JP2010112748A - Detection method, detecting sample cell and detecting kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To suppress the irregularity of signal intensity, to efficiently utilize an intensified electric field and to attain a high S/N ratio in a detection method for detecting the amount of a substance to be detected. <P>SOLUTION: In the detection method wherein a sample S is supplied to the sensor part 14 on a sensor chip 10 and irradiated with exciting light Lo to produce an intensified photoelectric field D on the sensor part 14, a fluorescent label F is excited and the amount of the substance A to be detected is detected on the basis of the quantity of the light Lf caused by this excitation; a blocking substance R, of which the properties are similar to those of the fluorescent substance F, for blocking the surface of the sensor part 14 and the fluorescent substance (fluorescent label) F obtained by including a plurality of fluorescent coloring matter molecules (f) by a light transmitting material 16 permitting the fluorescence produced from a plurality of the fluorescent coloring matter molecules (f) to transmit are used. Then, the non-specific adsorption of the fluorescent substance F onto the sensor part 14 is prevented by the blocking substance R. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a detection method for detecting a substance to be detected in a sample, a detection sample cell, and a detection kit.

  In bio-measurement and the like, the fluorescence method is widely used as a highly sensitive and easy measurement method. The fluorescence method is qualitative or qualitative by irradiating a sample considered to contain a substance to be detected that emits fluorescence when excited by light of a specific wavelength, by irradiating the excitation light of the specific wavelength and detecting the fluorescence emitted at this time. This is a method for quantitatively confirming the presence of a substance to be detected. In addition, when the substance to be detected is not a fluorescent material, the substance to be detected is labeled with a fluorescent label such as an organic fluorescent dye, and then the fluorescence is detected in the same manner. It is a method to confirm.

  In the above-described fluorescence method, there is a technique in which only a specific target substance can be efficiently detected while flowing a sample, and the target substance is fixed to the surface of the sensor unit by the following two methods and then fluorescence detection is performed. It is common. One of such techniques is, for example, when the substance to be detected is an antigen, the antigen is specifically bound to the primary antibody immobilized on the surface of the sensor unit, and then a fluorescent label is provided and the antigen and By binding a secondary antibody that specifically binds to the antigen, a binding state of primary antibody-antigen-secondary antibody is formed, and fluorescence from a fluorescent label attached to the secondary antibody is detected. This is the so-called sandwich method. The other is, for example, when the substance to be detected is an antigen, a secondary antibody in which an antigen and a fluorescent label are attached to the primary antibody immobilized on the surface of the sensor unit (unlike the above-described secondary antibody, Is a so-called competition method in which the primary antibody is bound to the primary antibody competitively and the fluorescence from the fluorescent label attached to the competitively bound secondary antibody is detected. .

  In addition, for the reason that the S / N ratio can be improved in fluorescence detection, an evanescent fluorescence method has been proposed in which a fluorescent label indirectly fixed to a sensor unit by the above-described method is excited by evanescent light. In the evanescent fluorescence method, excitation light is incident from the back surface of the sensor unit, and the fluorescent label is excited by evanescent light that oozes out to the surface of the sensor unit, and fluorescence generated from the fluorescent label is detected.

  On the other hand, in the evanescent fluorescence method, a method using the effect of electric field enhancement by plasmon resonance is proposed in Patent Document 1, Non-Patent Document 1, and the like in order to improve sensitivity. In this surface plasmon enhanced fluorescence method, a metal layer is provided in a sensor portion, surface plasmon is generated in the metal layer, and the S / N ratio is improved by increasing the fluorescence signal by the electric field enhancing action.

  Further, in the evanescent fluorescence method, as in the surface plasmon enhanced fluorescence method, as a method having the effect of enhancing the electric field of the sensor unit, a method using the electric field enhancement effect by the optical waveguide mode is proposed in Non-Patent Document 2. . In this optical waveguide mode enhanced fluorescence spectroscopy (OWF), a metal layer and an optical waveguide layer made of a dielectric or the like are sequentially formed on a sensor portion, and an optical waveguide mode is formed on the optical waveguide layer. The fluorescence signal is enhanced by the electric field enhancement effect.

  Further, in Patent Document 2 and Non-Patent Document 3, instead of detecting fluorescence from a fluorescent label as in the fluorescence method described above, the fluorescence is generated by newly inducing surface plasmon in the metal layer. A method for detecting synchrotron radiation (SPCE: Surface Plasmon-Coupled Emission) has been proposed.

As described above, in biomeasurement and the like, as a method for detecting a substance to be detected, irradiation of excitation light causes plasmon resonance and an optical waveguide mode, and the fluorescent label is excited by an electric field enhanced by these. Various methods for detecting light generated due to excitation of the fluorescent label have been proposed.
JP-A-10-307141 US Patent Application Publication No. 2005/0053974 W. Knoll et al., Analytical Chemistry 77 (2005), p.2426-2431 2007 Spring Japan Society of Applied Physics Proceedings No.3, P.1378 Thorsten Liebermann Wolfgang Knoll, "Surface-plasmon field-enhanced fluorescence spectroscopy" Colloids and Surfaces A 171 (2000) 115-130

  However, the effect of electric field enhancement by surface plasmon resonance and optical waveguide mode attenuates rapidly as the distance from the surface of the metal layer or optical waveguide layer increases, so the distance from this surface to the fluorescent label changes only slightly. There is a problem that a difference occurs and signal variation occurs.

  For example, FIG. 19 shows a schematic diagram of the vicinity of a sensor unit of a device that detects fluorescence due to an electric field enhancement effect by surface plasmon resonance. A gold film 102 is provided on the surface of the prism (substrate) 101, and the primary antibody B <b> 1 is immobilized on the gold film 102. When the sandwich assay is performed, the binding state of the primary antibody B1-antigen A-labeled secondary antibody BF is formed as described above. Here, the labeled secondary antibody BF is a secondary antibody provided with a fluorescent label (here, a fluorescent dye molecule f). Then, excitation light is incident on the interface between the prism 101 and the gold film 102 at an angle greater than the total reflection angle, thereby exciting surface plasmons on the gold film surface and enhancing the electric field on the gold film surface. As a result, the fluorescent label f is excited in an enhanced electric field and emits fluorescence.

  The graph in FIG. 19 shows the distance dependence of the electric field strength from the sensor part surface (gold film surface). As shown in the graph, the electric field strength rapidly decreases as the distance from the surface increases. At this time, the distance from the surface of the sensor unit to the fluorescent label f of the labeled secondary antibody BF may be as much as about 50 nm, and in such a case, the fluorescence intensity is attenuated by 30% or more. In addition, the primary antibody B1 is not always fixed upright on the surface of the sensor unit, and may fall down along the surface due to the flow of liquid or steric hindrance. Accordingly, the distance from the surface of the fluorescent label f varies accordingly, and this variation leads to variations in signal intensity.

  The present invention has been made in view of the above problems, and is a fluorescent label that can suppress the variation in signal intensity, can efficiently use the enhanced electric field, and can realize a high S / N ratio. It is an object of the present invention to provide a detection method for detecting light generated due to excitation of light.

  Furthermore, an object of the present invention is to provide a detection sample cell and a detection kit used in the detection method.

In order to solve the above problems, the detection method according to the present invention comprises:
On one surface of the dielectric plate, a sensor chip including a sensor unit having a laminated structure including a metal layer adjacent to the dielectric plate is used.
By bringing the sample into contact with the sensor unit, the fluorescent label binding substance composed of the fluorescent label and the binding substance labeled on the fluorescent label in an amount corresponding to the amount of the substance to be detected contained in the sample is transferred to the sensor unit. Combined on top
By irradiating the sensor unit with excitation light, an enhanced photoelectric field is generated on the sensor unit,
In the detection method of detecting the amount of the substance to be detected based on the amount of light generated by exciting the fluorescent label with the enhanced photoelectric field and the excitation,
As a fluorescent label, a plurality of first fluorescent dye molecules and a first particle made of a light-transmitting material that transmits fluorescence generated from the plurality of first fluorescent dye molecules and including the plurality of first fluorescent dye molecules Using a fluorescent substance composed of
Specific binding of the binding substance that does not contain the first fluorescent dye molecule as a blocking agent for preventing the fluorescent label binding substance from adsorbing to the sensor part due to nonspecific adsorption of the fluorescent label binding substance to the sensor part It is a blocking substance which does not have the property, It is characterized by using the blocking substance which has nonspecific adsorption property equivalent to the nonspecific adsorption property of a fluorescence label binding substance.

  Here, “laminated structure including a metal layer” means a structure including at least one layer including a metal layer. That is, the laminated structure may be only a metal layer. Here, the “layer” does not necessarily need to cover one surface, and may be, for example, one having a minute opening or one having a rough density.

  The “binding substance” means a substance that specifically binds to a specific target substance. For example, when an antigen is considered as a specific target substance, an antibody that specifically binds to the antigen is exemplified as a binding substance. In the above case, another antibody that competes with the antigen and specifically binds to the antibody is also a binding substance.

  The “fluorescent label binding substance” is bound on the sensor unit by an amount corresponding to the amount of the substance to be detected through the first binding substance that is fixed on the sensor part and specifically binds to the substance to be detected. It shall mean a binding substance labeled with a fluorescent label. That is, for example, when an assay by the sandwich method is performed, the fluorescent label binding substance is composed of a second binding substance that specifically binds to the substance to be detected and a fluorescent label that labels the second binding substance. The As a result, a sandwich structure of the first binding substance-the substance to be detected-the second binding substance is created, and the fluorescent label binding substance is bound on the sensor unit. Here, the binding site for the first binding substance of the substance to be detected is different from the binding site for the second binding substance. On the other hand, when performing an assay by the competition method, the fluorescently labeled binding substance is composed of a third binding substance that competes specifically with the substance to be detected and specifically binds to the first binding substance, and the third binding substance. And a fluorescent label for labeling. As a result, a first binding substance-third binding substance bond is created, and the fluorescent label binding substance is bound on the sensor unit.

  The “photoelectric field” means an electric field caused by evanescent light or near-field light generated by irradiation with excitation light.

  Generating an “enhanced photoelectric field” means that an enhanced photoelectric field is formed by enhancing the photoelectric field. The method for enhancing the photoelectric field may be enhancement by excitation of plasmon resonance or enhancement by excitation of the optical waveguide mode.

  “Light resulting from excitation” of a fluorescent label is light that is directly or indirectly generated by excitation of the fluorescent label, and is between the amount of light generated and the number of excited fluorescent labels. It shall mean that which has a correlation.

  “Detecting the amount of the substance to be detected” includes detection of the presence or absence of the substance to be detected, and means not only a qualitative amount but also a quantitative amount.

  “Non-specific adsorptivity of fluorescent label binding substance” with respect to the sensor part means that the fluorescent label binding substance is placed anywhere on the sensor part due to the interaction between the fluorescent label binding substance and the sensor part. It shall mean an adsorbable property. Here, the “interaction” is, for example, an action due to hydrogen bonding, electrostatic force, hydrophilicity / hydrophobicity or the like.

  The “specific binding property of the binding substance” constituting the fluorescent label binding substance refers to the binding substance (fluorescent label binding substance via the first binding substance that is fixed on the sensor unit and specifically binds to the detection target substance. ) Means a property capable of specifically binding on the sensor unit. That is, the specific binding property of the binding substance means the specific binding property of the second binding substance that specifically binds to the substance to be detected, for example, when assaying by the sandwich method, while the assay by the competitive method is performed. In this case, it means the specific binding property of the third binding substance that competes with the substance to be detected and specifically binds to the first binding substance.

  “Non-specific adsorptivity equivalent to non-specific adsorptivity of fluorescent label binding substance” of blocking substance means that the sensor part to which the fluorescent label binding substance can be adsorbed by the interaction between the blocking substance and the sensor part. It shall mean the property that the blocking substance can be adsorbed to the same or nearby location. That is, the non-specific adsorptivity of the blocking substance is equivalent to the non-specific adsorptivity of the fluorescently labeled binding substance means that the “location” on the sensor part where each substance adsorbs is the same or located in the vicinity. It means to do.

  The number of fluorescent dye molecules included in the light-transmitting material of the fluorescent substance may be one, but a plurality is more preferable. In the case where the fluorescent substance includes a plurality of fluorescent dye molecules, it is sufficient that at least one fluorescent dye molecule is encased in a light-transmitting material, and some of the other fluorescent dye molecules are made of the light-transmitting material. It may be exposed to the outside.

  Furthermore, in the detection method according to the present invention, it is preferable that the blocking substance has second particles having at least a surface made of a material equivalent to the light-transmitting material. Here, the “equivalent material” means the same material as the translucent material, or the molecular structure, side in the range in which the blocking substance has nonspecific adsorptivity equivalent to that of the fluorescently labeled binding substance. It shall mean a similar material from the viewpoint of chain, functional group and charged state. In this case, it is preferable that the blocking substance has a modifying substance which is surface-modified on the surface of the second particle and does not have the specific binding property. The second particles preferably have a particle size smaller than the particle size of the first particles. In this case, it is preferable that the blocking substance and the fluorescent label binding substance flow down on the sensor unit simultaneously.

  The second particles preferably have a plurality of second fluorescent dye molecules that emit fluorescence having a wavelength different from the fluorescence emitted by the first fluorescent dye molecules in the second particles, or The second particles preferably do not contain a fluorescent substance in the second particles.

Furthermore, as a method of “exciting a fluorescent label and detecting the amount of a substance to be detected based on the amount of light caused by this excitation”, the method may be to detect fluorescence from the fluorescent label. In addition, the emitted light may be detected by the fluorescence generated by newly inducing surface plasmons in the metal layer. Specifically, the following methods (1) to (4) may be mentioned.
(1) A method of exciting a plasmon in a metal layer by irradiation of excitation light, generating a photoelectric field enhanced by the plasmon, and detecting fluorescence generated from the fluorescent label by this excitation as light generated due to excitation of the fluorescent label .
(2) The plasmon is excited on the metal layer by the irradiation of the excitation light, and the photoelectric field enhanced by the plasmon is generated. A method of detecting radiated light from a newly induced plasmon that is radiated to the other surface opposite to the one surface of the dielectric plate by newly inducing plasmons.
(3) A sensor chip having a laminated structure including an optical waveguide layer is used to excite an optical waveguide mode in the optical waveguide layer by irradiation with excitation light, to generate an enhanced photoelectric field by the optical waveguide mode, A method of detecting fluorescence generated from a fluorescent label by this excitation as light generated by excitation.
(4) A sensor chip having a laminated structure including an optical waveguide layer is used to excite an optical waveguide mode in the optical waveguide layer by irradiation of excitation light, thereby generating an enhanced photoelectric field by the optical waveguide mode. As light generated by excitation, the fluorescence generated from the fluorescent label by this excitation induces a new plasmon in the metal layer, and is radiated to the other surface side facing the one surface with respect to the dielectric plate. A method for detecting radiation from induced plasmons.

  In (1) and (2), the excitation of plasmons uses a metal layer as a metal film, and the excitation light is incident on the interface between the metal film and the substrate at an angle greater than the total reflection angle from the back surface of the substrate. It is also possible to excite surface plasmons. On the other hand, the metal layer is composed of a metal microstructure having irregularities with a period smaller than the wavelength of the excitation light on the surface, or a plurality of metal nanorods having a size smaller than the wavelength of the excitation light. It is also possible to excite localized plasmons in the metal microstructure or the metal nanorod by irradiation. In addition, plasmon excitation may be performed using a minute opening of a metal film.

Furthermore, the detection sample cell according to the present invention is a detection sample cell used as a sensor chip in the detection method described above,
A base having a channel through which a liquid sample flows;
An inlet for injecting a liquid sample into a flow path provided upstream of the flow path;
An air hole provided on the downstream side of the flow channel for flowing the liquid sample injected from the injection port downstream;
A sensor chip provided in a flow path between the inlet and the air hole, and a dielectric plate provided as a part of the inner wall surface of the flow path, and a predetermined region on the sample contact surface of the dielectric plate A sensor chip portion including a provided sensor portion,
The sensor unit has a laminated structure including a metal layer adjacent to the dielectric plate, and the surface of the sensor unit is subjected to a blocking process with a blocking substance.

  The detection sample cell according to the present invention preferably includes a first binding substance that specifically binds to the substance to be detected and that is fixed on the sensor unit.

  Moreover, it is preferable to provide a fluorescent label binding substance disposed in the flow channel upstream of the sensor unit.

  The laminated structure preferably includes an optical waveguide layer.

  In addition, the detection sample cell according to the present invention is suitable for performing an assay by the sandwich method when the fluorescent label binding substance includes a second binding substance and a fluorescent label for labeling the second binding substance. On the other hand, when it comprises a third binding substance and a fluorescent label for labeling it, it is suitable for performing an assay by a competitive method.

Furthermore, the detection kit according to the present invention is a detection kit used in the detection method described above,
A base having a channel through which the liquid sample flows down, an inlet for injecting the liquid sample into the channel provided upstream of the channel, and an inlet provided downstream of the channel An air hole for flowing the liquid sample injected from the downstream side, and a sensor chip part provided in the flow channel between the injection port and the air hole, provided as a part of the inner wall surface of the flow channel A dielectric chip, a sensor chip portion including a sensor portion provided in a predetermined region of the sample contact surface of the dielectric plate, and a first binding substance that specifically binds to the substance to be detected, The sensor unit is a laminated structure including a metal layer adjacent to the dielectric plate, and the surface of the sensor unit is subjected to a blocking treatment with a blocking substance. Used as a sensor chip Use the sample cell, and flows down after falling of the liquid sample simultaneously or liquid sample in the flow path and is characterized in that it comprises an indicator solution containing a fluorescent label binding substance.

  The detection kit according to the present invention is suitable for performing an assay by the sandwich method when the fluorescent label binding substance contained in the labeling solution comprises a second binding substance and a fluorescent label. In the case where a third binding substance and a fluorescent label are provided, it is suitable for performing an assay by a competitive method.

Alternatively, the detection kit according to the present invention is a detection kit used in the detection method described above,
A base having a channel through which the liquid sample flows down, an inlet for injecting the liquid sample into the channel provided upstream of the channel, and an inlet provided downstream of the channel An air hole for flowing the liquid sample injected from the downstream side, and a sensor chip part provided in the flow channel between the injection port and the air hole, provided as a part of the inner wall surface of the flow channel A dielectric chip, a sensor chip portion including a sensor portion provided in a predetermined region of the sample contact surface of the dielectric plate, and a first binding substance that specifically binds to the substance to be detected, A detection sample cell used as a sensor chip, wherein the sensor unit is composed of a laminated structure including a metal layer adjacent to the dielectric plate,
A labeling solution containing a fluorescently labeled binding substance that flows into the channel simultaneously with the liquid sample or after the liquid sample flows, and a flow into the channel simultaneously with the labeling solution or before the labeling solution flows. A blocking solution containing a blocking substance is provided.

Alternatively, the detection kit according to the present invention is a detection kit used in the detection method described above,
A base having a channel through which the liquid sample flows down, an inlet for injecting the liquid sample into the channel provided upstream of the channel, and an inlet provided downstream of the channel An air hole for flowing the liquid sample injected from the downstream side, and a sensor chip part provided in the flow channel between the injection port and the air hole, provided as a part of the inner wall surface of the flow channel A dielectric chip, a sensor chip portion including a sensor portion provided in a predetermined region of the sample contact surface of the dielectric plate, and a first binding substance that specifically binds to the substance to be detected, A detection sample cell used as a sensor chip, and a liquid sample, wherein the sensor unit has a laminated structure including a metal layer adjacent to the dielectric plate. At the same time or after flowing down the liquid sample It is characterized by comprising a labeling and blocking combined solution containing a fluorescent label binding substance and a blocking substance.

  In the detection kit according to the present invention, the laminated structure preferably includes an optical waveguide layer on the metal layer.

  In the detection method according to the present invention, the plurality of first fluorescent dye molecules are made of a plurality of first fluorescent dye molecules and a light-transmitting material that transmits fluorescence generated from the plurality of first fluorescent dye molecules as the fluorescent label. The fluorescent substance comprised from the 1st particle | grains containing is used. Therefore, since it is possible to earn more signal strength, it is possible to suppress variation in signal strength and efficiently use the enhanced electric field. Furthermore, in the detection method according to the present invention, the blocking agent does not contain the first fluorescent dye molecule and does not have the specific binding property of the binding substance, and is a non-specific fluorescent label binding substance. A blocking substance having nonspecific adsorptivity equivalent to the adsorptivity is used. Therefore, nonspecific adsorption of the fluorescent label binding substance can be prevented by blocking the place where the fluorescent label binding substance can be adsorbed nonspecifically with this blocking substance. As a result, it is possible to suppress noise caused by nonspecific adsorption of the fluorescent label binding substance. That is, a signal with a good S / N ratio can be detected. As described above, it is possible to accurately detect the presence and / or amount of the substance to be detected.

  In addition, if the detection sample cell or detection kit of the present invention is used, the detection method of the present invention can be easily implemented, effectively using the enhanced electric field, suppressing variations in signal intensity, and The presence / absence and / or amount of the substance to be detected can be accurately detected with a high S / N ratio.

  Hereinafter, although an embodiment of the present invention is described using a drawing, the present invention is not limited to this.

"Detection method and detection device"
<First Embodiment>
For example, as shown in FIG. 1, the detection method according to the present invention is a sensor unit 14 including a metal layer 12 on one surface of a dielectric plate 11, and is subjected to a blocking process with a blocking substance (not shown). Using the sensor chip 10 having 14, the sample is brought into contact with the sensor unit 14, whereby an amount of the fluorescent label binding substance BF corresponding to the amount of the substance A to be detected contained in the sample is supplied to the sensor unit 14. The sensor portion 14 is irradiated with the excitation light Lo, the photoelectric field on the sensor portion 14 is enhanced by the irradiation of the excitation light Lo, and the fluorescence of the fluorescent label binding substance BF in the enhanced photoelectric field D This is a detection method for detecting the amount of the substance to be detected A based on the amount of light generated due to the excitation of the fluorescent substance F as a label.

  In this detection method, a sensor chip 10 including a dielectric plate 11 and a sensor unit 14 provided with a metal layer 12 in a predetermined region on one surface thereof is used.

  The sensor chip 10 is obtained by forming a metal film as a metal layer 12 in a predetermined region on one surface of a dielectric plate 11 such as a glass plate. The dielectric plate 11 is made of a transparent material such as transparent resin or glass. The dielectric plate 11 is preferably made of a resin. In this case, it is more preferable to use a resin such as polymethyl methacrylate (PMMA), polycarbonate (PC), or amorphous polyolefin (APO) containing cycloolefin. . The metal film 12 can be formed by a known vapor deposition method by forming a mask having an opening in a predetermined region on one surface of the plate 11. The thickness of the metal film 12 can be appropriately determined so that the surface plasmon is strongly excited by the material of the metal film 12 and the wavelength of the excitation light. For example, when laser light having a central wavelength at 780 nm is used as excitation light and a gold (Au) film is used as the metal film, the thickness of the metal film is preferably 50 nm ± 20 nm. More preferably, it is 47 nm ± 10 nm. The metal film is preferably composed mainly of at least one metal selected from the group consisting of Au, Ag, Cu, Al, Pt, Ni, Ti, and alloys thereof. The “main component” is defined as a component having a content of 90% by mass or more.

  The fluorescently labeled binding substance BF is a fluorescently labeled binding substance that binds on the sensor unit 14 by an amount corresponding to the amount of the substance A to be detected. As shown in FIG. 1, the fluorescent label binding substance BF is composed of a binding substance that specifically binds to the substance to be detected and a fluorescent label when assaying by the sandwich method. In addition, the fluorescent label binding substance BF is composed of a binding substance that competes with the substance to be detected and a fluorescent label when assaying by the competitive method described later. More specifically, when the sensor chip 10 is used in which the first binding substance B1 that specifically binds to the substance A to be detected is fixed to the sensor unit 14, the fluorescent label binding substance BF in the sandwich method is The second binding substance B2 that specifically binds to the substance A to be detected and the fluorescent label F that labels the second binding substance B2. On the other hand, in the same case, the fluorescently labeled binding substance BF in the competitive method labels the third binding substance that specifically binds to the first binding substance B1 by competing with the detection target substance A and the third binding substance. It consists of a fluorescent label. When the substance A to be detected is an antigen, a so-called primary antibody may be used as the first binding substance B1, and a so-called labeled secondary antibody in which a secondary antibody is labeled as the fluorescent label binding substance.

  Further, as shown in an enlarged view of a part of FIG. 1, the fluorescent label transmits the plurality of first fluorescent dye molecules f to the transparent material 16 that transmits the fluorescence generated from the plurality of first fluorescent dye molecules f. It is the fluorescent substance F included by these. The fluorescent substance F can be produced as follows.

First, polystyrene particles (Estapoor, φ500 nm, 10% Solid, carboxyl group, product number K1-050) are prepared to prepare 0.1% Solid inphosphate (polystyrene solution: pH 7.0).
Next, a first fluorescent dye molecule (Hayashibara Biochemical Laboratory, NK-2014, excitation wavelength: 780 nm) 0.3 mg of ethyl acetate solution (1 mL) is prepared.
The polystyrene solution and the fluorescent dye solution are mixed and impregnated while evaporating, and then centrifuged (15000 rpm, 4 ° C., 20 minutes twice) to remove the supernatant.

  Through the above-described steps, a fluorescent substance F in which the first fluorescent dye molecule f is encapsulated with polystyrene having a function of transmitting fluorescence generated from the first fluorescent dye molecule can be obtained. The particle size of the fluorescent substance F produced by impregnating polystyrene particles with the first fluorescent dye molecules by such a procedure is the same as that of the polystyrene particles (φ500 nm in the above example).

  Since the fluorescent substance F contains a plurality of first fluorescent dye molecules f, the amount of emitted fluorescence can be greatly increased compared to the case where a conventional fluorescent dye molecule f alone is used as a fluorescent label. it can. The fluorescent material F preferably has a particle size of 5300 nm or less from the viewpoint of diffusion time. Further, from the viewpoint of the amount of fluorescence and the closest packing density on the sensor part and from the viewpoint of disturbance of the surface plasmon, those of 100 nm to 700 nm are more preferred, and those of 130 nm to 500 nm are particularly preferred. Specific examples of the light-transmitting material 16 include polystyrene, SiO2, and the like, but can include the first fluorescent dye molecule f and can transmit the fluorescent Lf from the fluorescent dye molecule f to be emitted to the outside. If it is, it will not be restrict | limited.

  The blocking substance R is a blocking agent for preventing the fluorescent label binding substance from being adsorbed at an arbitrary location on the sensor part due to the interaction generated between the fluorescent label binding substance BF and the sensor part. The blocking substance does not contain the first fluorescent dye molecule, does not have the specific binding property of the binding substance constituting the fluorescent label binding substance, and is non-specific equivalent to the non-specific adsorption property of the fluorescent label binding substance. It has a adsorptive property. Here, the specific binding property of the binding substance constituting the fluorescent label binding substance refers to the binding substance (fluorescent label binding) via the first binding substance fixed on the sensor unit and specifically binding to the detection target substance. It is assumed that the substance is capable of specifically binding on the sensor unit. That is, the specific binding property of the binding substance means the specific binding property of the second binding substance that specifically binds to the substance to be detected, for example, when assaying by the sandwich method, while the assay by the competitive method is performed. In this case, it means the specific binding property of the third binding substance that competes with the substance to be detected and specifically binds to the first binding substance. In addition, the “nonspecific adsorption property equivalent to the nonspecific adsorption property of the fluorescent label binding substance” of the blocking substance means that the fluorescent label binding substance can be adsorbed by the interaction generated between the blocking substance and the sensor unit. It shall mean the property that the blocking substance can be adsorbed at the same location as or near the location on the sensor unit. That is, the non-specific adsorptivity of the blocking substance is equivalent to the non-specific adsorptivity of the fluorescently labeled binding substance means that the “location” on the sensor part where each substance adsorbs is the same or located in the vicinity. It means to do.

  Noise caused by non-specific adsorption of a biological substance with a label to the sensor part is a big problem that affects the detection limit in a biometric method. To deal with this problem, non-specification as described above is conventionally performed by using a biological substance (nucleic acid, biotin, BSA, casein, etc.) or a substance similar to the biological substance (hydrophilic polymer, etc.) as a blocking agent. Is prevented. This method has a certain effect when the label is approximately the same size as the biological substance (detected substance, binding substance, etc.). However, in the detection method according to the present invention, a fluorescent substance F having a size 10 times or more that of a biological substance is used as a fluorescent label. FIG. 2A is a schematic diagram showing the magnitude relationship between the fluorescent substance F and the biological substance. As shown in FIG. 2A, the biological material is at most about 10 nm, whereas the first particle 16 is, for example, 100 to 500 nm. As described above, when the fluorescent substance F is much larger than the biological substance, the non-specific adsorptivity of the fluorescent substance F is higher than the first particle 16 and the sensor part than the interaction between the binding substance B2 and the sensor part 14. Interaction with 14 becomes dominant. In this case, as shown in FIG. 3A, it is difficult to prevent nonspecific adsorption of the fluorescent label binding substance BF only by performing a blocking treatment using a blocking agent r such as a biological substance (BF in the figure). 'Indicates a non-specifically adsorbed fluorescently labeled binding substance.)

  Therefore, in the present embodiment, the blocking substance R according to the present embodiment is composed of the second particles 16 'and the modifying substance B2' for modifying the second particles 16 'as shown in FIG. 2B.

  The second particle 16 ′ is equivalent to the translucent material constituting the first particle 16 of the fluorescent substance F in order to achieve nonspecific adsorption equivalent to the nonspecific adsorption of the fluorescent label binding substance BF. It is preferable to be constituted by a material. Here, the “equivalent material” means the same material as the translucent material, or the molecular structure, side in the range in which the blocking substance has nonspecific adsorptivity equivalent to that of the fluorescently labeled binding substance. It shall mean a similar material from the viewpoint of chain, functional group and charged state. Based on the above, examples of the material of the second particle 16 ′ include, but are not limited to, polystyrene, polylactic acid, acrylic, polyethyleneimine, and SiO 2.

  The particle diameter of the second particle 16 ′ is preferably smaller than that of the first particle 16 in order to increase the movement / adsorption rate as compared with the fluorescent label binding substance BF. By doing so, even if the fluorescent label binding substance BF and the blocking substance R are simultaneously supplied onto the sensor part 14, nonspecific adsorption of the fluorescent label binding substance BF to the sensor part can be prevented.

  FIG. 2B shows a case where the second particle 16 ′ does not contain anything as another substance. The second particle 16 ′ is a fluorescent light emitted from the first fluorescent dye molecule f. It is good also as what has a some 2nd fluorescent dye molecule | numerator which emits the fluorescence of a different wavelength. In this way, the fluorescent signal from the blocking substance R and the fluorescent signal from the fluorescent substance F can be separated and detected. Accordingly, it is possible to perform measurement such as simultaneously detecting the fluorescence signal from the blocking substance R as a reference signal for data correction.

  For example, the modifying substance B2 ′ realizes non-specific adsorptivity equivalent to the non-specific adsorptivity of the fluorescently labeled binding substance BF. For example, the modifying substance B2 ′ has a different recognition antigen from the binding substance constituting the fluorescently labeled binding substance BF. Is a functional group imparting However, the modifying substance B2 'is not always essential.

  As shown in FIG. 3B, the blocking treatment is performed at a place where the fluorescent label binding substance BF can be adsorbed nonspecifically in order to prevent nonspecific adsorption of the fluorescent label binding substance BF to the sensor unit. This is a process of supplying and adsorbing (blocking agent r such as biological substance or blocking substance R). Usually, it is preferable to supply the blocking substance R before supplying the fluorescent label binding substance BF. However, when the particle size of the second particle 16 ′ is smaller than the particle size of the first particle, the blocking material R has a higher migration / adsorption rate than the fluorescent label binding material BF. And the fluorescent label binding substance BF may be supplied simultaneously. In addition, you may use the sensor chip 10 in which the blocking process was performed with the blocking substance R previously.

  The detection device of the present invention is for carrying out the above-described detection method, and includes a housing part 19 that houses the sensor chip 10, and an excitation light irradiation optical system 20 that irradiates the sensor part 14 with excitation light Lo. And a light detection means 30 for detecting an amount of light corresponding to the substance A to be detected that is generated by the irradiation of the excitation light Lo.

  In the present invention, an enhanced photoelectric field D is generated on the sensor unit 14 by irradiation with excitation light Lo, and light generated by excitation of a fluorescent label in the enhanced photoelectric field is detected. However, the method of enhancing the photoelectric field may be by surface plasmon resonance or localized plasmon resonance, or by excitation of an optical waveguide mode. Moreover, the fluorescence generated from the fluorescent label may be detected, or the emitted light generated by the fluorescence inducing a new surface plasmon in the metal layer may be detected. Specific embodiments will be described in the following embodiments. Note that the detection method and apparatus of the present embodiment are a fluorescence detection method and apparatus that enhances a photoelectric field by surface plasmon resonance and detects fluorescence excited in the enhanced photoelectric field.

  In the present embodiment, the sample holding unit 13 that holds the liquid sample S is provided on the sensor chip 10, and the sensor chip 10 and the sample holding unit 13 constitute a box-shaped cell that can hold the liquid sample S. Yes. In the case of measuring a small amount of liquid sample that remains on the sensor chip 10 with surface tension, the sample holder 13 may not be provided.

  The excitation light irradiation optical system 20 includes a light source 21 made of a semiconductor laser (LD) or the like that outputs excitation light Lo, and a prism 22 that is arranged so that one surface is in contact with the dielectric plate 11. The prism 22 guides the excitation light Lo into the dielectric plate 11 so that the excitation light Lo is totally reflected at the interface between the dielectric plate 11 and the metal film 12. The prism 22 and the dielectric plate 11 are in contact with each other through a refractive index matching oil. The light source 21 is arranged so that the excitation light Lo is incident on the sample contact surface of the sensor chip 10 from the other surface of the prism 22 at a specific angle that is greater than the total reflection angle and that causes surface plasmon resonance with the metal film. Furthermore, you may arrange | position a light guide member between the light source 21 and the prism 22 as needed. The excitation light Lo is incident on the interface with p-polarized light so as to induce surface plasmons.

  As the photodetector 30, a CCD, PD (photodiode), photomultiplier, c-MOS, or the like can be used as appropriate.

  The accommodating part 19 is configured such that when the sensor chip 10 is accommodated, the sensor part 14 of the sensor chip is disposed on the prism 22 so that fluorescence can be detected by the photodetector 30. The sensor chip 10 can be taken in and out in the direction of the arrow X in the figure with respect to the housing part 19.

Hereinafter, the operation of the detection method and the detection apparatus in the present embodiment will be described.
Here, as an example, a case where the antigen A is detected as a detection target substance contained in the sample S will be described.

  As the sensor chip 10, the primary antibody B 1 is modified as a first binding substance that specifically binds to the antigen A on the metal film 12 that is the sensor unit 14, and then the metal film 12 is coated with the blocking substance R. Prepare a material that has been subjected to blocking treatment.

  First, the sample S to be inspected is caused to flow through the sample holder 13 and the sample S is brought into contact with the metal film 12 of the sensor chip 10. Subsequently, similarly, a solution containing a secondary antibody B2 that is a second binding substance that specifically binds to the antigen A and a fluorescent label binding substance BF composed of the fluorescent substance F is flowed. In this case, the primary antibody B1 whose surface is modified on the metal film 12 and the secondary antibody B2 of the fluorescently labeled binding substance BF are those that bind to the antigen A and bind to different binding sites (epitopes). If antigen A is present in sample S, antigen A specifically binds to primary antibody B1, and further, secondary antibody B2 of fluorescently labeled binding substance BF binds to antigen A to form a sandwich. Thereafter, in order to separate the bound product from which the sandwich has not been formed and the unreacted fluorescently labeled binding substance BF, the buffer solution is poured to eliminate the unreacted fluorescently labeled binding substance.

  The above steps may be performed before the sensor chip 10 is set in the housing part 19 of the detection device 1 or after the setting. In addition, the timing of labeling the detection target substance (antigen A) is not particularly limited, and before the detection target substance (antigen A) is bound to the first binding substance (primary antibody B1), the fluorescent substance is previously applied to the sample. May be added.

  In the detection method according to the present embodiment, a plurality of first fluorescent dye molecules f and a light-transmitting material that transmits fluorescence Lf generated from the plurality of first fluorescent dye molecules f are used as fluorescent labels, and the plurality of first fluorescent dye molecules f is used. The fluorescent substance F composed of the first particles 16 including the fluorescent dye molecule f is used. That is, by using this fluorescent substance F, the substance A to be detected can be labeled as a group of a plurality of first fluorescent dye molecules f. Therefore, since it is possible to earn more signal strength, it is possible to suppress variation in signal strength and efficiently use the enhanced electric field. Furthermore, in the detection method according to the present invention, the blocking agent does not contain the first fluorescent dye molecule and does not have the specific binding property of the binding substance, and is a non-specific fluorescent label binding substance. A blocking substance having nonspecific adsorptivity equivalent to the adsorptivity is used. Therefore, nonspecific adsorption of the fluorescent label binding substance can be prevented by blocking the place where the fluorescent label binding substance can be adsorbed nonspecifically with this blocking substance. As a result, it is possible to suppress noise caused by nonspecific adsorption of the fluorescent label binding substance. That is, a signal with a good S / N ratio can be detected. As described above, it is possible to accurately detect the presence and / or amount of the substance to be detected.

Furthermore, when the metal layer 12 is provided on the surface of the sensor unit 14 as in the present embodiment, the influence of the metal quenching of the first fluorescent dye molecules by the metal layer 12 becomes strong. It is necessary to precisely control the distance between 12 and the first fluorescent dye molecule. The degree of influence of metal quenching is inversely proportional to the cube of the distance if the metal is a plane with a semi-infinite thickness, inversely proportional to the fourth power of the distance if the metal is an infinitely thin plate, and if the metal is a fine particle It becomes smaller in inverse proportion to the sixth power of the distance. Therefore, the distance between the metal layer 12 and the first fluorescent dye molecule f is preferably secured at least several nm or more, more preferably 10 nm or more. Such control is generally performed by providing a barrier layer such as a polymer film, a SiO ₂ film, a SAM film, or a CMD film on the metal layer, but it is cumbersome and cumbersome and is not practical enough. Not suitable. However, in the present invention, when the fluorescent substance contains a sufficient amount of the first fluorescent dye molecule, the metal layer 12 is necessarily formed even if a metal quenching prevention film is not provided on the metal layer 12. The distance from many first fluorescent dye molecules can be separated to some extent. This eliminates the trouble of forming a CMD film and a SAM film, which has been conventionally required for preventing metal quenching, effectively preventing metal quenching by a very simple method, and stabilizing fluorescence signals. Can be detected.

<Design change example of the first embodiment>
In each of the embodiments described above, for example, parallel light incident on the interface at a predetermined angle θ is incident as the excitation light Lo. However, as the excitation light, a fan beam having an angular width Δθ centered on the angle θ ( Focused light) may be used. In the case of a fan beam, it is incident on the interface between the prism and the metal film on the prism at an incident angle in the range of angle θ−Δθ / 2 to θ + Δθ / 2, and there is a resonance angle within this angle range. For example, surface plasmons can be excited in the metal film. Before and after supplying the sample onto the metal film, the refractive index of the solvent on the metal film changes, thereby changing the resonance angle at which surface plasmons are generated. When parallel light is used as excitation light as in the above-described embodiment, it is necessary to adjust the incident angle of parallel light every time the resonance angle changes. However, by using a fan beam having a width that is incident on the interface, it is possible to cope with a change in the resonance angle without adjusting the incident angle. In addition, it is more preferable that the fan beam has a flat distribution with little intensity change due to the incident angle.

<Second Embodiment>
A detection method and apparatus according to the second embodiment will be described with reference to FIG. FIG. 4 is an overall view showing a schematic configuration of the detection apparatus according to the second embodiment. The detection method and the detection apparatus according to the present embodiment are a detection method and an apparatus that enhances a photoelectric field by localized plasmon resonance and detects fluorescence excited in the enhanced photoelectric field. In the following, the same components as those in the first embodiment are denoted by the same reference numerals.

  The fluorescence detection device 2 shown in FIG. 4 is different from the fluorescence detection device 1 of the first embodiment described above in the sensor chip 10 ′ and the excitation light irradiation optical system 20 ′ used.

  The sensor chip 10 ′ is a metal layer 12 ′ provided on the dielectric plate 11 and subjected to blocking treatment with a blocking substance (not shown). It has a structure or a plurality of metal nanorods. Here, the metal microstructure has a concavo-convex structure smaller than the wavelength of the excitation light Lo on the surface. On the other hand, the metal nanorod has a size smaller than the wavelength of the excitation light. When the metal layer 12 ′ that generates such localized plasmons is provided, it is not necessary to make the excitation light incident on the interface between the metal layer 12 ′ and the dielectric plate 11 so as to be totally reflected. The excitation light irradiation optical system 20 ′ is configured to irradiate the excitation light Lo from above the dielectric plate.

  The excitation light irradiation optical system 20 ′ includes a light source 21 made of a semiconductor laser (LD) or the like that outputs excitation light Lo, and a half mirror 23 that reflects the excitation light Lo and guides it to the sensor chip 10 ′. . The half mirror 23 reflects the excitation light Lo and transmits the fluorescence Lf.

  A specific example of the sensor chip 10 'will be described with reference to FIGS. In addition, in the sensor chip demonstrated below, although description about the blocking process by a blocking substance is abbreviate | omitted for convenience, blocking process is performed also in these sensor chips.

  A sensor chip 10A shown in FIG. 5A includes a dielectric plate 11 and a metal microstructure 73 composed of a plurality of metal particles 73a fixed in an array on a predetermined region of the plate 11. The arrangement pattern of the metal particles 73a can be designed as appropriate, but is preferably substantially regular. In such a configuration, the average diameter and pitch of the metal particles 73a are designed to be smaller than the wavelength of the excitation light Lo.

  A sensor chip 10B shown in FIG. 5B is composed of a dielectric plate 11 and a metal microstructure 74 that is provided in a predetermined region on the plate and includes a metal pattern layer in which fine metal wires 74a are patterned in a lattice pattern. Has been. The pattern of the metal pattern layer can be designed as appropriate and is preferably substantially regular. In such a configuration, the average line width and pitch of the fine metal wires 74a are designed to be smaller than the wavelength of the excitation light Lo.

  A sensor chip 10C shown in FIG. 5C is grown in a plurality of fine holes 77a of a metal oxide body 77 formed in the process of anodizing a metal 76 such as Al as described in JP-A-2007-171003. The metal fine structure 75 is composed of a plurality of mushroom-like metals 75a. Here, the metal oxide body 77 corresponds to a dielectric plate. In this metal microstructure 75, a part of a metal body (Al, etc.) is anodized to form a metal oxide body (Al2O3, etc.), and inside the plurality of fine holes 77a of the metal oxide body 77 formed in the process of anodization. The metal 75a can be grown by plating or the like.

  In the example shown in FIG. 5C, the head of the mushroom-like metal 75a is in the form of particles, and when viewed from the surface of the sample plate, the metal fine particles are arranged. In such a configuration, the head of the mushroom-shaped metal 75a is a convex portion, and the average diameter and pitch thereof are designed to be smaller than the wavelength of the measurement light L.

  The metal layer 12 ′ that generates localized plasmons upon irradiation with excitation light is obtained by anodizing a metal body described in JP-A-2006-332067, JP-A-2006-250924, and the like. Various forms of metal microstructures using the resulting microstructure can be used.

  Furthermore, the metal layer that generates localized plasmons may be formed of a metal film having a roughened surface. Examples of the roughening method include an electrochemical method using oxidation reduction and the like. Moreover, you may comprise a metal layer by the some metal nanorod arrange | positioned on the sample plate. The metal nanorods have a minor axis length of about 3 nm to 50 nm and a major axis length of about 25 nm to 1000 nm, and the major axis length is smaller than the wavelength of the excitation light. The metal nanorod is described in, for example, Japanese Patent Application Laid-Open No. 2007-139612.

  The metal microstructure or metal nanorod used as the metal layer 12 ′ is at least one metal selected from the group consisting of Au, Ag, Cu, Al, Pt, Ni, Ti, and alloys thereof. The main component is preferred.

  Also in the detection method according to the present embodiment, a plurality of first fluorescent dye molecules f and a plurality of first fluorescent dye molecules f and a light-transmitting material that transmits fluorescence Lf generated from the plurality of first fluorescent dye molecules f are used as fluorescent labels. The fluorescent substance F composed of the first particles 16 including the fluorescent dye molecule f is used. Further, in the detection method according to the present invention, the blocking agent does not contain the first fluorescent dye molecule and does not have the specific binding property of the binding substance, and the non-specific fluorescent label binding substance A blocking substance having nonspecific adsorptivity equivalent to the adsorptivity is used. Therefore, the same effect as the first embodiment can be obtained.

  Furthermore, the detection method according to the present embodiment includes a metal microstructure or a plurality of metal nanorods that generate so-called localized plasmons when irradiated with excitation light. In such a case, the excitation light does not need to be incident on the interface between the metal layer 12 ′ and the dielectric plate 11 so as to be totally reflected. Therefore, a low-cost detection method can be performed with a simple optical system.

<Third Embodiment>
A detection method and apparatus according to the third embodiment will be described with reference to FIG. FIG. 6 is an overall view showing a schematic configuration of the detection apparatus of the third embodiment. The detection method and apparatus according to the present embodiment enhances a photoelectric field by surface plasmon resonance, and fluorescence excited in the enhanced photoelectric field induces plasmons in the metal layer, thereby causing a metal with respect to the dielectric plate. A detection method and apparatus for detecting light emitted from a newly induced plasmon radiated to a surface opposite to a layer forming surface.

  The emitted light detection device 3 shown in FIG. 6 is different from the fluorescence detection device of the first embodiment in the arrangement of the photodetectors. In the present radiation light detection device 3, the photodetector 30 radiates the surface of the dielectric plate to the surface opposite to the metal layer formation surface by newly inducing surface plasmons in the metal layer. It arrange | positions so that the emitted light Lp from the newly induced plasmon may be detected.

A radiation light detection method of the present embodiment using the detection device 3 will be described.
In the present embodiment, the surface of the sensor unit 14 is subjected to a blocking process with a blocking substance (not shown). Thereby, nonspecific adsorption | suction to the sensor part 14 of the fluorescence label binding substance BF is prevented. As described above, the excitation light Lo is irradiated by the excitation light irradiation optical system 20 in a state where the influence of non-specific adsorption of the fluorescent label binding substance BF is reduced as in the first embodiment. The excitation light Lo is incident on the interface between the dielectric plate 11 and the metal film 12 by the excitation light irradiation optical system 20 at a specific incident angle that is equal to or greater than the total reflection angle, thereby entering the sample S on the metal film 12. Evanescent light oozes out, and surface plasmons are excited in the metal film 12 by the evanescent light. This surface plasmon causes an electric field distribution on the surface of the metal film 12, and an electric field enhancement region D is formed. Then, the fluorescent dye molecule f in the fluorescent substance F is excited to generate fluorescence Lf. At this time, the fluorescence is enhanced by the effect of the electric field enhancement by the surface plasmon. The fluorescence Lf generated on the metal film 12 newly induces surface plasmon on the metal film 12, and the surface plasmon causes the emitted light Lp to be emitted at a specific angle to the side opposite to the metal film forming surface of the sensor chip 10. The By detecting the emitted light Lp by the photodetector 30, it is possible to detect the presence and / or amount of the detection target substance bound to the fluorescent label binding substance.

  The emitted light Lp is generated when the fluorescence is combined with surface plasmons having a specific wave number of the metal film. The wave number to be combined is determined according to the wavelength of the fluorescence, and the emission angle of the emitted light is determined according to the wave number. . Since the wavelength of the excitation light Lo is usually different from the wavelength of the fluorescence, the surface plasmon excited by the fluorescence has a wave number different from that of the surface plasmon generated by the excitation light Lo, and is different from the incident angle of the excitation light Lo. The emitted light Lp is emitted at an angle.

  Also in the detection method according to the present embodiment, a plurality of first fluorescent dye molecules f and a plurality of first fluorescent dye molecules f and a light-transmitting material that transmits fluorescence Lf generated from the plurality of first fluorescent dye molecules f are used as fluorescent labels. The fluorescent substance F composed of the first particles 16 including the fluorescent dye molecule f is used. Further, in the detection method according to the present invention, the blocking agent does not contain the first fluorescent dye molecule and does not have the specific binding property of the binding substance, and the non-specific fluorescent label binding substance A blocking substance having nonspecific adsorptivity equivalent to the adsorptivity is used. Therefore, the same effect as the first embodiment can be obtained.

  Furthermore, in the present embodiment, since light caused by fluorescence generated on the sensor surface is detected from the back side of the sensor, the distance that the fluorescence Lf passes through a solvent having a large light absorption can be reduced to about several tens of nm. Therefore, for example, light absorption in blood can be almost ignored, and measurement can be performed without pretreatment of centrifuging blood to remove colored components such as erythrocytes, or making it serum or plasma through a blood cell filter It becomes.

<Fourth Embodiment>
A detection method and apparatus according to the fourth embodiment will be described with reference to FIG. FIG. 7 is an overall view showing a schematic configuration of a detection apparatus according to the fourth embodiment. The detection method and apparatus of the present embodiment uses a sensor chip including an optical waveguide layer on a metal layer, excites an optical waveguide mode in the optical waveguide layer by irradiation of excitation light, enhances an electric field by the optical waveguide mode, A detection method and apparatus for detecting fluorescence excited in an enhanced electric field.

  The configuration of the detection device 4 shown in FIG. 7 is the same as the configuration of the detection device of the first embodiment, but the sensor chip used is different, and the electric field enhancement mechanism is different depending on the sensor chip.

  The sensor chip 10 ″ includes an optical waveguide layer 12b on the metal layer 12a. Further, a blocking process (not shown) is performed on the optical waveguide layer 12b. The layer of the optical waveguide layer 12b. The thickness is not particularly limited, and can be determined in consideration of the wavelength of the excitation light Lo, the incident angle, the refractive index of the optical waveguide layer 12b, etc. so that the optical waveguide mode is induced. Similarly to the case of using laser light having a central wavelength of 780 nm as the excitation light Lo and using one layer of silicon oxide film as the optical waveguide layer 12b, the wavelength is preferably about 500 to 600 nm. 12b may have a laminated structure including an internal optical waveguide layer made of an optical waveguide material such as one or more dielectrics. The laminated structure includes an internal optical waveguide layer and an internal optical waveguide layer sequentially from the metal layer side. It is preferred parts are alternately laminated structure of a metal layer.

The fluorescence detection method of this embodiment using the fluorescence detection device 4 will be described.
In the present embodiment, the surface of the sensor unit 14 is subjected to a blocking process with a blocking substance (not shown). Thereby, nonspecific adsorption | suction to the sensor part 14 of the fluorescence label binding substance BF is prevented. As described above, the excitation light Lo is irradiated by the excitation light irradiation optical system 20 in a state where the influence of non-specific adsorption of the fluorescent label binding substance BF is reduced as in the first embodiment. The excitation light Lo is incident on the interface between the dielectric plate 11 and the metal layer 12a by the excitation light irradiation optical system 20 at a specific incident angle equal to or greater than the total reflection angle, so that the evanescent light spreads on the metal layer 12a. The evanescent light is coupled with the optical waveguide mode of the optical waveguide layer 12b, and the optical waveguide mode is excited. As a result, an electric field distribution on the optical waveguide layer 12b is generated, and the electric field enhancement region D is formed. Then, the fluorescent dye molecule f in the fluorescent substance F is excited to generate fluorescence Lf. At this time, the fluorescence is enhanced by the effect of the electric field enhancement by the surface plasmon. By detecting this fluorescence Lf by the photodetector 30, it is possible to detect the presence and / or amount of the substance to be detected bound to the fluorescent label binding substance.

  Also in the detection method according to the present embodiment, a plurality of first fluorescent dye molecules f and a plurality of first fluorescent dye molecules f and a light-transmitting material that transmits fluorescence Lf generated from the plurality of first fluorescent dye molecules f are used as fluorescent labels. The fluorescent substance F composed of the first particles 16 including the fluorescent dye molecule f is used. Further, in the detection method according to the present invention, the blocking agent does not contain the first fluorescent dye molecule and does not have the specific binding property of the binding substance, and the non-specific fluorescent label binding substance A blocking substance having nonspecific adsorptivity equivalent to the adsorptivity is used. Therefore, the same effect as the first embodiment can be obtained.

  Furthermore, the electric field distribution generated by the excitation of the optical waveguide mode has a more gradual degree of electric field attenuation with the distance from the surface than the electric field distribution generated by the normal surface plasmon fluorescence method. When a fluorescent substance having a large diameter encapsulating one fluorescent dye molecule is used, a larger fluorescence amount increasing effect can be obtained than when the electric field enhancement by the normal surface plasmon fluorescence method is used.

<Fifth Embodiment>
A detection method and apparatus according to a fifth embodiment will be described with reference to FIG. FIG. 8 is an overall view showing a schematic configuration of the detection apparatus of the fifth embodiment. The detection method and apparatus of the present embodiment uses a sensor chip including an optical waveguide layer on a metal layer, excites an optical waveguide mode in the optical waveguide layer by irradiation of excitation light, enhances an electric field by the optical waveguide mode, Fluorescence excited in the enhanced electric field induces a new plasmon in the metal layer, and the emitted light from the newly induced plasmon emitted from the side opposite to the metal layer forming surface of the dielectric plate It is the detection method and apparatus which detect.

  The detection device 5 of the present embodiment shown in FIG. 8 has the same configuration as the detection device 3 of the third embodiment, and the sensor chip used in the detection method of the present embodiment is the detection method of the fourth embodiment. It is the same as the sensor chip used.

The fluorescence detection method of this embodiment using the detection device 5 will be described.
In the present embodiment, the surface of the sensor unit 14 is subjected to a blocking process with a blocking substance (not shown). Thereby, nonspecific adsorption | suction to the sensor part 14 of the fluorescence label binding substance BF is prevented. As described above, the excitation light Lo is irradiated by the excitation light irradiation optical system 20 in a state where the influence of non-specific adsorption of the fluorescent label binding substance BF is reduced as in the first embodiment. The excitation light Lo is incident on the interface between the dielectric plate 11 and the metal layer 12a by the excitation light irradiation optical system 20 at a specific incident angle equal to or greater than the total reflection angle, so that the evanescent light spreads on the metal layer 12a. The evanescent light is coupled with the optical waveguide mode of the optical waveguide layer 12b, and the optical waveguide mode is excited. As a result, an electric field distribution on the optical waveguide layer 12b is generated, and the electric field enhancement region D is formed. Then, the fluorescent dye molecule f in the fluorescent substance F is excited to generate fluorescence Lf. At this time, the fluorescence Lf is enhanced by the effect of electric field enhancement by surface plasmons. The fluorescence Lf generated on the optical waveguide layer 12b newly induces a surface plasmon on the metal film 12, and the surface plasmon causes the emitted light Lp to be emitted at a specific angle to the side opposite to the metal film formation surface of the sensor chip 10 ″. By detecting the emitted light Lp by the photodetector 30, it is possible to detect the presence and / or amount of the target substance labeled with the fluorescent label F.

  Also in the detection method according to the present embodiment, a plurality of first fluorescent dye molecules f and a plurality of first fluorescent dye molecules f and a light-transmitting material that transmits fluorescence Lf generated from the plurality of first fluorescent dye molecules f are used as fluorescent labels. The fluorescent substance F composed of the first particles 16 including the fluorescent dye molecule f is used. Further, in the detection method according to the present invention, the blocking agent does not contain the first fluorescent dye molecule and does not have the specific binding property of the binding substance, and the non-specific fluorescent label binding substance A blocking substance having nonspecific adsorptivity equivalent to the adsorptivity is used. Therefore, the same effect as the first embodiment can be obtained.

  Further, in the present embodiment, the light caused by the fluorescence generated on the sensor surface is detected from the back side of the sensor, so the distance that the fluorescence Lf passes through the solvent having a large light absorption can be reduced to about several tens of nm. The same effect as that of the second embodiment can be obtained.

  Furthermore, since the electric field enhancement by excitation of the optical waveguide mode is used, the effect of increasing the amount of fluorescence can be obtained as in the fourth embodiment.

(Example of design change in the first to fifth embodiments)
In the first to fifth embodiments, as the fluorescent label, as shown in FIG. 9, the surface of the light transmitting material 16 further provided with a metal coating Mc having a thickness that transmits fluorescence is used. Also good. The metal coating Mc may cover the entire surface of the translucent material 16, or may cover the entire surface, and may be provided so that a part of the surface is exposed. As a material of the metal coating Mc, the same metal material as that of the above-described metal layer can be used. In this case, it is preferable to provide a similar metal film for the blocking substance.

  When the metal coating Mc is provided on the surface of the fluorescent material, the surface plasmon or local plasmon generated in the metal layers 12 and 12 ′ of the sensor chip 10, 10 ′ is the whispering gallery mode of the metal coating Mc of the fluorescent material F. And the fluorescent dye molecule f in the fluorescent substance F can be excited with higher efficiency. The whispering gallery mode is an electromagnetic wave mode that localizes and circulates on the surface of a microsphere such as a fluorescent material having a diameter of about 5300 nm or less used here.

An example of a metal coating method on a fluorescent material is given.
First, a quenching-resistant fluorescent material is prepared by the above-described procedure, and polyethyleneimine (PEI) (Epomin, Nippon Shokubai Co., Ltd.) is modified on the surface.
Next, Pd nanoparticles having an average particle diameter of 15 nm (average particle diameter of 19 nm, Tokuru Head Office) are adsorbed on the PEI on the particle surface.
By immersing polystyrene particles adsorbed with Pd nanoparticles in an electroless gold plating solution (HAuCl4, Kojima Chemical Co., Ltd.), a gold film is produced on the surface of polystyrene particles using electroless plating using Pd nanoparticles as a catalyst. To do.

"Sample cell for detection"
A detection sample cell used as a sensor chip of the detection method of the present invention will be described.
<Detection Sample Cell of First Embodiment>
FIG. 10A is a plan view showing the configuration of the detection sample cell 50 of the first embodiment, and FIG. 10B is a side sectional view of the detection sample cell 50.

  The detection sample cell 50 includes a base 51 made of a dielectric plate, a spacer 53 that holds the liquid sample S on the base 51 and forms a flow path 52 of the liquid sample S, and an inlet for injecting the sample S. 54a and an upper plate 54 made of a glass plate provided with an air hole 54b serving as a discharge port for discharging the sample flowing down the flow path 52, and the sample between the inlet 54a and the air hole 54b of the flow path 52 Sensor portions 58 and 59 composed of metal layers 58a and 59a provided on a predetermined region of the base 51 serving as a contact surface are provided. Further, a membrane filter 55 is provided at a location from the inlet 54 a to the flow path 52, and a waste liquid reservoir 56 is formed at a portion connected to the air hole 54 b downstream of the flow path 52.

  In this embodiment, the base 51 is composed of a dielectric plate and also serves as the dielectric plate of the sensor unit. However, as the base, only the part where the sensor unit is composed is composed of a dielectric plate. May be used.

  The detection sample cell 50 can be similarly used as a sensor chip in the detection apparatus and method of any of the first to fifth embodiments described above.

  The detection sample cell 50 can be subjected to a blocking process with a blocking substance on the sensor portion.

  The detection sample cell 50 can be used by appropriately fixing a first binding substance that specifically binds to the substance to be detected to the sensor unit according to the substance to be detected.

  Further, the detection sample cell 50 can include a fluorescent label binding substance BF disposed in the flow channel upstream of the sensor unit.

<Sample Cell for Detection of Second Embodiment>
FIG. 11 shows a side cross section of a detection sample cell according to a second embodiment suitable for an assay by the sandwich method. In the detection sample cell 50A of the present embodiment, a secondary antibody (second binding substance) B2 that specifically binds to an antigen that is a substance to be detected from the upstream side of the flow path 52 on the base 51 and the 2 Labeled secondary antibody adsorption area 57 in which labeled secondary antibody BF (fluorescent label binding substance) consisting of fluorescent substance F whose surface is modified by secondary antibody B2 is physically adsorbed, specifically binds to the antigen to be detected. Primary antibody B0 that specifically binds to the labeled secondary antibody BF without binding to the antigen A that is the target substance, the first measurement area 58 to which the primary antibody (first binding substance) B1 is fixed Are fixed in order. Further, the first and second measurement areas 58 and 59 are subjected to a blocking process with the blocking substance R. In this example, an example in which two measurement areas are provided in the sensor unit is described, but only one measurement area may be provided.

  Au (gold) films 58a and 59a are formed as metal layers in the first measurement area 58 and the second measurement area 59 on the base 51, respectively. A primary antibody B1 is further immobilized on the Au film 58a in the first measurement area 58, and a primary antibody B0 different from the primary antibody B1 is further immobilized on the Au film 59a in the second measurement area 59. ing. The surfaces of the Au film 58a and the Au film 59a are subjected to a blocking process with a blocking substance R. The first measurement area 58 and the second measurement area 59 have the same configuration except that different primary antibodies are provided. The primary antibody B0 fixed in the second measurement area 59 does not bind to the antigen A but directly binds to the labeled secondary antibody BF. As a result, fluctuation factors relating to the reaction such as the amount and activity of the labeled secondary antibody flowing through the flow path and fluctuation factors relating to the electric field enhancement such as the excitation light irradiation optical system 20, the gold (Au) films 58a and 59a, and the liquid sample S are detected. And can be used for calibration. Note that a known amount of labeling substance may be immobilized in advance in the second measurement area instead of the primary antibody B0. The labeling substance may be the same type as the fluorescent substance surface-modified with the secondary antibody, or may be a fluorescent substance having a different wavelength and size. Furthermore, metal fine particles may be used. In this case, only the fluctuation factors relating to the surface plasmon enhancement such as the excitation light irradiation optical system 20, the gold films 58a and 59a, and the liquid sample S can be detected and used for calibration. Which of the labeled secondary antibody BF and the known amount of the labeled substance is fixed in the second measurement area 59 may be appropriately determined depending on the calibration purpose and method.

  The detection sample cell 50A can be similarly used in place of the sensor chip in any of the detection apparatuses and methods of the first to fifth embodiments described above. The detection sample cell 50 is movable in the X direction relative to the excitation light irradiation optical system 20 and the detector 30 in the housing unit 19, and detects fluorescence or radiation light from the first measurement area 58. After the measurement, the second measurement area 59 is moved to the detection position to detect fluorescence or radiated light from the second measurement area 59.

  In the detection method of the present invention, refer to FIG. 12 for the procedure for performing an assay by the sandwich method to determine whether blood (whole blood) contains an antigen, which is a substance to be detected, using the detection sample cell 50A of the present embodiment. To explain.

step 1: Blood (whole blood) So to be examined is injected from the injection port 54a. Here, the case where the antigen A which is a to-be-detected substance is contained in this blood So is demonstrated. In FIG. 12, blood So is indicated by a shaded area.
step 2: The blood So is filtered by the membrane filter 55, and large molecules such as red blood cells and white blood cells become residues.
Step 3: Plasma S (blood separated by the membrane filter 55) leaks into the flow path 52 by capillary action. Alternatively, in order to accelerate the reaction and shorten the detection time, a pump may be connected to the air hole 54b, and the plasma S may be caused to flow down by pump suction and push-out operations. In FIG. 12, plasma S is indicated by a hatched area.
step4: a fluorescent substance plasma exuded S and the secondary antibody B ₂ is applied to the flow passage 52 is mixed, the antigen A in plasma S bind with the labeling secondary antibodies BF.
step5: Plasma S gradually flows into the air hole 54b side along the flow path 52, the antigen A bound to the labeling secondary antibodies BF are primary 1 are fixed on the first measurement area 58 antibody _{B 1} bound, so-called sandwich antigen a is sandwiched with primary antibody B ₁ and the labeling secondary antibodies BF are formed with. At this time, the non-specific adsorption effect on the sensor part of the labeled secondary antibody BF is reduced by the blocking substance R.
Step 6: A part of the labeled secondary antibody BF that has not bound to the antigen A binds to the primary antibody B ₀ fixed on the second measurement area 59. Furthermore, even if the labeled secondary antibody BF that has not bound to the antigen A or the primary antibody B ₀ may remain on the measurement area, the subsequent plasma S plays a role of washing, and is suspended and not on the plate. The labeled secondary antibody BF that has been specifically adsorbed is washed away.

Thus, by injecting the blood So from the inlet 54a, from step1 to sandwich antigen A on the first measurement area 58 is sandwiched between the primary antibody _{B 1} and the labeling secondary antibodies BF are formed Step6 Thereafter, the presence or absence of the antigen and / or its concentration can be detected by detecting a fluorescence signal or a radiated light signal (hereinafter referred to as a detection signal) from the first measurement area 58. Thereafter, the sample cell 50 is moved in the X direction so that the detection signal from the second measurement area 59 can be detected, and the detection signal from the second measurement area 59 is detected. Detection signals from the second measurement area 59 is the primary antibody B ₀ that bind with the labeling secondary antibodies BF was fixed, flows down the amount of labeled secondary antibodies BF, fluorescence signal that reflects the reaction conditions, such as active By correcting the detection signal from the first measurement area 58 using this detection signal as a reference, a more accurate detection result can be obtained. In addition, as described above, even when a known amount of a labeling substance (fluorescent substance, metal fine particles) is fixed in advance in the second measurement area 59, the optical signal from the second measurement area 59 is similarly received. As a reference, the detection signal from the first measurement area 58 can be corrected.

  In the sample cell for detection according to the present embodiment, a plurality of first fluorescent dye molecules and a plurality of first fluorescent dye molecules that transmit fluorescence generated from the plurality of first fluorescent dye molecules are used as fluorescent labels. A fluorescent material composed of first particles including fluorescent dye molecules is used. That is, by using this fluorescent substance, the substance to be detected can be labeled as a group of a plurality of first fluorescent dye molecules. Therefore, since it is possible to earn more signal strength, it is possible to suppress variation in signal strength and efficiently use the enhanced electric field. Furthermore, the detection sample cell according to the present invention is a blocking substance that does not contain the first fluorescent dye molecule and does not have the specific binding property of the binding substance as a blocking agent, A blocking substance having nonspecific adsorptivity equivalent to the specific adsorptivity is used. Therefore, nonspecific adsorption of the fluorescent label binding substance can be prevented by blocking the place where the fluorescent label binding substance can be adsorbed nonspecifically with this blocking substance. As a result, it is possible to suppress noise caused by nonspecific adsorption of the fluorescent label binding substance. That is, a signal with a good S / N ratio can be detected. As described above, it is possible to accurately detect the presence and / or amount of the substance to be detected.

<Sample Cell for Detection of Third Embodiment>
FIG. 13 shows a sample cell for detection according to a third embodiment suitable for the assay by the competition method. In the detection sample cell 50B of the present embodiment, the secondary antibody C3 (the third antibody) that specifically binds to a primary antibody described later without binding to the antigen A from the upstream side of the flow path 52 on the base 51 in the detection sample cell 50B. A labeled secondary antibody adsorption area 57 ′ in which a labeled secondary antibody CF (fluorescent label binding material) comprising the secondary antibody C 3 and a fluorescent material F whose surface has been modified is physically adsorbed, a substance to be detected The first measurement area 58 ′ to which the primary antibody C1 (first binding substance) that specifically binds by binding to the antigen A and the secondary antibody C3 is immobilized, and the antigen A as the detection target substance A second measurement area 59 ′ to which the primary antibody C0 that specifically binds to the labeled secondary antibody CF without being bound is fixed is provided in order. Further, the first and second measurement areas 58 ′ and 59 ′ are subjected to a blocking process with a blocking substance. In this example, an example in which two measurement areas are provided in the sensor unit is described, but only one measurement area may be provided.

  Gold (Au) films 58a and 59a are formed as metal layers in the first measurement area 58 and the second measurement area 59 on the base 51, respectively. A primary antibody C1 is further immobilized on the Au film 58a in the first measurement area 58 ′, and a primary antibody C0 different from the primary antibody C1 is further immobilized on the Au film 59a in the second measurement area 59 ′. ing. The surfaces of the Au film 58a and the Au film 59a are subjected to a blocking process with a blocking substance R. The first measurement area 58 'and the second measurement area 59' have the same configuration except that different primary antibodies are provided. The antigen A and the labeled secondary antibody CF are competitively bound to the primary antibody C1 fixed in the first measurement area 58 '. The primary antibody C0 fixed in the second measurement area 59 'does not bind to the antigen A but directly binds to the labeled secondary antibody CF. As a result, fluctuation factors relating to the reaction such as the amount and activity of the labeled secondary antibody flowing through the flow path and fluctuation factors relating to the electric field enhancement such as the excitation light irradiation optics 20, the gold (Au) films 58a and 59a, and the liquid sample S are detected. Can be used for calibration. In the second measurement area, a known amount of labeling substance may be immobilized in advance instead of the primary antibody C0. The labeling substance may be the same type as the fluorescent substance surface-modified with the secondary antibody, or may be a fluorescent substance having a different wavelength and size. In this case, only the fluctuation factors relating to the surface plasmon enhancement such as the excitation light irradiation optics 20, the gold (Au) films 58a and 59a, and the liquid sample S can be detected and used for calibration. Which of the labeled secondary antibody CF and the known amount of the labeled substance is fixed in the second measurement area 59 can be appropriately selected depending on the calibration purpose and method.

  The detection sample cell 50B can be similarly used in place of the sensor chip in the detection apparatus and method of any of the first to fifth embodiments described above, similarly to the detection sample cell 50A described above.

  In the detection method of the present invention, refer to FIG. 14 for the procedure for performing an assay by the sandwich method to determine whether or not an antigen that is a substance to be detected is contained in blood (whole blood) using the detection sample cell 50B of the present embodiment. To explain.

step 1: Blood (whole blood) So to be examined is injected from the injection port 54a. Here, the case where the antigen A which is a to-be-detected substance is contained in this blood So is demonstrated. In FIG. 14, blood So is indicated by a shaded area.
step 2: The blood So is filtered by the membrane filter 55, and large molecules such as red blood cells and white blood cells become residues.
Step 3: Plasma S (blood separated by the membrane filter 55) leaks into the flow path 52 by capillary action. Alternatively, in order to accelerate the reaction and shorten the detection time, a pump may be connected to the air hole 54b, and the plasma S may be caused to flow down by pump suction and push-out operations. In FIG. 14, the plasma S is indicated by a hatched area.
Step 4: The plasma S that has oozed into the flow path 52 and the fluorescent substance F to which the labeled secondary antibody CF has been added are mixed.
step 5: The plasma S gradually flows along the flow path 52 toward the air hole 54b, the antigen A and the labeled secondary antibody CF compete with each other, and are fixed on the first measurement area 58 ′. It binds to the antibody _{C 1.} At this time, the influence of non-specific adsorption on the sensor part of the labeled secondary antibody CF is reduced by the blocking substance R.
step6: 'Some on the primary antibody C1 with unbound labeled secondary antibody CF, the second measurement area 59' first measurement area 58 coupled with and fixed on the primary antibody C ₀ To do. Even if further primary antibody C ₁ or not bound to C _0-labeled secondary antibody CF remains on the measurement area, a subsequent plasma S is responsible for washing, floating and non-on plate The labeled secondary antibody CF that has been specifically adsorbed is washed away.

  In this way, blood is injected from the inlet, and after Step 1 to Step 6 until antigen A and secondary antibody C3 competitively bind to primary antibody C1 on measurement area 58 ′, first measurement area 58 ′ and By detecting the detection signal from the second measurement area 59 ′, the presence and / or concentration of the antigen can be detected. Thereafter, the detection sample cell 50 is moved in the X direction so that the detection signal from the second measurement area 59 can be detected, the detection signal from the second measurement area 59 is detected, and this detection signal is used as a reference. By correcting the detection signal from the first measurement area, a more accurate detection result can be obtained.

  In the competition method, if the concentration of the substance A to be detected is high, the amount of the third binding substance C3 that binds to the first binding substance C1 is small, that is, the number of the quenching-proof fluorescent substance F on the metal layer is small. Therefore, if the concentration of the substance A to be detected is low, the amount of the third binding substance C3 bound to the first binding substance C1 is large, that is, the quenching-preventing fluorescent substance on the metal film. The fluorescence intensity increases because the number of The competitive method is suitable for detection of a low molecular weight substance because it can be measured if the substance to be detected has one epitope.

  The detection sample cell according to the present embodiment also includes a plurality of first fluorescent dye molecules and a plurality of first fluorescent dye molecules that transmit fluorescence generated from the plurality of first fluorescent dye molecules as the fluorescent label. A fluorescent material composed of first particles including fluorescent dye molecules is used. Further, the detection sample cell according to the present invention is also a blocking substance that does not contain the first fluorescent dye molecule as a blocking agent and does not have the specific binding property of the binding substance. A blocking substance having nonspecific adsorptivity equivalent to the specific adsorptivity is used. Therefore, the same effect as in the second embodiment can be obtained.

(Example of design change of sample cell for detection)
FIG. 15 shows a cross-sectional view of a sample cell for detection used in a detection method and apparatus using electric field enhancement by the optical waveguide mode. Although substantially the same as the configuration of the detection sample cell of the first embodiment shown in FIG. 10, optical waveguide layers 58b and 59b are further provided on the metal layers 58a and 59a of the sensor unit. In this case, the blocking treatment with the blocking substance is performed on the optical waveguide layers 58b and 59b.

  This detection sample cell can also be used by appropriately fixing the first binding substance to the sensor section and the fluorescent label binding substance upstream of the sensor section.

"Detection kit"
<First Embodiment>
The detection kit of the first embodiment used in the detection method of the present invention will be described.

FIG. 16A is a schematic diagram showing the configuration of the detection kit 60a.
The detection kit 60a includes a detection sample cell 61 whose sensor part has been subjected to blocking treatment with a blocking substance, and a fluorescent label binding substance BF that flows down into the flow path at the same time as the liquid sample or after the liquid sample flows down. And a labeling solution 63. Here, for example, in FIG. 16A, the labeling solution 63 is put in an ampoule 62 and sealed.

  The detection sample cell 61 differs from the detection sample cell 50A of the second embodiment described above only in that the detection sample cell does not include a physical adsorption area in which the fluorescent label binding substance BF is physically adsorbed. Has substantially the same configuration as the detection sample cell 50A.

  In the detection method of the present invention, referring to FIG. 17 for the procedure for performing an assay using the sandwich method to determine whether or not an antigen as a detection substance is contained in blood (whole blood) using the detection kit 60a of the present embodiment. I will explain.

step 1: Blood (whole blood) So to be examined is injected from the injection port 54a. Here, the case where the antigen A which is a to-be-detected substance is contained in this blood So is demonstrated. In FIG. 17, blood So is indicated by a shaded area.
step 2: The blood So is filtered by the membrane filter 55, and large molecules such as red blood cells and white blood cells become residues. Subsequently, plasma S (blood separated by the membrane filter 55) oozes into the flow path 52 by capillary action. Alternatively, in order to accelerate the reaction and shorten the detection time, a pump may be connected to the air hole, and the plasma S may be caused to flow down by pump suction and push-out operations. In FIG. 17, the plasma S is indicated by a hatched area.
step3: Plasma S gradually flows into the air hole 54b side along the flow path 52, the antigen A in plasma S binds that primary antibody _{B 1} which is fixed on the first measurement area 58.
step4: 2 antibody _{B 2} injects labeling solution 63 containing a fluorescent substance F which is modified from the supply port 54a.
step5: 2 antibody _{B 2} is modified fluorescent substance F is exuded into the flow path 52 by capillary action. Alternatively, in order to accelerate the reaction and shorten the detection time, a pump may be connected to the air hole, and the labeling solution 63 may be caused to flow down by pump suction and push-out operations.
step6: fluorescent substance F gradually flows to the downstream side, labeled secondary antibodies BF are bound to the antigen A, so-called sandwich antigen A is sandwiched with primary antibody B ₁ and the labeling secondary antibodies BF are formed. A portion of the secondary antibody B2 that has not bound to the antigen A binds to the primary antibody B0 fixed on the second measurement area 59. At this time, the non-specific adsorption effect on the sensor part of the labeled secondary antibody BF is reduced by the blocking substance R. Furthermore, even if a labeled secondary antibody (fluorescent substance) that did not bind to antigen A or primary antibody B0 may remain on the measurement area, the subsequent plasma plays a role of washing, floats on the plate, and The labeled secondary antibody adsorbed nonspecifically is washed away.

  In this manner, after blood is injected from the injection port, and after step 1 to step 6 until the antigen is combined with the primary antibody and the secondary antibody, the fluorescent label is bound on the sensor unit by electrostatic interaction in the detection device. By attracting the substance and detecting a detection signal from the first measurement area 58, the presence and / or concentration of the antigen can be detected. Thereafter, the detection sample cell 61 is moved in the X direction so that the detection signal from the second measurement area 59 can be detected. Similarly, the fluorescent label binding substance is drawn on the sensor unit, and the second measurement area 59 The detection signal is detected. The detection signal from the second measurement area 59 that fixes the primary antibody B0 that binds to the labeled secondary antibody BF is a detection signal that reflects reaction conditions such as the amount and activity of the labeled secondary antibody flowing down. A detection result with higher accuracy can be obtained by correcting the detection signal from the first measurement area using this detection signal as a reference. In addition, a known amount of a labeling substance (fluorescent substance, metal fine particles) is fixed in advance in the second measurement area 59, and the detection signal from the first measurement area with the fluorescence signal from the second measurement area 59 as a reference. May be corrected.

  An example of a method for modifying the secondary antibody to the fluorescent substance F and a method for producing a labeling solution will be described.

50 mM MES buffer and 5.0 mg / mL anti-hCG monoclonal antibody (Anti-hCG 5008 SP-5, Medix Biochemica) solution in the fluorescent substance solution (fluorescent substance diameter φ500 nm, excitation wavelength 780 nm) prepared by the above-mentioned procedure Add and stir. This modifies the antibody to the fluorescent substance.
Next, a 400 mg / mL aqueous solution of WSC (Product No. 01-62-0011, Wako Pure Chemical Industries) is added and stirred at room temperature.
Furthermore, after adding 2 mol / L Glycine aqueous solution and stirring, particle | grains are settled by centrifugation.
Finally, the supernatant is removed, PBS (pH 7.4) is added, and the surface-modified fluorescent substance is redispersed by an ultrasonic cleaner. After further centrifugation and removal of the supernatant, 500 μL of 1% BSA in PBS (pH 7.4) is added to re-disperse the surface-modified fluorescent substance to obtain a labeling solution.

  In the detection kit according to the present embodiment, a plurality of first fluorescent dye molecules and a plurality of first fluorescent dye molecules that are made of a light-transmitting material that transmits fluorescence generated from the plurality of first fluorescent dye molecules are used as fluorescent labels. A fluorescent material composed of first particles including dye molecules is used. That is, by using this fluorescent substance, the substance to be detected can be labeled as a group of a plurality of first fluorescent dye molecules. Therefore, since it is possible to earn more signal strength, it is possible to suppress variation in signal strength and efficiently use the enhanced electric field. Furthermore, in the detection kit according to the present invention, the blocking agent does not contain the first fluorescent dye molecule and does not have the specific binding property of the binding substance, and is non-specific for the fluorescent label binding substance. A blocking substance having a non-specific adsorptive property equivalent to the static adsorptive property is used. Therefore, nonspecific adsorption of the fluorescent label binding substance can be prevented by blocking the place where the fluorescent label binding substance can be adsorbed nonspecifically with this blocking substance. As a result, it is possible to suppress noise caused by nonspecific adsorption of the fluorescent label binding substance. That is, a signal with a good S / N ratio can be detected. As described above, it is possible to accurately detect the presence and / or amount of the substance to be detected.

<Second Embodiment>
The detection kit of the second embodiment used in the detection method of the present invention will be described.

FIG. 16B is a schematic diagram showing the configuration of the detection kit 60b.
The detection kit 60b includes a detection sample cell 61 that has not been subjected to blocking treatment, a labeling solution 63 containing a fluorescent label binding substance BF that is flowed into the flow path at the same time as the liquid sample or after the flow of the liquid sample, and a label A blocking solution 65 containing a blocking substance R is provided which flows down into the flow path at the same time as the solution for use 63 or before the flow of the labeling solution. Here, for example, in FIG. 16B, the labeling solution 63 is put in an ampoule 62, and the blocking solution 65 is put in an ampoule 64 and sealed.

  The detection sample cell 61 is not provided with a physical adsorption area in which the fluorescent label binding substance BF is physically adsorbed in the detection sample cell, and the blocking process using the blocking substance is not performed in the second implementation described above. Unlike the detection sample cell 50A, the other configuration is substantially the same as that of the detection sample cell 50A.

  In the detection kit 60b according to this embodiment, since the blocking process has not been performed on the sensor portion in advance, the blocking solution 65 flows down before the labeling solution 63, or the blocking solution 65 and the labeling solution are used. The measurement is performed by flowing down the solution 63 simultaneously. Here, when flowing down simultaneously, it is preferable that the particle size of the 2nd particle | grains which comprise a blocking substance is smaller than the particle size of the 1st particle | grains which comprise a fluorescent substance.

  The detection kit according to the present embodiment also includes a plurality of first fluorescent dye molecules and a plurality of first fluorescent dyes that are made of a plurality of first fluorescent dye molecules and a light-transmitting material that transmits fluorescence generated from the plurality of first fluorescent dye molecules. A fluorescent material composed of first particles including dye molecules is used. Further, in the detection kit according to the present invention, the blocking agent does not contain the first fluorescent dye molecule and does not have the specific binding property of the binding substance, and is non-specific for the fluorescent label binding substance. A blocking substance having a non-specific adsorptive property equivalent to the static adsorptive property is used. Therefore, the same effect as the first embodiment can be obtained.

<Third Embodiment>
A detection kit according to a third embodiment used in the detection method of the present invention will be described.

FIG. 16C is a schematic diagram showing the configuration of the detection kit 60c.
The detection kit 60c is used for labeling including a detection sample cell 61 that is not subjected to blocking treatment, and a fluorescent label-binding substance BF and a blocking substance R that are flowed into the flow path at the same time as the liquid sample or after the liquid sample flows down And a dual-purpose solution 67 for blocking. Here, for example, in FIG. 16C, the combined solution 67 is put in an ampoule 66 and sealed.

  The detection sample cell 61 is not provided with a physical adsorption area in which the fluorescent label binding substance BF is physically adsorbed in the detection sample cell, and the blocking process using the blocking substance is not performed in the second implementation described above. Unlike the detection sample cell 50A, the other configuration is substantially the same as that of the detection sample cell 50A.

  In the detection kit 60c according to the present embodiment, since the blocking process is not performed on the sensor unit in advance, the measurement is performed by flowing down the combined solution 67. Here, it is preferable that the particle diameter of the second particle constituting the blocking substance is smaller than the particle diameter of the first particle constituting the fluorescent substance.

  The detection kit according to the present embodiment also includes a plurality of first fluorescent dye molecules and a plurality of first fluorescent dyes that are made of a plurality of first fluorescent dye molecules and a light-transmitting material that transmits fluorescence generated from the plurality of first fluorescent dye molecules. A fluorescent material composed of first particles including dye molecules is used. Further, in the detection kit according to the present invention, the blocking agent does not contain the first fluorescent dye molecule and does not have the specific binding property of the binding substance, and is non-specific for the fluorescent label binding substance. A blocking substance having a non-specific adsorptive property equivalent to the static adsorptive property is used. Therefore, the same effect as the first embodiment can be obtained.

(Example of design change of detection kit)
The detection kit according to the present invention is suitable for a detection apparatus and method for performing an assay by a competitive method by using the detection sample cell shown in FIG. 13 having an optical waveguide layer as an attached detection sample cell. Become.

  In addition, the detection kit according to the present invention uses a detection sample cell shown in FIG. 15 having an optical waveguide layer as an attached detection sample cell, so that a detection apparatus and method using electric field enhancement by an optical waveguide mode are used. It is suitable for.

<Example>
FIG. 18A is a diagram showing a state of non-specific adsorption of a fluorescently labeled binding substance on a gold film when there is no blocking treatment with a blocking substance. On the other hand, FIG. 18B is a diagram showing a state of nonspecific adsorption of a fluorescently labeled binding substance on a gold film when there is a blocking treatment with a blocking substance. As can be seen from the figure, the noise was improved by an order of magnitude. From these figures, it was demonstrated that the effect of non-specific adsorption of the fluorescently labeled binding substance on the sensor part can be reduced by performing a blocking treatment with a blocking substance.

Schematic configuration diagram (SPF) showing a detection apparatus according to the first embodiment of the present invention. Schematic showing the magnitude relationship between fluorescent label binding substances and biological substances Schematic showing the blocking substance used in the present invention The figure which shows the mode of nonspecific adsorption to the sensor part of the fluorescence label binding substance The figure which shows a mode that nonspecific adsorption of the fluorescence label binding substance is blocked Schematic configuration diagram (LPF) showing a fluorescence detection apparatus according to a second embodiment of the present invention. The schematic block diagram which shows the sensor part of the sensor chip used for the fluorescence detection apparatus by 2nd Embodiment of this invention. The schematic block diagram (SPCE) which shows the detection apparatus by 3rd Embodiment of this invention. Schematic block diagram showing a detection device according to a fourth embodiment of the present invention (waveguide-fluorescence) Schematic configuration diagram (waveguide-radiated light) showing a detection apparatus according to a fifth embodiment of the present invention. Schematic showing a fluorescent substance with a metal coating (A) Top view and (B) Side sectional view showing a sample cell for detection according to a first embodiment of the present invention. Side sectional view showing a sample cell for detection according to a second embodiment of the present invention. The figure which shows the assay procedure by the sandwich method using the sample cell for a detection of 2nd Embodiment. Side sectional view showing a sample cell for detection according to a third embodiment of the present invention. The figure which shows the assay procedure by the competition method using the sample cell for a detection of 3rd Embodiment. Cross-sectional side view showing an example of design change of detection sample cell Schematic diagram showing the configuration of the fluorescence detection kit according to the present invention Schematic diagram showing the configuration of a fluorescence detection kit of another embodiment (part 1) Schematic diagram showing the configuration of a fluorescence detection kit of another embodiment (part 2) Diagram showing assay procedure by sandwich method using fluorescence detection kit Diagram showing the amount of non-specific adsorption without blocking treatment Figure showing the amount of non-specific adsorption with blocking treatment Conceptual diagram showing the detection method in the conventional example

Explanation of symbols

1, 2, 3, 4, 5 Detector 10, 10 ′, 10 ″ Sensor chip 11 Dielectric plate 12 Metal layer (metal film)
12 'metal layer (metal microstructure)
16 1st particle (translucent material)
16 '2nd particle 20, 20' Excitation light irradiation optical system 30 Photo detector 35 Magnetic field applying means 50, 50A, 50B, 61 Sample cell for detection 51 Dielectric plate 52 Channel 53 Spacer 54 Upper plate 57 Adsorption of fluorescent substance Area 58, 59 Detection area 60 Detection kit 63 Detection labeling solution _BF , _CF Labeled secondary antibody (fluorescent label binding substance)
F Fluorescent substance f Fluorescent dye molecule Lo Excitation light Lf Fluorescence Lp Radiation light Mc Metal coating R Blocking substance r Blocking agent by biological substance

Claims (19)

Using a sensor chip including a sensor unit having a laminated structure including a metal layer adjacent to the dielectric plate on one surface of the dielectric plate,
By bringing the sample into contact with the sensor unit, a fluorescent label binding substance comprising a fluorescent label and a binding substance labeled with the fluorescent label in an amount corresponding to the amount of the target substance contained in the sample, Combined on the sensor,
By irradiating the sensor unit with excitation light, an enhanced photoelectric field is generated on the sensor unit,
In the detection method of exciting the fluorescent label with the enhanced photoelectric field and detecting the amount of the substance to be detected based on the amount of light generated due to the excitation,
The fluorescent label includes a plurality of first fluorescent dye molecules and a light-transmitting material that transmits fluorescence generated from the first fluorescent dye molecules, and includes the first fluorescent dye molecules. A fluorescent material composed of particles of
As a blocking agent for preventing the fluorescent label binding substance from adsorbing to the sensor part due to the nonspecific adsorption property of the fluorescent label binding substance to the sensor part, the binding does not include the first fluorescent dye molecule A detection method characterized by using a blocking substance that does not have a specific binding property of a substance and has a nonspecific adsorption property equivalent to the nonspecific adsorption property of the fluorescent label binding substance.   The detection method according to claim 1, wherein the blocking substance has second particles having at least a surface made of a material equivalent to the light-transmitting material.   The detection method according to claim 2, wherein the blocking substance has a modified substance that is surface-modified on the surface of the second particle and does not have the specific binding property.   The detection method according to claim 2 or 3, wherein the second particles have a particle size smaller than the particle size of the first particles.   The detection method according to claim 4, wherein the blocking substance and the fluorescent label binding substance are simultaneously allowed to flow down on the sensor unit.   The second particle has a plurality of second fluorescent dye molecules that emit fluorescence having a wavelength different from that of the fluorescence emitted by the first fluorescent dye molecule in the second particle. The detection method according to any one of claims 2 to 5.   The detection method according to claim 2, wherein the second particles do not contain a substance that emits fluorescence in the second particles. Exciting plasmons on the metal layer by irradiation with the excitation light, generating the enhanced photoelectric field by the plasmons,
The detection method according to any one of claims 1 to 7, wherein fluorescence generated from the fluorescent label by the excitation is detected as the light generated due to the excitation of the fluorescent label. Exciting plasmons on the metal layer by irradiation with the excitation light, generating the enhanced photoelectric field by the plasmons,
As the light generated due to the excitation of the fluorescent label, the fluorescence generated from the fluorescent label by the excitation induces a new plasmon in the metal layer, so that the other surface is opposed to the one surface with respect to the dielectric plate. The detection method according to claim 1, further comprising detecting emitted light from the newly induced plasmon emitted to the side. As the sensor chip, using the sensor chip in which the laminated structure includes an optical waveguide layer,
Exciting an optical waveguide mode in the optical waveguide layer by irradiation of the excitation light, and generating the enhanced photoelectric field by the optical waveguide mode,
The detection method according to any one of claims 1 to 7, wherein fluorescence generated from the fluorescent label by the excitation is detected as the light generated due to the excitation of the fluorescent label. As the sensor chip, the laminated structure includes an optical waveguide layer, but also uses the sensor chip,
Exciting an optical waveguide mode in the optical waveguide layer by irradiation of the excitation light, and generating the enhanced photoelectric field by the optical waveguide mode,
As the light generated due to the excitation of the fluorescent label, the fluorescence generated from the fluorescent label by the excitation induces a new plasmon in the metal layer, so that the other surface is opposed to the one surface with respect to the dielectric plate. The detection method according to claim 1, further comprising detecting emitted light from the newly induced plasmon emitted to the side. A detection sample cell used as the sensor chip in the detection method according to any one of claims 1 to 4 and 6 to 11,
A base having a channel through which a liquid sample flows;
An inlet for injecting the liquid sample into the channel provided upstream of the channel;
An air hole provided on the downstream side of the flow path for flowing the liquid sample injected from the injection port to the downstream side;
A sensor chip portion provided in the flow path between the inlet and the air hole, the dielectric plate provided as a part of the inner wall surface of the flow path, and a sample contact of the dielectric plate A sensor chip portion including a sensor portion provided in a predetermined area of the surface,
The sample cell for detection, wherein the sensor unit has a laminated structure including a metal layer adjacent to the dielectric plate, and the surface of the sensor unit is subjected to blocking treatment with the blocking substance. .   The sample cell for detection according to claim 12, comprising a first binding substance that specifically binds to the substance to be detected, the substance being fixed on the sensor unit.   The sample cell for detection according to claim 13, further comprising the fluorescent label binding substance disposed in the flow path upstream of the sensor unit.   The sample cell for detection according to claim 12, wherein the laminated structure includes an optical waveguide layer. A detection kit used in the detection method according to any one of claims 1 to 4 and 6 to 11,
A base having a channel through which the liquid sample flows, an inlet for injecting the liquid sample into the channel provided on the upstream side of the channel, and provided on the downstream side of the channel An air hole for flowing the liquid sample injected from the injection port to the downstream side, and a sensor chip portion provided in the flow path between the injection port and the air hole, A sensor chip provided with a dielectric plate provided as a part of the inner wall surface of the path and a sensor unit provided in a predetermined region of the sample contact surface of the dielectric plate, specifically binds to the substance to be detected. A first binding material comprising: the first binding material fixed on the sensor unit, wherein the sensor unit has a laminated structure including a metal layer adjacent to the dielectric plate; and A block made of the blocking substance on the surface of the sensor unit. A detection sample cell used as the sensor chip, and the fluorescent label binding substance that flows down into the flow path at the same time as the liquid sample or after the liquid sample flows down. A detection kit comprising a labeling solution containing the detection kit. A detection kit used in the detection method according to claim 1,
A base having a channel through which the liquid sample flows, an inlet for injecting the liquid sample into the channel provided on the upstream side of the channel, and provided on the downstream side of the channel An air hole for flowing the liquid sample injected from the injection port to the downstream side, and a sensor chip portion provided in the flow path between the injection port and the air hole, A dielectric plate provided as a part of the inner wall surface of the path, a sensor chip provided with a sensor unit provided in a predetermined region of the sample contact surface of the dielectric plate, and the fluorescent label binding substance on the sensor unit. A first binding material for fixing to the sensor portion, the first binding material being fixed on the sensor portion, wherein the sensor portion includes a metal layer adjacent to the dielectric plate As the sensor chip Detection sample cell to be use,
The labeling solution containing the fluorescent label binding substance that flows into the flow path at the same time as the liquid sample or after the liquid sample flows, and the flow at the same time as the labeling solution or before the flow of the labeling solution A detection kit comprising a blocking solution containing the blocking substance that flows down into a channel. A detection kit used in the detection method according to claim 5,
A base having a channel through which the liquid sample flows, an inlet for injecting the liquid sample into the channel provided on the upstream side of the channel, and provided on the downstream side of the channel An air hole for flowing the liquid sample injected from the injection port to the downstream side, and a sensor chip portion provided in the flow path between the injection port and the air hole, A dielectric plate provided as a part of the inner wall surface of the path, a sensor chip provided with a sensor unit provided in a predetermined region of the sample contact surface of the dielectric plate, and the fluorescent label binding substance on the sensor unit. A first binding material for fixing to the sensor portion, the first binding material being fixed on the sensor portion, wherein the sensor portion includes a metal layer adjacent to the dielectric plate As the sensor chip Sample cell for detection to be used, and a combined labeling and blocking solution containing the fluorescent label binding substance and the blocking substance that is flowed into the flow path at the same time as the liquid sample or after the liquid sample flows A detection kit comprising:   The detection kit according to claim 16, wherein the laminated structure includes an optical waveguide layer.