitive and quantitative detection of a trace amount of analyte, such as a protein or DNA, would allow a quick understanding of a patient's condition and the subsequent his/her treatment. For this reason, there is a need for a method of detecting a trace amount of analyte highly sensitively and quantitatively.

As a highly sensitive method of detecting an analyte, surface plasmon-field enhanced fluorescence spectroscopy (hereinafter abbreviated as “SPFS”) is known. SPFS utilizes surface plasmon resonance (hereinafter abbreviated as “SPR”) generated by irradiating a metal film with light under specific conditions (see, Patent Literature (hereinafter abbreviated as PTL) 1, for example).

In SPFS, a ligand (e.g., primary antibody) that can specifically bind to an analyte is first immobilized above a metal film, thereby forming a reaction site for specifically capturing an analyte. When a sample containing an analyte is provided to the reaction site, the analyte binds to the ligand in the reaction site. Then, when another ligand (e.g., secondary antibody) labeled with a fluorescent substance is provided to the reaction site, the analyte bound to the ligand in the reaction site is labeled with the fluorescent substance. When the metal film is irradiated with excitation light in this state, the fluorescent substance that labels the analyte is excited by enhanced electric fields due to SPR to emit fluorescence. Thus, the detection of emitted fluorescence allows the detection of the presence or an amount of the analyte. SPFS can detect an analyte highly sensitively since a fluorescent substance is excited by enhanced electric fields due to SPR.

During the detection of fluorescence, however, the presence of an unreacted fluorescent substance that does not label an analyte above a metal film results in background noise. Accordingly, in order to detect an analyte accurately, it is preferable to remove an unreacted fluorescent substance in advance by washing.

SPFS is broadly categorized into prism coupling (PC)-SPFS and grating coupling (GC)-SPFS in accordance with a means for coupling excitation light with surface plasmon. PC-SPFS utilizes a prism formed on one surface of a metal film. In this method, excitation light and surface plasmon are coupled by total reflection of excitation light at an interface between the prism and the metal film. Although PC-SPFS is a mainstream method today, it has a challenge in downsizing a detection apparatus since a prism is used and an incident angle of excitation light on a metal film is large.

In contrast, GC-SPFS couples excitation light with surface plasmon utilizing a diffraction grating (see PTL 2, for example). GC-SPFS can downsize a detection apparatus compared with PC-SPFS, since a prism is not used and an incident angle of excitation light on a diffraction grating is small.

CITATION LIST

Patent Literature

SUMMARY OF INVENTION

Technical Problem

As described above, although GC-SPFS has the advantage that a detection apparatus can be downsized compared with PC-SPFS, GC-SPFS has not yet been vigorously studied compared with PC-SPFS. Accordingly, a detection apparatus and a detection method utilizing GC-SPFS have room for improvement in detection sensitivity.

In addition, both GC-SPFS and PC-SPFS may fail to detect an analyte accurately due to the effect of background noise caused by an unreacted fluorescent substance. When washing is performed to eliminate the effect of background noise, there is a problem in which a binding state between an analyte and a metal film (or primary antibody) varies during washing, and thus a real-time measurement of a reaction process is impossible.

An object of the present invention is to provide a detection chip for use in detecting the presence or an amount of an analyte accurately utilizing SPFS even if an unreacted fluorescent substance is present above a metal film, as well as a detection method using the detection chip.

Solution to Problem

To achieve at least one of the aforementioned objects, a detection chip according to an embodiment of the present invention includes: a housing section for housing a liquid; a metal film disposed in a bottom portion of the housing section so that one surface of the metal film faces inside the housing section; a first reaction site where a ligand for capturing an analyte is immobilized, the first reaction site being disposed on the one surface of the metal film; and a second reaction site where the ligand is immobilized, the second reaction site being disposed in a different region from the first reaction site on the one surface of the metal film, in which when a liquid is housed inside the housing section, a depth of the liquid above the first reaction site is different from a depth of the liquid above the second reaction site.

To achieve at least one of the aforementioned objects, a detection method according to an embodiment of the present invention for detecting an analyte utilizing surface plasmon resonance, includes: a first step of binding an analyte labeled with a fluorescent substance to the ligand in the first reaction site and in the second reaction site inside a housing section of the detection chip according to the present invention; a second step of irradiating the metal film positioned under the first reaction site with excitation light so as to generate surface plasmon resonance while the liquid is present inside the housing section in a first depth of the liquid above the first reaction site, and detecting fluorescence emitted from the fluorescent substance present above the first reaction site; a third step of irradiating the metal film positioned under the second reaction site with excitation light so as to generate surface plasmon resonance while the liquid is present inside the housing section in a second depth different from the first depth of the liquid above the second reaction site, and detecting fluorescence emitted from the fluorescent substance present above the second reaction site; and a fourth step of calculating a signal value indicating the presence or an amount of an analyte based on a detected value obtained in each of the second step and the third step.

Advantageous Effects of Invention

According to the present invention, a detection chip and a detection method for detecting an analyte utilizing SPFS can accurately and easily detect an analyte. Moreover, according to the present invention, an analyte can be detected in real time.

DESCRIPTION OF EMBODIMENTS

Embodiments of the present invention will be described in detail hereinafter with reference to the accompanying drawings.

FIG. 1Ais a sectional view of detection chip10according to an embodiment, andFIG. 1Bis a plan view of detection chip10. The sectional view ofFIG. 1Acorresponds to a sectional view at the A-A line inFIG. 1B.FIG. 2is a perspective view of diffraction grating13of detection chip10according to the embodiment. As illustrated inFIGS. 1A, 1B, and 2, a height direction of detection chip10is defined as z-direction, an alignment direction of a periodic structure of diffraction grating13as x-direction, and a perpendicular direction to both z-direction and x-direction as y-direction.

As illustrated inFIGS. 1A and 1B, detection chip10includes substrate11, metal film12formed on substrate11, and frame14disposed on substrate11. Housing section15for housing a liquid is formed by disposing frame14on substrate11. Although details will be described hereinafter, detection chip10according to the embodiment houses a liquid in different depths (first depth h1and second depth h2) when the liquid is housed inside housing section15. Metal film12includes diffraction grating13, and ligand16(e.g., primary antibody) is immobilized on diffraction grating13. Thus, a surface of diffraction grating13also functions as a reaction site for binding ligand16and an analyte.

Substrate11is a support member for metal film12. The shape of substrate11is not limited. In the embodiment, substrate11has one step, and thus two terrace surfaces are formed on substrate11. The orientation and the height of the step are not limited as long as the optical paths of excitation light α and fluorescence β are not obstructed. For example, a wall surface of the step may be parallel to yz-plane or parallel to xz-plane. Materials for substrate11are not limited as long as they have enough mechanical strength to support metal film12. Examples of the materials for substrate11include inorganic materials, such as glass, quartz, and silicon, and resins, such as an acrylic resin, polymethyl methacrylate, a polycarbonate, polystyrene, and a polyolefin.

Metal film12is disposed on a bottom portion of housing section15with one surface faced inside housing section15. In the embodiment, metal film12is disposed on substrate11so as to be exposed to inside housing section15both on a terrace surface on the upper side and on a terrace surface on the lower side. As already mentioned, metal film12includes diffraction grating13. Because of this, surface plasmon, which is generated in metal film12upon irradiation of metal film12with light at a specific incident angle, and evanescent waves, which are generated by diffraction grating13, are coupled, thereby generating surface plasmon resonance (SPR). The thickness of metal film12is not limited. The thickness of metal film12is, for example, 30 to 500 nm, preferably 100 to 300 nm. Materials for metal film12are not limited as long as metals can generate surface plasmon. Examples of the materials for metal film12include gold, silver, aluminum, platinum, copper, and an alloy thereof.

Diffraction grating13generates evanescent waves when metal film12is irradiated with light. The shape of diffraction grating13is not limited as long as evanescent waves can be generated. For example, diffraction grating13may be a one-dimensional diffraction grating or a two-dimensional diffraction grating. As illustrated inFIG. 2, in the embodiment, diffraction grating13is a one-dimensional diffraction grating, and is formed on a surface of metal film12as a plurality of parallel protruded strips (and recessed strips) at a specific spacing. Also, the cross-sectional shape of diffraction grating13is not limited. Examples of the cross-sectional shapes of diffraction grating13include a square waveform, a sinusoidal waveform, and a sawtooth shape. In the embodiment, the cross-sectional shape of diffraction grating13is a square waveform. The optical axis of excitation light α described hereinafter is parallel to xz-plane. The pitch and the depth of grooves (recessed strips) of diffraction grating13are not limited as long as evanescent waves can be generated, and can be set appropriately in accordance with a wavelength of light to be irradiated with. For example, the pitch of the grooves of diffraction grating13is preferably in a range of 100 nm to 2,000 nm, and the depth of the grooves of diffraction grating13is preferably in a range of 10 nm to 1,000 nm.

Ligand16for capturing an analyte is immobilized above diffraction grating13. A region where ligand16is immobilized above diffraction grating13(metal film12) is herein particularly referred to as “a reaction site.” Ligand16specifically binds to an analyte. Thus, an analyte is immobilized on metal film12(diffraction grating13). In the embodiment, ligand16is almost evenly immobilized on both the terrace surfaces on the upper side and on the lower side. In the embodiment, as illustrated inFIG. 1A, when a liquid is housed in chamber section15, first reaction site17, above which the depth of the liquid is first depth h1, and second reaction site, above which the depth of the liquid is second depth h2, are disposed on one surface of metal film12. Second reaction site18is disposed in a different region from first reaction site17. First reaction site17is disposed on a terrace surface on the upper side, and second reaction site18is disposed in a terrace surface on the lower side. In first reaction site17and in second reaction site18, a same type of ligand is immobilized in a same concentration.

The types of ligand16are not limited as long as analytes can be captured. For example, ligand16is an antibody (primary antibody) that can specifically bind to an analyte, a fragment thereof, an enzyme that can specifically bind to an analyte, or the like.

During use, diffraction grating13comes into contact with a liquid, such as a buffer, for reaction, washing, or other operations. Accordingly, diffraction grating13is typically disposed in space where a liquid can be housed. In the embodiment, diffraction grating13is disposed in a bottom portion of housing section15.FIGS. 3A and 3Bare schematic sectional views illustrating other modes of detection chip10according to the embodiment. As illustrated inFIGS. 1A and 1B, diffraction grating13may be disposed on an inner surface (e.g., bottom surface) of a well, which houses a liquid, or may be disposed on an inner surface (e.g., bottom surface) of a channel (flow cell), which can feed a liquid continuously, as illustrated inFIGS. 3A and 3B. For example, detection chip10illustrated inFIGS. 1A and 1Bis suitable for a general measurement of an analyte (non-real-time measurement), as well as a mass transfer analysis (real-time measurement; see Embodiment 2) between the bulk and a surface of metal film12, and a measurement of enhanced electric field space scale (z-axis direction). For example, detection chip10illustrated inFIGS. 3A and 3Bis suitable for a general measurement of an analyte (non-real-time measurement) as well as a reaction constant analysis (real-time measurement; see Embodiment 2) of molecules (analyte) relative to another molecule (ligand) immobilized on a surface of metal film12. In such a case, a step may be formed so that the wall surface is formed perpendicularly to the flow direction of a liquid inside the channel as illustrated inFIG. 3A, or formed along the flow direction of a liquid inside the channel as illustrated inFIG. 3B.

As illustrated inFIG. 1B, frame14is a plate having a through hole, and is disposed on substrate11. Inner surfaces of the through hole constitute side surfaces of housing section15. The thickness of frame14is not limited, and is set in accordance with a volume of liquid to be housed in housing section15.

Housing section15is disposed on metal film12(or substrate11), and houses a liquid. In the embodiment, as illustrated inFIG. 1A, housing section15includes a first bottom surface and a second bottom surface each disposed at a different height. The aforementioned first reaction site17is disposed on metal film12above the first bottom surface, and second reaction site18is disposed on metal film12above the second bottom surface. Housing section15can house a liquid in first depth h1above first reaction site17(first bottom surface), and the liquid in second depth h2above second reaction site18(second bottom surface). The depth of a liquid to be housed inside housing section15is not limited, but first depth h1and second depth h2are preferably in a range of 10 μm to 1 cm. When a ratio of second depth h2to first depth h1(h2/h1) is m, m is preferably in a range of 0.1 to 10 excluding 0.9 to 1.1. The shape, the size, or the like of housing section15is not limited as long as a desired volume of a liquid can be housed, and can be appropriately set in accordance with the use. As described above, housing section15is formed by disposing frame14on substrate11, but a method for forming housing section15is not limited to this. Other examples of a method for forming housing section15include disposing a lid having recessed portions formed on the lower surface on substrate11(seeFIGS. 3A and 3B, for example).

The type of liquid to be housed in housing section15is not limited. Examples of the types of liquid include a sample containing an analyte, a labeling solution containing a fluorescent substance, and a buffer. Generally, the refractive index and the dielectric constant of the liquid are comparable with the refractive index and the dielectric constant of water. The types of sample and analyte are not limited. Examples of the samples include bodily fluids, such as blood, serum, plasma, urine, nostril mucus, saliva, and semen, and a dilute solution thereof. Examples of the analytes include a nucleic acid (DNA, RNA, or the like), a protein (a polypeptide, an oligopeptide, or the like), an amino acid, a carbohydrate, a lipid, and a modified molecule thereof.

(Manufacturing Method of Detection Chip)

In the following, a manufacturing method of detection chip10according to the embodiment will be described. The manufacturing method of detection chip10is not limited. An example of the manufacturing method of detection chip10according to the embodiment will be described hereinafter.

Detection chip10according to the embodiment, for example, can be manufactured by performing1) a first step of preparing frame14and substrate11in which a step and a plurality of grooves have been formed,2) a second step of forming metal film12and reaction sites (first reaction site17and second reaction site18) on substrate11, and3) a third step of mutually fixing frame14and substrate11in which the reaction sites have been formed. In the following, each step will be described.

In the first step, prepared are frame14and substrate11in which a step and a plurality of grooves have been formed. Specifically, a step and a plurality of grooves are formed on planar substrate11formed from a resin. A plurality of grooves constitute diffraction grating13. A method for forming a step and grooves on substrate11is not limited, and can be appropriately selected from known methods. For example, a step may be formed on substrate11by molding or lithography. Also, grooves may be formed on substrate11by pressing with an uneven original plate.

In the second step, metal film12and reaction sites are formed on substrate11. Specifically, metal film12may be formed in at least part of a region on substrate11where grooves have been formed, and a ligand may be immobilized on metal film12. Thus, metal film12including diffraction grating13can be disposed on substrate11. A formation method of metal film12is not limited. Examples of the formation method of metal film12include sputtering, vapor deposition, and plating.

Moreover, an immobilization method of ligand16is not limited. For example, a self-assembled monolayer (hereinafter referred to as “SAM”) or a polymer film, to which ligand16is bound, may be formed on diffraction grating13. Examples of SAM include films made of a substituted aliphatic thiol, such as HOOC(CH2)11SH. Examples of materials for the polymer film include polyethylene glycol and MPC polymer. Alternatively, a polymer having a reactive group (or a functional group that can be converted into a reactive group) that can bind to ligand16may be immobilized on diffraction grating13, followed by binding of ligand16to the polymer.

The order of the formation of metal film12and the formation of diffraction grating13is not limited to the aforementioned method. For example, after forming metal film12on planar substrate11, protruded/recessed shapes may be imparted to metal film12.

In the third step, substrate11and frame14are fixed. A method for fixing frame14on substrate11is not limited. For example, examples of a method for fixing substrate11and frame14include bonding using a double-stick tape, an adhesive, or the like, laser welding, and ultrasonic welding.

Through the above procedure, detection chip10according to the embodiment can be manufactured. The order of performing the second step and the third step is not limited to the aforementioned one. For example, after fixing frame14on substrate11, metal film12and reaction sites may be formed.

(Detection Method of Analyte)

In the following, a detection method of an analyte using detection chip10according to the embodiment will be described. For example, an analyte can be detected using detection apparatus (SPFS apparatus)100described hereinafter.

FIG. 4is a schematic view illustrating a configuration of detection apparatus (SPFS apparatus)100for detecting an analyte using detection chip10. The configuration of SPFS apparatus100will be described first, followed by the description of a detection method of an analyte using detection chip10and SPFS apparatus100.

Excitation light irradiation section110irradiates metal film12(diffraction grating13) of detection chip10with excitation light α having a certain wavelength and quantity of light. In this step, excitation light irradiation section110irradiates metal film12(diffraction grating13) with p-polarized light relative to a surface of metal film12so as to generate diffracted light that can couple with surface plasmon in metal film12. The optical axis of excitation light α extends along the alignment direction of a periodic structure of diffraction grating13(x-axis direction inFIG. 4). Accordingly, the optical axis of excitation light α is parallel to xz-plane (seeFIG. 4). Since excitation light α is p-polarized light relative to a surface of metal film12, the oscillation direction of the electric field of excitation light α is parallel to xz-plane including the optical axis of excitation light α and a normal line to a surface of metal film12.

Excitation light irradiation section110includes at least light source111. Excitation light irradiation section110may further include a collimator lens, an excitation light filter, and/or the like. Light source111emits excitation light α toward diffraction grating13of detection chip10. The types of light source111are not limited. Examples of the types of light source111include a light emitting diode, a mercury lamp, and other laser light sources. In the embodiment, light source111is a laser diode. The wavelength of excitation light α emitted from light source111is, for example, in a range of 400 nm to 1,000 nm. The size of an irradiation spot is preferably about 1 mm ø, for example.

Incident angle θ1of excitation light α on metal film12(seeFIG. 4) is preferably an angle in which the intensity of enhanced electric fields generated by SPR is highest, and consequently the intensity of fluorescence β from a fluorescent substance is also highest. Incident angle θ1of excitation light α is appropriately selected in accordance with a pitch of grooves of diffraction grating13, a wavelength of excitation light α, the type of component metal of metal film12, and/or the like. Since an optimal incident angle θ1of excitation light α varies in accordance with changes in various conditions, SPFS apparatus100preferably includes a first angle adjustment section (not shown) configured to adjust incident angle θ1by relatively rotating the optical axis of excitation light α and detection chip10. The first angle adjustment section, for example, may rotate excitation light irradiation section110or detection chip10around the intersection between the optical axis of excitation light α and metal film12.

As illustrated inFIG. 4, excitation light irradiation section110irradiates diffraction grating13(metal film12) with excitation light α at a specific incident angle θ1. In the irradiated region, surface plasmon generated in metal film12and evanescent waves generated by diffraction grating13are coupled, thereby generating SPR. When a fluorescent substance is present in the irradiated region, the fluorescent substance is excited by enhanced electric fields generated by SPR, and thus fluorescence β is emitted. As described above, in GC-SPFS, different from PC-SPFS, fluorescence β is emitted with directivity in a particular direction. For example, emission angle θ2of fluorescence β is approximated by 2θ1. Under conditions for generating SPR, reflected light γ of excitation light α scarcely occurs.

Fluorescence detection section120detects at least twice fluorescence β emitted from a fluorescent substance above metal film12(diffraction grating13). More specifically, fluorescence detection section120detects at least once fluorescence β emitted from a fluorescent substance above first reaction site17while the depth of a liquid above first reaction site17is first depth h1, and detects at least once fluorescence β emitted from a fluorescent substance above second reaction site18while the depth of a liquid above second reaction site18is second depth h2. Fluorescence detection section120is disposed so as to sandwich a normal line to the surface of metal film12, which passes through the intersection between the optical axis of excitation light α and metal film12, between fluorescence detection section120and excitation light irradiation section110.

Fluorescence detection section120includes at least light receiving sensor121. Fluorescence detection section120may further include a condensing lens group, an aperture stop, a fluorescence filter, or the like. Light receiving sensor121detects fluorescence β emitted from a fluorescent substance present above metal film12, and thus detects a fluorescence image above metal film12. The type of light receiving sensor121, although not limited, is a photomultiplier tube with high sensitivity and S/N ratio, for example, and may be an avalanche photodiode (APD), a photodiode (PD), a CCD image sensor, or the like.

In GC-SPFS, fluorescence β from a fluorescent substance immobilized above metal film12is found to be emitted with directivity in a particular direction from diffraction grating13(reaction site). In this case, an angle of the optical axis of fluorescence detection section120from the normal line (to the surface of metal film12) is preferably an angle with maximum intensity of fluorescence β (fluorescence peak angle). Accordingly, SPFS apparatus100preferably includes a second angle adjustment section (not shown) configured to adjust an angle of the optical axis of fluorescence detection section120by relatively rotating the optical axis of fluorescence detection section120and detection chip10. For example, the second angle adjustment section may rotate fluorescence detection section120or detection chip10around the intersection between the optical axis of fluorescence detection section120and metal film120.

Conveyance section130moves a position of detection chip10. Conveyance section130includes conveyance stage131and chip holder132. Chip holder132is fixed to conveyance stage131and holds detection chip10detachably. The shape of chip holder132is a shape that can holds detection chip10without obstructing the optical paths of excitation light α and fluorescence β. Conveyance stage131moves chip holder132in one direction and in the opposite direction. The shape of conveyance stage131is also a shape without obstructing the optical paths of excitation light α and fluorescence β. Conveyance stage131is driven by a stepping motor and/or the like, for example.

Control section140controls the operation of excitation light irradiation section110(light source111and first angle adjustment section), fluorescence detection section120(light receiving sensor121and second angle adjustment section), and conveyance section130(conveyance stage131). Also, control section140functions as a processing section for processing output signals (detected results) from fluorescence detection section120. Specifically, the processing section calculates, based on two or more detected values at fluorescence detection section120(light receiving sensor121), a signal value indicating the presence or an amount of an analyte and a noise value as needed. Control section140is, for example, a computer that includes an arithmetic apparatus, a control apparatus, a storage apparatus, an input apparatus, and an output apparatus, and executes software.

In the following, the detection operation of SPFS apparatus100(detection method according to the embodiment) will be described.FIG. 5is a flow chart illustrating an example of an operational procedure of SPFS apparatus100. Described will be an example in which a primary antibody is used as ligand16, and an analyte is labeled with a fluorescent substance by binding a secondary antibody labeled with the fluorescent substance to the analyte captured by the primary antibody.

First, the detection is prepared (step S110). Specifically, detection chip10is prepared, and then detection chip10is installed in chip holder132of SPFS apparatus100. When a humectant is present above metal film12of detection chip10, the humectant is removed by washing above metal film12so that a primary antibody captures an analyte properly.

Then, the analyte in a sample and the primary antibody are bound to each other (primary reaction: step S120). Specifically, the sample is provided to above metal film12so that the sample comes into contact with the primary antibody. When an analyte is present in the sample, at least part of the analyte binds to the primary antibody.

Then, the analyte bound to the primary antibody is labeled with a fluorescent substance (secondary reaction: step S130). Specifically, a fluorescent labeling solution containing a secondary antibody labeled with a fluorescent substance is provided to above metal film12so that the analyte bound to the primary antibody comes into contact with the fluorescent labeling solution. The fluorescent labeling solution is a buffer containing a secondary antibody labeled with a fluorescent substance, for example. When an analyte is bound to a primary antibody, at least part of the analyte is labeled with a fluorescent substance. In this case, a liquid (e.g., buffer or fluorescent labeling solution) is housed in first depth h1above first reaction site17and in second depth h2above second reaction site18. As described hereinafter, SPFS apparatus100according to the embodiment can detect an analyte even without removal of a free secondary antibody. It is preferable, however, to wash above metal film12with a buffer or the like after labeling with a fluorescent substance to remove a free secondary antibody.

The order of the primary reaction and the secondary reaction is not limited to the aforementioned one. For example, after binding an analyte to a secondary antibody, a liquid containing the complex may be provided to above metal film12. Alternatively, a sample and a fluorescent labeling solution may be provided to above metal film12simultaneously.

Then, fluorescence β emitted from the fluorescent substance above first reaction site17(metal film12) is detected while diffraction grating13(metal film12) positioned under first reaction site17is irradiated with excitation light α (step S140). Specifically, control section140operates excitation light irradiation section110to irradiate diffraction grating13where first reaction site17is disposed with excitation light α so as to generate SPR, and simultaneously records detected value Iaat light receiving sensor121. During this operation, a liquid is housed in first depth h1above first reaction site17.

Then, reaction sites as detection targets are switched (step S150). Specifically, control section140operates conveyance stage131to move detection chip10. This enables excitation light irradiation section110to irradiate second reaction site18with excitation light α.

Then, fluorescence β emitted from the fluorescent substance above second reaction site18(metal film12) is detected while diffraction grating13(metal film12) positioned under second reaction site18is irradiated with excitation light α (step S160). Specifically, control section140operates excitation light irradiation section110to irradiate diffraction grating13where second reaction site18is disposed with excitation light α so as to generate SPR, and simultaneously records output value Ibat light receiving sensor121. During this operation, a liquid is housed in second depth h2above second reaction site18.

Even when inside housing section15is washed by replacing the fluorescent labeling solution inside housing section15with a secondary antibody-free buffer after the secondary reaction (step S130), part of the secondary antibody bound to the analyte is released in the buffer. Alternatively, when washing is not performed after the secondary reaction (step S130), the fluorescent labeling solution is left untouched inside housing section15. Accordingly, in either the cases, detected value Iain step S140and detected value Ibin step S160contain a fluorescence β component emitted from the fluorescent substance (mainly the fluorescent substance labeling the analyte captured by the primary antibody), which is excited by enhanced electric fields due to SPR, and a fluorescence β component emitted from the fluorescent substance (mainly the free fluorescent substance in the liquid inside housing section15), which is excited by light other than that excited by enhanced electric fields due to SPR (excitation light α and extraneous light).

Finally, control section (processing section)140calculates a signal value indicating the presence or an amount of the analyte based on detected values obtained at fluorescence detection section120in steps S140to S160(step S170). A calculation method of the signal value will be described hereinafter.

FIGS. 6A and 6Bare schematic views for explaining a detection principle of an analyte at SPFS apparatus100.FIG. 6Aillustrates a state in which a liquid is present in first depth h1above first reaction site17, andFIG. 6Billustrates a state in which a liquid is present in second depth h2above second reaction site18. InFIGS. 6A and 6B, white stars represent a fluorescent substance.

As illustrated inFIG. 6A, in a state in which a liquid is present in first depth h1, fluorescence β emitted from first reaction site17contains light generated while being influenced by enhanced electric fields due to SPR and light generated without being influenced by enhanced electric fields due to SPR. In other words, a detected value during the detection of fluorescence β contains component I1of light generated while being influenced by enhanced electric fields due to SPR (signal component) and component I2of light generated without being influenced by enhanced electric fields due to SPR (noise component). In this case, I1is mainly attributed to fluorescence β from a fluorescent substance that labels an analyte captured by a primary antibody, and I2is mainly attributed to fluorescence β from a free fluorescent substance in a liquid inside housing section15. Accordingly, I1is a signal value indicating the presence or an amount of an analyte, and I2is a noise value. Therefore, in a state in which the depth of a liquid above first reaction site17is first depth h1, detected value Iaat fluorescence detection section120is represented by the following equation 1.
[1]
I1+I2=Ia(Equation 1)

Further, as illustrated inFIG. 6B, in a state in which a liquid is present in second depth h2, fluorescence β emitted from second reaction site18contains light generated while being influenced by enhanced electric fields due to SPR and light generated without being influenced by enhanced electric fields due to SPR. Meanwhile, a distance from a surface of diffraction grating13where the effects of enhanced electric fields due to SPR are exerted is constant regardless of the depth of a liquid housed inside housing section15. Thus, the magnitude of I1is the same both in a state in which a liquid is present in first depth h1and in a state in which a liquid is present in second depth h2.

Meanwhile, when a ratio of second depth h2to first depth h1is m, present above reaction site18are a liquid in a volume m times a volume above first reaction site17, and a fluorescent substance in an amount m times an amount above first reaction site17. Compared with the depth of a liquid (several μm to several cm), a distance where the effects of enhanced electric fields are exerted, which is 100 nm or less, is small enough to be ignored. Accordingly, m can be approximated as a height ratio of a region of a liquid in depth h2without being influenced by enhanced electric fields due to SPR to a region of a liquid in depth h1without being influenced by enhanced electric fields due to SPR. Thus, fluorescence β emitted while a liquid is present in second depth h2contains m times I2contained in fluorescence β emitted while a liquid is present in first depth h1. Therefore, in a state in which the depth of a liquid above second reaction site18is second depth h2in step S160, detected value Ibat fluorescence detection section120is represented by the following equation 2.
[2]
I1+m×I2=Ib(Equation 2)

Control section (processing section)140calculates I1represented by the following equation 3 as a signal value indicating the presence or an amount of an analyte based on detected values Iaand Ibrepresented by equation 1 and equation 2, respectively.

Moreover, control section (processing section)140can further calculate, as needed, noise value I2represented by the following equation 4, which indicates neither the presence nor an amount of an analyte.

Through the above procedure, the presence or an amount of an analyte in a sample can be detected. In the present invention, measurements of blank values may not necessarily be performed since noise can be eliminated through the above procedure.

As described above, by using detection chip10according to the embodiment, background noise can be eliminated, and the presence or an amount of an analyte can be detected highly sensitively, easily, and accurately even if an unreacted fluorescent substance is present above metal film12.

In addition, since the detection method according to the embodiment can eliminate noise component contained in fluorescence β, an analyte can be detected without removal of a free secondary antibody after performing the secondary reaction (step S130).

In the embodiment, the order of the first detection step of fluorescence β (step S140) and the second detection step of fluorescence β (step S160) is not limited to the aforementioned one. In other words, fluorescence β emitted from above first reaction site17may be detected after detecting fluorescence β emitted from above second reaction site18.

Detection chip10, whose housing section15has a first bottom surface and a second bottom surface, is described in the embodiment. The detection chip according to the present invention, however, is not limited to this mode, and may be detection chip10a,10b, or10cillustrated inFIGS. 7A to 7C, for example.FIG. 7Ais a sectional view illustrating a configuration of detection chip10aaccording to Modification 1,FIG. 7Bis a sectional view illustrating a configuration of detection chip10baccording to Modification 2, andFIG. 7Cis a sectional view illustrating a configuration of detection chip10caccording to Modification 3.

As illustrated inFIG. 7A, in detection chip10aaccording to Modification 1, a bottom surface of housing section15acontains a sloping surface. Thus, in detection chip10a, first reaction site17and second reaction site18are disposed in predetermined different regions on the sloping surface. Also, as illustrated inFIG. 7B, detection chip10baccording to Modification 2 further includes lid19bto be disposed in the upper portion of housing section15b. The shape of lid19bis not limited as long as a liquid can be housed in first depth h1and in second depth h2inside housing section15. In the embodiment, lid19bhas a step. Because of this, when lid19bis disposed on frame14, housing section15bincludes a first upper surface and a second upper surface each disposed at a different height. In this case, first reaction site17is disposed on metal film12in a position facing the first upper surface, and second reaction site18is disposed on metal film12in a position facing the second upper surface. Further, as illustrated inFIG. 7C, detection chip10caccording to Modification 3 further includes lid19c, which is disposed in the upper portion of housing section15cand contains a sloping surface relative to a bottom surface of housing section15c. In detection chip10c, first reaction site17and second reaction site18are disposed in predetermined different regions on metal film12in positions facing the sloping surface. Any detection chip10a,10b, or10ccan be used in the same manner as detection chip10.

As detection chips10band10caccording to Modifications 2 and 3, the detection chip according to the present invention may have a lid. The lid may preferably be formed from a material transparent to excitation light α and fluorescence β. Examples of the materials for the lid include resins. As long as a portion where excitation light α and fluorescence β pass through is transparent to excitation light α and fluorescence β, the other portion of the lid may be formed from opaque materials. The lid is joined with a frame through, for example, bonding with a double-stick tape, an adhesive, or the like, laser welding, ultrasonic welding, or crimping with a clamping member.

Further, although detection chip10according to the embodiment and detection chip10baccording to Modification 2 have two terrace surfaces on the bottom or the upper surface of housing section15, the number of terrace surfaces is not limited to this, and may be three or more, for example.

Detection chip10′ according to Embodiment 2 has the same configuration as that of detection chip10according to Embodiment 1, and SPFS apparatus100′ using detection chip10′ according to Embodiment 2 also has the same configuration as that of SPFS apparatus100according to Embodiment 1. Meanwhile, the detection method according to Embodiment 2 is different from the detection method according to Embodiment 1 in that the former performs real-time measurements. Accordingly, the description of the configurations of detection chip10′ and SPFS apparatus100′ is omitted, and only an operational procedure of SPFS apparatus100′ will be described.

SPFS apparatus100′ according to the embodiment continually irradiates diffraction grating13positioned under first reaction site17and second reaction site18with excitation light α, and continually detects fluorescence β emitted from a fluorescent substance. The term “continual” herein refers to not only continuous operation, but also intermittent operation. Accordingly, the phrase “continual irradiation with excitation light” means irradiation with excitation light α for proper time at proper frequency that allows detection of changes in analyte over time. The phrase “continual detection of fluorescence” herein means detection of fluorescence β for proper time at proper frequency that allows detection of changes in analyte over time.

For example, continual irradiation with excitation light α may be continuous irradiation with excitation light α or may be intermittent irradiation with excitation light α. From the viewpoint of preventing fading of a fluorescent substance, continual irradiation with excitation light α is preferably intermittent irradiation with excitation light α. In this case, an interval for irradiation with excitation light α may be constant or inconstant (predetermined). Alternatively, an interval for irradiation with excitation light α may be automatically determined based on certain conditions, such as automatic calculation by a program, may be empirically determined by a preliminary experiment or the like, or may be predetermined by a user.

Also, an interval for irradiation with excitation light α may be determined in accordance with detected results of fluorescence intensity. For example, when a detected value of fluorescence intensity is small, an interval for irradiation with excitation light α may be shortened further, whereas when a detected value of fluorescence intensity is large, an interval for irradiation with excitation light α may be extended further. Alternatively, when a detected value of fluorescence intensity largely varies over time, an interval for irradiation with excitation light α may be shortened further, whereas when a detected value of fluorescence intensity slightly varies over time, an interval for irradiation with excitation light α may be extended further. Such adjustment of an interval for irradiation with excitation light α can be performed by, for example, appropriate setting of a threshold value for detected values of fluorescence intensity, and feedback control based on detected values of fluorescence intensity. From the viewpoint of closely observing changes in analyte over time, such adjustment of an interval for irradiation with excitation light α is preferable.

The same applies to the timing of continual detection of fluorescence β.

FIG. 8is a flow chart showing an example of an operational procedure of SPFS apparatus100′ according to Embodiment 2. In this example, a primary antibody as a ligand is immobilized above metal film12. As a ligand for fluorescent labeling, a secondary antibody labeled with a fluorescent substance is used.

First, the detection is prepared (step S110′). Specifically, detection chip10′ is prepared, and then detection chip10′ is installed in chip holder132of SPFS apparatus100′. In the same manner as Embodiment 1, above metal film12is washed as needed.

Then, an analyte in a sample is reacted with a primary antibody (primary reaction: step S120′). Specifically, a sample is provided to above metal film12so that the sample comes into contact with the primary antibody. When an analyte is present in the sample, at least part of the analyte binds to the primary antibody. After that, above metal film12is washed with a buffer or the like to remove a substance unbound to the primary antibody.

Then, the analyte bound to the primary antibody is labeled with a fluorescent substance (secondary reaction: step S130′). Specifically, a fluorescent labeling solution containing a secondary antibody labeled with a fluorescent substance is provided to above metal film12so that the analyte bound to the primary antibody comes into contact with the fluorescent labeling solution. The fluorescent labeling solution is, for example, a buffer containing a secondary antibody labeled with a fluorescent substance. When an analyte is bound to a primary antibody, at least part of the analyte is labeled with the fluorescent substance. In the same manner as Embodiment 1, SPFS apparatus100′ can measure an analyte without removal of a free secondary antibody. In this case, a liquid (e.g., buffer or fluorescent labeling solution) is housed in first depth h1above first reaction site17and in second depth h2above second reaction site18.

The order of the primary reaction and the secondary reaction is not limited to the aforementioned one. For example, after binding an analyte to a secondary antibody, a liquid containing the complex may be provided to above metal film12. Alternatively, a sample and a fluorescent labeling solution may be provided to above metal film12simultaneously.

Then, fluorescence β emitted from the fluorescent substance above first reaction site17(metal film12) is detected while metal film12positioned under first reaction site17is irradiated with excitation light α (step S140′), detection chip10′ is moved so that excitation light irradiation section110irradiates metal film12positioned under second reaction site18with excitation light α (step S150′), fluorescence β emitted from the fluorescent substance above second reaction site18(metal film12) is detected while metal film12positioned under second reaction site18is irradiated with excitation light α (step S160′), and a signal value indicating the presence or an amount of the analyte is calculated based on results of fluorescence detection in steps S140′ and S150′ (step S170′). These steps S140′ to S170′ as one cycle are repeated specific times (step S180′). Through this step, fluorescence β emitted from the fluorescent substance present above first reaction site17while a liquid is present in first depth h1, and fluorescence β emitted from a fluorescent substance present above second reaction site18while a liquid is present in second depth h2are alternately detected a plurality of times, thereby obtaining respective detected values Iaand Ibat fluorescence detection section120continually (intermittently) while calculating signal values indicating the presence or an amount of the analyte continually (intermittently).

Specifically, in step S140′, control section140operates light source111to emit excitation light α continuously or intermittently at a specific interval (i.e., “continually”). The phrase “at a specific interval” herein refers to, for example, an interval for switching reaction sites as a detection target described hereinafter. At the same time, control section140operates fluorescence detection section120to detect fluorescence β emitted from the fluorescent substance present above metal film12continually. The timing of the continual detection of fluorescence β may be synchronized with or different from the timing of irradiation with excitation light α over time.

As described in Embodiment 1, fluorescence β emitted from above first reaction site17while a liquid is present in first depth h1contains light generated while being influenced by enhanced electric fields due to SPR and light generated without being influenced by enhanced electric fields due to SPR. In other words, detected value Iaduring the detection of fluorescence β contains component I1of light generated while being influenced by enhanced electric fields due to SPR (signal component) and component I2of light generated without being influenced by enhanced electric fields due to SPR (noise component) (seeFIG. 6A). Therefore, in step140′ in a state in which the depth of a liquid above first reaction site17is first depth h1, detected value Iaat fluorescence detection section120is represented by the following equation 5.
[5]
I1+I2=Ia(Equation 5)

In step160′, control section140operates light source111to keep emitting excitation light α continually. At the same time, control section140operates fluorescence detection section120to detect fluorescence β from metal film12continually.

As described in Embodiment 1, fluorescence β emitted from second reaction site18while a liquid is present in second depth h2also contains light generated while being influenced by enhanced electric fields due to SPR and light generated without being influenced by enhanced electric fields due to SPR. In other words, detected value Ibduring the detection of fluorescence β contains component I1of light generated while being influenced by enhanced electric fields due to SPR (signal component) and component I2of light generated without being influenced by enhanced electric fields due to SPR (noise component) (seeFIG. 6B). Also in Embodiment 2, noise component I2in detection of fluorescence β while a liquid is presence in second depth h2contains noise component m (h2/h1) times noise component in detection of fluorescence β while a liquid is present in first depth h1. Therefore, in step S160′ in a state in which the depth of a liquid above second reaction site18is second depth h2, detected value Ibat fluorescence detection section120is represented by the following equation 6.
[6]
I1+m×I2=Ib(Equation 6)

The order of the first fluorescence detection (step S140′) and the second fluorescence detection (step S160′) is not limited to the aforementioned one. In other words, the detection of fluorescence β emitted from above first reaction site17(first fluorescence detection) may be performed after the detection of fluorescence β emitted from above second reaction site18(second fluorescence detection).

In step S170′, control section (processing section)140calculates signal value which is represented by the following equation 7, and indicates the presence or an amount of the analyte based on detected values Iaand Ibobtained at fluorescence detection section120represented by equation 5 and equation 6, respectively (step S180′). Noise value I2can be calculated as needed.

in which m is a ratio of the second depth h2to the first depth h1, and is a real number excluding 1.

In step S180′, for example, control section140counts the number of the second detection of fluorescence β (C2), adjusts so that first reaction site17becomes a detection target again when C2does not reach a set value (e.g., N times), and performs the first detection of fluorescence β by returning to step S140′. When C2reaches N times, the detection is terminated.

Although the step of calculating a signal value (step S170′) is performed every time when the fluorescence detection (step S140′ and S160′) is performed, the step is not limited to this. For example, a signal value may be calculated after C2reaches N times and the detection of fluorescence β is terminated.

Thus, signal values that vary over time can be calculated in real time.

As described above, even if an unreacted fluorescent substance is present above metal film12, by using detection chip10′ according to the embodiment, background noise can be eliminated, and the presence or an amount of an analyte can be detected in real time. Therefore, in the detection of an analyte, real-time measurements can be performed highly sensitively, easily, and accurately by using detection chip10′ according to the embodiment.

Moreover, since SPFS apparatus100′ can eliminate noise component contained in fluorescence β, an analyte can be detected without removal of a free secondary antibody after performing the secondary reaction (step S130′).

Therefore, according to the detection method of each aforementioned embodiment, an analyte in an unpurified sample, such as a crude product in biosynthesis of a novel biomolecule or a raw sample collected in a clinical test, can be detected easily and highly accurately over time.

Although a mode without measurements of blank values is described in each aforementioned embodiment, blank values may be measured as needed before the secondary reaction (step S130or S130′). In this case, specifically, blank values Ia′ and Ib′ are obtained in advance through the same procedure as in steps S140to S160or S140′ to S160′ while a fluorescent substance is absent in a liquid inside housing section15. In this case, according to the aforementioned calculation method, signal value I1and noise value I2, as needed, are calculated in step S170or S170′ after blank values Ia′ and Ib′ are subtracted from respective detected values Iaand Ib.

Although the detection method utilizing GC-SPFS is described in each aforementioned embodiment, the detection method according to the embodiment may also utilize PC-SPFS. In this case, a detection chip includes a prism formed from a dielectric, and metal film12is disposed on the prism instead of substrate11. In addition, metal film12lacks diffraction grating13. Further, a rear surface of metal film12corresponding to reaction sites (first reaction site17and second reaction site18) is irradiated with excitation light α through the prism.

Although an example in which detection chip10or10′ is irradiated with excitation light α from the side of metal film12is described in the embodiment, detection chip10or10′ may be irradiated from the side of substrate11.

Furthermore, detection chips10ato10caccording to the modifications can be used for the detection method according to Embodiment 2 (real-time measurements).

This application is entitled to and claims the benefit of Japanese Patent Application No. 2014-249044, filed on Dec. 9, 2014, the disclosure of which including the specification, drawings and abstract is incorporated herein by reference in its entirety.

INDUSTRIAL APPLICABILITY

The detection chip and the detection method according to the present invention can detect an analyte highly reliably, and thus are useful for clinical tests, for example.

Moreover, the detection chip and the detection method according to the present invention can detect an analyte highly reliably even without washing a metal film surface after a labeling solution or the like is provided. Thus, the detection chip and the detection method according to the present invention are expected to shorten the detection time, as well as to contribute to development, widespread use, and advancement of a downscalable quantitative immunoassay apparatus and an extremely simple quantitative immunoassay system.

REFERENCE SIGNS LIST