JP5450993B2 - Detection method, detection sample cell and detection kit - Google Patents

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 such technique 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 attached. A secondary antibody that specifically binds to the antigen, and further binds to the antigen to form a primary antibody-antigen-secondary antibody binding state, and the fluorescence from the fluorescent label attached to the secondary antibody This is a so-called sandwich method for detection. 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 order to improve sensitivity in the evanescent fluorescence method, a method using the effect of electric field enhancement by plasmon resonance has been proposed in Patent Document 1, Non-Patent Document 1, and the like. In the surface plasmon enhanced fluorescence method, in order to generate plasmon resonance, a metal layer is provided in the sensor unit, surface plasmon is generated in the metal layer, and the S / N ratio is increased by increasing the fluorescence signal by the electric field enhancing action. It is to improve.

  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.

However, in the method described above is still 10 ⁵ level in terms of the number of molecules, at present, far from the detection of a single molecule.

Therefore, as shown in Non-Patent Document 4, Raman spectroscopy using a metal probe that can be detected at a single molecule level has attracted attention. In this method, light is incident on the tip of the metal probe to generate a localized plasmon at the tip, thereby utilizing a local electric field enhancement field generated between the substrate and the probe. This effectively increases the scattering cross section of the Raman process by the molecules directly under the probe, and theoretically, it is considered that an increase in intensity of several tens to 10 ⁶ times the incident light intensity can be obtained (non- Patent Document 4 pp. 277 Section 2.2.1).
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 Yasushi Inoue, Kei Kawada, Spectroscopic Research, Vol. 51, No. 6, p. 276-285 (2002)

  However, the Raman signal is greatly influenced by the state and environment in which a substance to be detected such as a solvent is placed, and vibrations of impurities appear in the spectrum. Therefore, the Raman signal is useful as a qualitative analysis but cannot be expected to be quantitative. In addition, since the local strong electric field due to the metal probe has a width of about several tens of nanometers at most and the region that can be measured is small, the metal probe needs to be scanned, which makes measurement difficult. Furthermore, in general, a Raman spectroscopic device is large and expensive, and its operability is not good.

  The present invention has been made in view of the above problems, and provides a detection method that enables high-sensitivity detection at a single molecule level and has a wide detection range by a detection method that is easier and less expensive than Raman spectroscopy. It is for the purpose.

  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, a detection method according to the present invention uses 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, an amount of the fluorescent label binding substance according to the amount of the substance to be detected contained in the sample is bound on the sensor unit,
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 of the fluorescent label binding substance with the enhanced photoelectric field and detecting the amount of the detected substance based on the amount of light generated due to this excitation,
As a fluorescent label, a fluorescent substance comprising a plurality of fluorescent dye molecules and one or more fluorescence enhancing fine particles by a light-transmitting material that transmits fluorescence generated from the plurality of fluorescent dye molecules is used.

  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 “fluorescent label binding substance” means a fluorescently labeled binding substance that binds on the sensor unit by an amount corresponding to the amount of the substance to be detected. For example, when an assay by the sandwich method is performed, it is composed of a binding substance that specifically binds to the substance to be detected and a fluorescent label. When an assay by the competitive method is performed, a binding substance that competes with the substance to be detected It consists of a fluorescent label.

  “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.

  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” shall mean forming an enhanced photoelectric field 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.

  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.

  “Fluorescence-enhanced fine particles” mean those that enhance the fluorescence generated from the fluorescent label by the interaction of the fluorescence-enhanced fine particles with the above-described photoelectric field, metal layer, or other fluorescent-enhanced fine particles in the vicinity. And

  Furthermore, in the detection method according to the present invention, the fluorescence enhancing fine particles are preferably scattering fine particles.

  Further, the fluorescence enhancing fine particles are preferably metal fine particles, and more preferably metal nanorods.

  And it is preferable that the particle diameter of fluorescence enhancement fine particle is 10-200 nm.

  Here, the “particle diameter” of the metal fine particles means the maximum diameter of the fine particles.

  “Nanorod” means a nanoparticle having a rod-like (rod-like) shape, and the length ratio (aspect ratio) between the vertical axis and the horizontal axis is approximately 1 to 20.

  Furthermore, as a method of “exciting the fluorescent label of the fluorescent label binding substance and detecting the amount of the substance to be detected based on the amount of light generated due to this excitation”, a method for detecting fluorescence from the fluorescent label Alternatively, it may be one that detects the emitted light that is generated when the fluorescence induces a new surface plasmon 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 a photoelectric field enhanced by the plasmon is generated, and the fluorescence generated from the fluorescent label by this excitation is generated in the metal layer as light generated by the excitation of the fluorescent label. 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, for plasmon excitation, 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.

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 is characterized by having a laminated structure including a metal layer adjacent to the dielectric plate.

  The detection sample cell according to the present invention preferably includes a first binding substance for fixing the fluorescent label binding substance on the sensor part, which is fixed on the sensor part.

  Further, any one of the second binding substance that specifically binds to the detected substance and the third binding substance that competes with the detected substance and specifically binds to the first binding substance, It is preferable to provide a fluorescent label binding substance, which is a fluorescent label binding substance composed of this one binding substance and a modified fluorescent substance, and disposed in the flow channel upstream of the sensor unit.

  The sample cell for detection according to the present invention is suitable for performing an assay by the sandwich method when the fluorescent label binding substance is composed of a second binding substance and a fluorescent label. When it consists of a substance and a fluorescent label, it is suitable for performing an assay by a competitive method.

  The laminated structure preferably includes an optical waveguide layer.

Furthermore, the detection kit according to the present invention is a detection kit used in the detection method described above,
From 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 injected liquid sample downstream, and a sensor chip portion provided in the flow path between the injection hole and the air hole, provided as a part of the inner wall surface of the flow path A sensor chip portion having a dielectric plate and a sensor portion provided in a predetermined region of the sample contact surface of the dielectric plate, and a second fixed on the sensor portion for fixing the fluorescent label binding substance on the sensor portion. And a sample cell for detection used as a sensor chip comprising a metal layer adjacent to a dielectric plate, and a sensor portion that flows at the same time as or after the liquid sample flows down A labeling solution that flows down the road The binding of either the second binding substance that specifically binds to the substance to be detected and the third binding substance that specifically binds to the first binding substance in competition with the substance to be detected It is characterized by comprising a labeling solution containing a fluorescent label binding substance composed of a substance and a fluorescent substance in which this one binding substance is modified.

  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.

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

  Here, in the detection sample cell and the detection kit, the “fluorescent substance” refers to a plurality of fluorescent dye molecules and one or more fluorescent enhancement fine particles that transmit fluorescence generated from the plurality of fluorescent dye molecules, as described above. It is comprised by optical material.

  In the fluorescence detection method according to the present invention, the detection is performed in a state where the fluorescence-enhanced fine particles are present in the vicinity of the fluorescent dye molecules in the fluorescent substance that is a fluorescent label. Therefore, the fluorescence generated from the fluorescent label can be enhanced by the interaction between the fluorescence enhancing fine particles and the photoelectric field, the metal layer, or other fluorescent enhancing fine particles in the vicinity. The light generated due to the excitation of the fluorescent label can be directly or indirectly enhanced by the enhancement of the fluorescence. As a result, a stable signal with a good S / N can be detected, and the presence and / or amount of the substance to be detected can be accurately detected.

  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>
In the detection method of the present invention, for example, as shown in FIG. 1, a sensor chip 10 having a sensor part 14 including a metal layer 12 on one surface of a dielectric plate 11 is used, and a sample is brought into contact with the sensor part 14. Thus, the fluorescent label binding substance BF in an amount corresponding to the amount of the substance A to be detected contained in the sample is bound on the sensor unit 14, the sensor unit 14 is irradiated with the excitation light Lo, and the excitation light Lo The photoelectric field on the sensor unit 14 is enhanced by irradiation, and based on the amount of light generated in the enhanced photoelectric field D due to the excitation of the fluorescent label of the fluorescent label binding substance BF, A detection method for detecting an amount, comprising a plurality of fluorescent dye molecules f and one or more fluorescence-enhancing fine particles M as fluorescent labels by a translucent material 16 that transmits fluorescence generated from the plurality of fluorescent dye molecules f. Fluorescent substance F It is characterized in that there.

  The detection device 1 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 the 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 irradiation with 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 D is detected. However, the method of enhancing the photoelectric field D may be by surface plasmon resonance or localized plasmon resonance, or by excitation of an optical waveguide mode (see the fourth embodiment). Further, the fluorescence Lf generated from the fluorescent label may be detected, or the emitted light Lp generated by the fluorescence Lf inducing a new surface plasmon in the metal layer 12 may be detected (see the third embodiment). ). Specific embodiments will be described in the following embodiments. Note that the detection method and apparatus of this embodiment are a fluorescence detection method and apparatus for detecting the fluorescence Lf excited in the enhanced photoelectric field D by enhancing the photoelectric field D by surface plasmon resonance.

  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 dielectric plate 11. The thickness of the metal film 12 is preferably determined as appropriate so that the surface plasmon is strongly excited by the material of the metal film 12 and the wavelength of the excitation light Lo. For example, when laser light having a center wavelength of 780 nm is used as the excitation light Lo and a gold (Au) film is used as the metal film 12, the thickness of the metal film 12 is preferably 50 nm ± 20 nm. More preferably, it is 47 nm ± 10 nm. The metal film 12 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 fluorescent label binding substance BF is a fluorescently labeled binding substance that binds to 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 first binding substance B2 that specifically binds to the substance A to be detected and a fluorescent label when assaying by the sandwich method. In the case of performing an assay by the competition method described later, the assay is composed of a third binding substance that competes with the substance A to be detected and a fluorescent label. More specifically, when a 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, in the sandwich method, the substance A to be detected is used. And a fluorescent label binding substance BF comprising a second binding substance B2 that specifically binds to the fluorescent substance and a fluorescent label in which the binding substance is modified. On the other hand, in the same case, in the competition method, a fluorescent label binding comprising a third binding substance that specifically binds to the first binding substance in competition with the substance to be detected, and a fluorescent label in which the binding substance is modified. Substance is used. When the substance A to be detected is an antigen, a so-called primary antibody can be used as the first binding substance.

  The fluorescent substance F is a light-transmitting material that transmits a plurality of fluorescent dye molecules f and one or more fluorescence enhancing fine particles M through the fluorescence generated from the plurality of fluorescent dye molecules f, as shown in an enlarged view in part of FIG. 16 is included. The fluorescent substance F can be produced as follows.

First, two examples of the method for forming the light transmitting material 16 enclosing the fluorescence enhancing fine particles M will be described. The first method is to form the translucent material 16 from a SiO2 film, and is roughly composed of the following steps (1) to (3).
(1) Synthesis of gold colloid to become gold fine particles (2) Replacement of gold colloid surface dispersant (citric acid → siloxane)
By adding APS ((3-aminopropyl) trimethoxysilane) aqueous solution (2.5 ml, 1 mmol) to 500 ml (milliliter) of 5 × = 10 ⁻⁴ mol colloidal gold colloid solution, and stirring vigorously for 15 minutes, colloidal gold Displace surface citric acid.
(3) SiO2 modification of gold colloid surface 20 ml of 0.54 wt% aqueous solution of sodium silicate adjusted to pH 10-11 is added to the gold colloid aqueous solution in step (2) and vigorously stirred. After 24 hours, a SiO2 film having a thickness of about 4 nm is formed. 170 ml of ethanol is added to a solution concentrated to 30 ml by centrifugation. When 0.6 ml of NH 4 OH (28%) is further added dropwise, 80 μl (micro liter) of TES (tetraethoxysilane) is added, and the mixture is slowly stirred for 24 hours, the light-transmitting material 16 composed of a 20 nm thick SiO 2 coating is obtained. It is formed.

  Next, as a second method, a case where the translucent material 16 is formed from a polymer coating will be described. This method is roughly divided into the following steps (1) to (2).

(1) Redispersion of gold nanoparticles as gold fine particles into DMF 1 ml of an aqueous dispersion containing citric acid-stabilized gold nanoparticles having an average particle diameter of about 30 nm at a maximum of about 360 pmol (= 7 × 10 ⁻¹¹ wt%) Prepare and centrifuge, then discard 0.95 ml of supernatant. The remaining dark red, viscous precipitate is redispersed in 1 ml DMF (N, N-dimethylformamide). It should be noted that excess citrate ions inhibit particle encapsulation. When using small particles, it is better to wash with water before adding DMF.
(2) Encapsulation of gold nanoparticles DMF dispersion containing about 648 pmol (= 7 × 10 ⁻¹¹ wt%) of citric acid-stabilized gold nanoparticles having an average particle size of about 30 nm obtained in the step (1) To 1 ml of the solution, add 10 μl of a DMF solution (about 10 ⁻² g / ml) of a polystyrene-polyacrylic acid block copolymer (about 100-mer polystyrene and about 13-mer polyacrylic acid), and Add 200 μl of water at a flow rate of 3 μl / min and stir vigorously. When the solution is stirred for 10 minutes, the color of the solution gradually changes to purple. Then, 5 μl of a 1% by weight dodecanethiol DMF solution is added and stirred for 24 hours. Thereafter, 3 ml of water is further added by a syringe pump at a flow rate of 2 ml / h.
The DMF is then removed by dialysis over 24 hours. Next, 72 μl of EDC solution (0.1 wt% with respect to water: 24 nmol) was added all at once while stirring, and after stirring for 30 minutes, 144 μl of EDODEA solution (0.1 wt% with respect to water: 96 nmol) was added all at once. Add and stir.
Thereafter, the reagent is removed by dialysis over 24 hours, followed by centrifugation at 4000 G for 30 minutes, and the supernatant corresponding to 80% by volume is discarded. Next, add the same volume of water as the discarded supernatant and centrifuge in the same manner. By repeating this operation from centrifugation to centrifugation three times or more, a film made of a cross-linked product of polystyrene-polyacrylic acid block copolymer is formed as a translucent material 16 around gold nanoparticles (gold fine particles). The

Then, the fluorescent substance F including the fluorescence enhancing fine particles M created in the above process is impregnated with the fluorescent dye molecule f. This process is as follows. A 0.1% Solid in phosphate (polystyrene solution: pH 7.0) is prepared by adjusting the fluorescent substance F including the fluorescence enhancing fine particles M prepared in the above process.
Next, a 0.3 mg ethyl acetate solution (1 mL) of fluorescent dye molecules (Hayashibara Biochemical Laboratories, NK-2014, excitation wavelength: 780 nm) 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 steps, a fluorescent substance F can be obtained in which the fluorescent dye molecule f and the one or more fluorescence enhancing fine particles M are encapsulated with polystyrene having a function of transmitting the fluorescence generated from the fluorescent dye molecule f. In such a procedure, the particle size of the fluorescent material F produced by impregnating the polystyrene particles with the fluorescent dye molecule f is the same as the particle size of the polystyrene particles (φ150 nm in the above example).

  Since the fluorescent substance F includes a plurality of fluorescent dye molecules f, the amount of emitted fluorescence can be significantly increased as compared with the case where a conventional fluorescent dye molecule f alone is used as a fluorescent label. 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 and SiO 2, but those that can encapsulate the fluorescent dye molecule f and can transmit the fluorescent Lf from the fluorescent dye molecule f to be emitted to the outside. There is no particular limitation.

  The fluorescence enhancing fine particles M enhance the fluorescence Lf by the interaction with the photoelectric field, the metal layer, or other fluorescent enhancing fine particles in the vicinity. The fluorescence enhancing fine particles M are not particularly limited, but are preferably scattering fine particles made of a scattering material. Examples of the scattering fine particles include inorganic fine particles such as quantum dots and organic fine particles made of a scattering polymer. Furthermore, the fluorescence enhancing fine particles M are preferably metal fine particles, and more preferably metal nanorods. This is because in the case of metal nanorods, the filling rate can be increased when a plurality of fine particles are filled. The particle size of the metal fine particles is preferably smaller than the wavelength of the excitation light Li, and particularly preferably 40 to 200 nm, in order to effectively induce localized plasmons. In addition, the particle diameter said here means the maximum diameter of microparticles | fine-particles.

  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 resonates with the metal film 12 at the surface plasmon resonance. . 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 10 is disposed on the prism 22 and the photodetector 30 can detect fluorescence. 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.

The sensor chip 10 is prepared by modifying the primary antibody B1 on the metal film 12 as a first binding substance that specifically binds to the antigen A.
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 label F is allowed to flow. 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 bind to different sites (epitopes) with respect to the antigen A as the substance to be detected. Use things. In addition, as the fluorescent label F, a fluorescent substance F enclosing the fluorescent dye molecule f and one fluorescent enhancement fine particle M is used (FIG. 1). 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 the unreacted fluorescently labeled binding substance BF, a buffer solution is passed to eliminate the unreacted fluorescently labeled binding substance BF.

  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. Further, the timing of labeling the detection substance (antigen A) is not particularly limited, and before the detection substance (antigen A) is bound to the first binding substance (primary antibody B1), the sample is labeled with a fluorescent label in advance. May be added.

  In the present embodiment, the fluorescent substance F enclosing the fluorescent dye molecule f and one fluorescence enhancing fine particle M is used as a fluorescent label. Then, excitation light Lo is irradiated by the excitation light irradiation optical system 20 toward a predetermined region of the dielectric plate 11 of the sensor chip 10. 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 forms an enhanced photoelectric field D on the surface of the metal film 12. At this time, the fluorescent photoelectric molecule D in the fluorescent substance F is excited by the enhanced photoelectric field D, and fluorescence Lf is generated. Here, the fluorescence Lf is enhanced by the effect of electric field enhancement by surface plasmons. Then, by detecting the fluorescence Lf by the photodetector 30, it is possible to detect the presence and / or amount of the detection target substance A bound to the fluorescent label binding substance BF.

  Furthermore, in the present invention, since the fluorescence enhancing fine particles M are present in the fluorescent substance F, the fluorescent Lf is efficiently generated from the fluorescent dye molecules f. The fluorescence Lf can be detected with sensitivity. The contribution of the fluorescence enhancing fine particles M to the increase in the fluorescence amount is largely due to the four effects shown in FIGS. 2A to 2D.

(1) Effect of Scattered Light of Evanescent Light Scattered by Fluorescence-Enhanced Fine Particle M As shown in FIG. 2A, by using the scattered light SL from the fluorescence-enhanced fine particle M as secondary excitation light, Furthermore, it can illuminate efficiently. As described above, the photoelectric field D rapidly attenuates as the distance from the surface of the sensor unit 14 increases. Therefore, the fluorescent dye molecules f that are separated from the surface of the sensor unit 14 cannot absorb sufficient light for excitation. That is, the fluorescence enhancing fine particles M contribute to an increase in the amount of fluorescence by generating the above scattered light as secondary excitation light for exciting such fluorescent dye molecules f.

(2) Effect of near-field light generated in the vicinity of the surface of the fluorescence enhancing fine particle M As shown in FIG. 2B, for example, when the fluorescence enhancing fine particle M is a metal fine particle, the interaction between the photoelectric field D and the metal fine particle M Localized plasmons are generated, and as a result, the near-field light NL enhanced by the localized plasmons oozes out around the metal fine particles M. The fluorescent dye molecules f in the vicinity of the fluorescence enhancing fine particles M are excited by the near-field light NL. That is, the fluorescence enhancing fine particles M contribute to an increase in the amount of fluorescence by generating such near-field light NL. The particle size of the metal fine particles M can be appropriately selected so that localized plasmons can be induced according to the wavelength of the excitation light Lo.

(3) Effect of Hot Spot HS1 Generated Between Fluorescence-Enhanced Fine Particle M and Metal Layer 12 As shown in FIG. 2C, for example, when fluorescence-enhanced fine particle M is a metal fine particle, a metal film is formed in the vicinity of metal fine particle M. When 12 is present, a hot spot HS1 is generated in the gap between the metal fine particle M and the metal film 12. A “hot spot” is a local strong electric field region formed as a result of concentration of electrostatic force (electric field concentration) on a substance having a microstructure. Therefore, the fluorescence Lf from the fluorescent dye molecule f present in the hot spot HS1 can be enhanced. That is, the fluorescence enhancing fine particles M contribute to an increase in the amount of fluorescence by causing such a hot spot HS1.

(4) Effect of Hot Spot HS2 Generated Between Plurality of Fluorescence Enhancement Fine Particles M As shown in FIG. 2D, for example, when the fluorescence enhancement fine particles M are metal fine particles, a plurality of metal fine particles M are present in the vicinity. The hot spot HS2 is generated in the gap between the metal fine particle M and the metal fine particle M. Therefore, the fluorescence Lf from the fluorescent dye molecule f existing in the hot spot HS2 can be enhanced. That is, the fluorescence enhancing fine particles M contribute to an increase in the amount of fluorescence by causing such hot spots HS2.

  Furthermore, the synergistic effect due to the effects (1) to (4) actually contributes to an increase in the amount of fluorescence. When both hot spots HS1 and HS2 and the localized plasmon of (2) occur simultaneously, the electric field enhanced by the localized plasmon of (2) concentrates, forming a huge electric field, As a result, a synergistic effect of inducing the partner's localized plasmon more strongly is generated, so that it is much stronger than in other cases (for example, when particles exist alone or non-metallic particles are close to each other). A photoelectric field D is formed.

  As described above, according to the method of the present invention, the fluorescence generated from the fluorescent label can be enhanced by the effect of a hot spot or the like generated in the gap between the metal fine particles and the metal fine particles or the gap between the metal fine particles and the metal film. As a result, high-sensitivity detection at a single molecule level is possible even when using a fluorescence method that is easier and less expensive than Raman spectroscopy. On the other hand, according to the method of the present invention, since the plurality of fluorescence enhancing fine particles M are widely dispersed on the detection unit, the hot spot can be formed over a wide range. As a result, measurement over a wide range can be performed in a short time compared to Raman spectroscopy in which the enhanced photoelectric field is limited to the vicinity of the tip of the metal probe.

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 fluorescent dye molecule f by the metal layer 12 becomes strong. It is necessary to precisely control the distance from the fluorescent dye molecule f. 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 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 F contains a sufficiently large amount of the fluorescent dye molecule f, it is inevitably much more than the metal layer 12 without providing a metal quenching prevention film on the metal layer 12. The distance from the fluorescent dye molecule f can be increased 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)
In the first embodiment described above, parallel light incident on the interface at a predetermined angle θ is incident as the excitation light Lo. However, the excitation light has an angle θ as schematically shown in FIG. A fan beam (focused light) having an angular width Δθ at the center may be used. In the case of a fan beam, the light beam is incident on the interface between the prism 122 and the metal film 112 on the prism at an incident angle in the range of angle θ−Δθ / 2 to θ + Δθ / 2, and the resonance angle is within this angle range. If there is, surface plasmon can be excited in the metal film 112. Before and after supplying the sample onto the metal film, the refractive index of the medium on the metal film changes, so that the resonance angle at which surface plasmons are generated changes. Therefore, 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 wide incident angle incident on the interface as shown in FIG. 3, it is possible to cope with a change in 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. The above also applies to the second to fifth embodiments described later.

<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, which is a metal microstructure having a concavo-convex structure smaller than the wavelength of the excitation light Lo on the surface. Or a plurality of metal nanorods having a size smaller than the wavelength of the excitation light. When the metal layer 12 ′ that generates such localized plasmons is provided, the excitation light Lo 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, here, the excitation light irradiation optical system 20 ′ is configured to irradiate the excitation light Lo from above the dielectric plate 11.

  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.
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 dielectric 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 includes a metal microstructure 74 composed of a dielectric plate 11 and a metal pattern layer provided in a predetermined region on the dielectric plate and in which metal thin wires 74a are patterned in a lattice pattern. It consists of 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 are designed to be smaller than the wavelength of the excitation light Lo.

  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.

The fluorescence detection method of this embodiment using the fluorescence detection apparatus 2 will be described.
The process of preparing the sensor chip and causing the antigen-antibody reaction is the same as in the first embodiment, and thus the description thereof is omitted. The same applies to the following embodiments.
In the present embodiment, the fluorescent substance F enclosing the fluorescent dye molecule f and one fluorescence enhancing fine particle M is used as a fluorescent label. Then, the excitation light Lo is irradiated by the excitation light irradiation optical system 20 toward a predetermined region of the dielectric plate 11 of the sensor chip 10 ′. The excitation light Lo emitted from the light source 21 is reflected by the half mirror 23 and is incident on the sample contact surface of the sensor chip 10 ′. By irradiation with the excitation light Lo, localized plasmons are excited on the surface of the metal layer 12 ′. An enhanced photoelectric field D is formed on the metal layer 12 ′ by this localized plasmon. Then, the fluorescent dye molecule f in the fluorescent substance F in the enhanced photoelectric field is excited to generate fluorescence Lf. By detecting this fluorescence Lf with 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 this embodiment, since the fluorescence enhancing fine particles M are present in the fluorescent substance F, the fluorescent Lf is efficiently generated from the fluorescent dye molecules f. Therefore, the sensitivity is higher than that of the normal surface plasmon fluorescence method. In addition, it is possible to detect the fluorescence Lf, and it is possible to obtain the same effect as in the first embodiment.

<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. This is to detect the emitted light from the newly induced plasmon emitted to the surface opposite to the 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 synchrotron radiation detection device 3, newly induced plasmon is emitted to the surface opposite to the metal layer forming surface with respect to the dielectric plate by the fluorescence newly inducing surface plasmon in the metal layer. The photodetector 30 is arranged so as to detect the radiated light Lp.

A radiation light detection method of the present embodiment using the detection device 3 will be described.
In the present embodiment, the fluorescent substance F enclosing the fluorescent dye molecule f and one fluorescence enhancing fine particle M is used as a fluorescent label. Then, similarly to the first embodiment, the excitation light Lo is irradiated by the excitation light irradiation optical system 20. 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 forms an enhanced photoelectric field D on the surface of the metal film 12. Then, the fluorescent dye molecule f in the fluorescent substance F in the enhanced photoelectric field D is excited to generate fluorescence Lf. At this time, fluorescence is enhanced by the effect of electric field enhancement by surface plasmons. 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 present embodiment, the presence of the fluorescence enhancing fine particles M in the fluorescent substance F efficiently generates the fluorescence Lf from the fluorescent dye molecule f, and detects the emitted light Lp resulting from the enhanced fluorescence Lf. The same effects as those of 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 having an optical waveguide layer on a metal layer, excites an optical waveguide mode in the optical waveguide layer by irradiation of excitation light, and enhances a photoelectric field by the optical waveguide mode. A detection method and apparatus for detecting fluorescence excited in an enhanced photoelectric 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. The layer thickness of the optical waveguide layer 12b is not particularly limited, and the excitation light Lo can be induced so that the optical waveguide mode is induced. It can be determined in consideration of the wavelength, the incident angle, the refractive index of the optical waveguide layer 12b, etc. For example, similarly to the above, a laser beam having a center wavelength of 780 nm is used as the excitation light Lo, and one layer as the optical waveguide layer 12b. In the case of using a silicon oxide film, the thickness is preferably about 500 to 600 nm The optical waveguide layer 12b has a laminated structure including an internal optical waveguide layer made of an optical waveguide material such as one or more dielectrics. This laminated structure is preferably an alternating laminated structure of an internal optical waveguide layer and an internal metal layer in order from the metal layer side.

The fluorescence detection method of this embodiment using the fluorescence detection device 4 will be described.
In the present embodiment, the fluorescent substance F enclosing the fluorescent dye molecule f and one fluorescence enhancing fine particle M is used as a fluorescent label. Then, similarly to the first embodiment, the excitation light Lo is irradiated by the excitation light irradiation optical system 20. 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 combined with the optical waveguide mode of the optical waveguide layer 12b, and the optical waveguide mode is excited, whereby an enhanced photoelectric field D is formed on the optical waveguide layer 12b. Then, the fluorescent dye molecule f in the fluorescent substance F in the enhanced photoelectric field 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 this embodiment, since the fluorescence enhancing fine particles M are present in the fluorescent substance F, the fluorescent Lf is efficiently generated from the fluorescent dye molecules f. Therefore, the sensitivity is higher than that of the normal surface plasmon fluorescence method. In addition, the fluorescence Lf can be detected, and the same effect as in the first embodiment can be obtained.

  Further, the enhanced photoelectric field D generated by the excitation of the optical waveguide mode has a gentler degree of electric field attenuation with the distance from the surface than the enhanced photoelectric field generated by the normal surface plasmon fluorescence method. When a fluorescent substance F having a large diameter encapsulating a plurality of fluorescent dye molecules f is used as a fluorescent label, 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 fluorescent substance F enclosing the fluorescent dye molecule f and one fluorescence enhancing fine particle M is used as a fluorescent label. Then, similarly to the first embodiment, the excitation light Lo is irradiated by the excitation light irradiation optical system 20. 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 combined with the optical waveguide mode of the optical waveguide layer 12b, and the optical waveguide mode is excited, whereby an enhanced photoelectric field D is formed on the optical waveguide layer 12b. Then, the fluorescent dye molecule f in the fluorescent substance F in the enhanced photoelectric field D 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 present embodiment, the presence of the fluorescence enhancing fine particles M in the fluorescent material F efficiently generates the fluorescence Lf from the fluorescent dye molecule f, and detects the emitted light Lp resulting from this enhanced fluorescence. The same effect as that of 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 photoelectric field enhanced by the excitation of the optical waveguide mode is used, the effect of increasing the amount of fluorescence can be obtained as in the fourth embodiment.

  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. May be. 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.

  When the metal coating Mc is provided on the surface of the fluorescent material F, the surface plasmon or localized plasmon generated in the metal layers 12 and 12 ′ of the sensor chip 10 and 10 ′, or generated in the optical waveguide layer 12b of the sensor chip 10 ″. The optical waveguide mode is coupled to the whispering gallery mode of the metal coating Mc of the fluorescent material F, and the fluorescent dye molecule f in the fluorescent material F can be excited more efficiently. Is an electromagnetic wave mode that localizes and circulates on the surface of a microsphere such as the fluorescent substance F having a diameter of about 5300 nm or less used here.

An example of a metal coating method on the fluorescent material F is shown below.
First, a 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 made of metal layers 58a and 59a respectively provided on predetermined regions of the base 51 serving as contact surfaces 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 used as a sensor chip in the detection apparatus and method of any one of the first to third embodiments described above. In addition, according to the substance to be detected, the detection sample cell 50 can be used by appropriately fixing the first binding substance that specifically binds to the substance to be detected to the sensor unit. And a second binding substance that specifically binds to the detected substance, and a third binding substance that competes with the detected substance and specifically binds to the first binding substance; In addition, the fluorescent labeling binding substance may be provided with a fluorescent labeling binding substance that is composed of a fluorescent substance in which the one binding substance is modified, and is disposed in the flow channel upstream of the sensor unit.

  In the detection sample cell according to the present embodiment, since the fluorescent substance F containing the fluorescent dye molecule f and one fluorescence enhancing fine particle M is provided in advance as a fluorescent label, the effects of the detection apparatus and method according to the present invention are as follows. Similarly, light generated due to excitation of a fluorescent label can be easily enhanced. As a result, a stable signal with a good S / N can be detected by detecting the substance to be detected using the detection sample cell according to this embodiment, and the presence and / or amount of the substance to be detected can be determined. It is possible to easily detect with high accuracy.

<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, the labeled secondary antibody adsorption area 57, the first sensor unit 58, and the second sensor unit 59 are sequentially provided on the base 51 from the upstream side of the flow path 52. Is provided. Here, in the labeled secondary antibody adsorption area 57, a secondary antibody (second binding substance) B2 that specifically binds to an antigen as a substance to be detected, and a fluorescent substance whose surface is modified with the secondary antibody B2 A labeled secondary antibody BF (fluorescent label binding substance) composed of F is arranged by physical adsorption. A primary antibody (first binding substance) B1 that specifically binds to an antigen that is a substance to be detected is fixed to the first sensor unit 58. In addition, a primary antibody B0 that specifically binds to the labeled secondary antibody BF without being bound to the antigen A that is the detection target is fixed to the second sensor unit 59. In this example, an example in which two sensor units are provided in the flow path 52 is described, but only one sensor unit may be provided. The fluorescent substance F contains the fluorescent dye molecule f and one fluorescence enhancing fine particle M.

  Gold (Au) films 58a and 59a are formed as metal layers on the first sensor portion 58 and the second sensor portion 59 on the base 51, respectively. A primary antibody B1 is further fixed on the Au film 58a of the first sensor unit 58, and a primary antibody B0 different from the primary antibody B1 is further fixed on the Au film 59a of the second sensor unit 59. ing. The first sensor unit 58 and the second sensor unit 59 have the same configuration except that different primary antibodies are provided. The primary antibody B0 fixed to the second sensor unit 59 does not bind to the antigen A, but directly binds to the labeled secondary antibody BF. As a result, the fluctuation factors relating to the reaction such as the amount and activity of the labeled secondary antibody BF flowing through the flow path, and the fluctuation relating to the enhancement of the photoelectric field such as the excitation light irradiation optical system 20, the gold (Au) films 58a and 59a, the liquid sample S, etc. Factors can be detected and used for calibration. It should be noted that a known amount of labeling substance may be immobilized in advance on the second sensor unit instead of the primary antibody B0. The labeling substance may be the same type as the fluorescent substance F surface-modified with the secondary antibody B2, or may be a fluorescent substance F 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 to the second sensor unit 59 may be appropriately determined depending on the calibration purpose and method.

  The detection sample cell 50A can be used as a sensor chip in any of the detection apparatuses and methods of the first to third 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 photodetector 30 in the accommodating portion 19, and the fluorescence or radiation light from the first sensor portion 58 is detected. After the detection measurement, the second sensor unit 59 is moved to the detection position to detect fluorescence or radiated light from the second sensor unit 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: The blood S (plasma) separated by the membrane filter 55 oozes out 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.
step 4: The plasma S that has oozed into the flow path 52 and the labeled secondary antibody BF disposed in the flow path 52 are mixed, and the antigen A in the plasma S binds to the labeled secondary antibody BF.
step 5: The plasma S gradually flows along the flow path 52 toward the air hole 54b, and the antigen A bound to the labeled secondary antibody BF is mixed with the primary antibody B1 fixed on the first sensor unit 58. The so-called sandwich is formed in which the antigen A is sandwiched between the primary antibody B1 and the labeled secondary antibody BF.
step 6: A part of the labeled secondary antibody BF that has not bound to the antigen A binds to the primary antibody B0 fixed on the second sensor unit 59. Furthermore, even if the labeled secondary antibody BF that has not bound to the antigen A or the primary antibody B0 may remain on the sensor part, the subsequent plasma S plays a role of washing, and is suspended and nonspecific on the plate. The adsorbed labeled secondary antibody BF is washed away.

  In this way, blood So is injected from the injection port 54a, and steps 1 to 6 are performed until a sandwich in which the antigen A is sandwiched between the primary antibody B1 and the labeled secondary antibody BF is formed on the first sensor unit 58. 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 sensor unit 58. Thereafter, the sample cell is moved in the X direction so that the detection signal from the second sensor unit 59 can be detected, and the detection signal from the second sensor unit 59 is detected. The detection signal from the second sensor unit 59 that fixes the primary antibody B0 that binds to the labeled secondary antibody BF is a fluorescence signal that reflects reaction conditions such as the amount and activity of the labeled secondary antibody BF. It is considered that the detection signal is used as a reference, and the detection signal from the first sensor unit 58 is corrected using the detection signal as a reference, so that 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 to the second sensor unit 59, the optical signal from the second sensor unit 59 is similarly received. As a reference, the detection signal from the first sensor unit 58 can be corrected.

  The sample cell for detection according to the present embodiment also includes a fluorescent substance F enclosing the fluorescent dye molecule f and one fluorescence enhancing fine particle M as a fluorescent label in advance. Therefore, also in this embodiment, the same effect as the detection sample cell of the first embodiment can be obtained.

<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 labeled secondary antibody adsorption area 57 ′, the first sensor unit 58 ′, and the second sensor unit 59 ′ are disposed upstream of the flow path 52 on the base 51. It is provided in order from the side. Here, in the labeled secondary antibody adsorption area 57 ′, a secondary antibody C3 (third binding substance) that specifically binds to a primary antibody C1 described later without binding to an antigen as a substance to be detected; A labeled secondary antibody CF (fluorescent label binding substance) comprising the secondary antibody C3 and a fluorescent substance F whose surface is modified is disposed by physical adsorption. A primary antibody C1 (first binding substance) that specifically binds to the antigen to be detected and the secondary antibody C3 is fixed to the first sensor unit 58 ′. In addition, a primary antibody C0 that specifically binds to the labeled secondary antibody CF without being bound to the antigen that is the substance to be detected is fixed to the second sensor unit 59 ′. In this example, an example in which two sensor units are provided in the flow path 52 is described, but only one sensor unit may be provided. The fluorescent substance F contains the fluorescent dye molecule f and one fluorescence enhancing fine particle M.

  Gold (Au) films 58a and 59a are formed as metal layers on the first sensor portion 58 'and the second sensor portion 59' on the base 51, respectively. A primary antibody C1 is further immobilized on the Au film 58a of the first sensor unit 58 ′, and a primary antibody C0 different from the primary antibody C1 is further immobilized on the Au film 59a of the second sensor unit 59 ′. ing. The first sensor portion 58 'and the second sensor portion 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 to the first sensor unit 58 '. The primary antibody C0 fixed to the second sensor portion 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 CF flowing through the flow path, and fluctuation factors relating to the enhancement of the photoelectric field such as the excitation light irradiation optical system 20, the gold (Au) films 58a and 59a, the liquid sample S, etc. Can be detected and used for calibration. In addition, not the primary antibody C0 but a known amount of labeling substance may be immobilized in advance on the second sensor unit. 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 optical system 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 to the second sensor unit 59 'can be appropriately selected depending on the calibration purpose and method.

  Similarly to the detection sample cell 50A described above, the detection sample cell 50B can be used as a sensor chip in any of the detection apparatuses and methods of the first to third embodiments 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: The blood S (plasma) separated by the membrane filter 55 oozes out 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 labeled secondary antibody CF disposed in the flow path 52 are mixed.
step 5: The plasma S gradually flows along the flow path 52 toward the air hole 54b, and the antigen A and the labeled secondary antibody CF compete with each other and are immobilized on the first sensor unit 58 ′. It binds to antibody C1.
Step 6: A part of the labeled secondary antibody CF that did not bind to the primary antibody C1 on the first sensor unit 58 ′ binds to the primary antibody C0 fixed on the second sensor unit 59 ′. . Furthermore, even if the labeled secondary antibody CF that is not bound to the primary antibody C1 or C0 may remain on the sensor part, the subsequent plasma S plays a role of washing, floating on the plate, and nonspecific adsorption Wash away the labeled secondary antibody CF.

  As described above, blood is injected from the injection port, and after Step 1 to Step 6 until the antigen A and the labeled secondary antibody CF are competitively bound to the primary antibody C1 on the sensor unit 58 ′, the first sensor unit By detecting the detection signals from 58 ′ and the second sensor unit 59 ′, the presence and / or concentration of the antigen can be detected. Thereafter, the detection sample cell is moved in the X direction so that the detection signal from the second sensor unit 59 ′ can be detected, the detection signal from the second sensor unit 59 ′ is detected, and this detection signal is used as a reference. By correcting the detection signal from the first sensor unit 58 ′, a more accurate detection result can be obtained.

  In the competition method, as described above, when the concentration of the substance A to be detected is high, the amount of the secondary labeled secondary antibody CF that binds to the primary antibody C1 decreases, and the number of fluorescent substances F on the metal layer decreases. Therefore, the fluorescence intensity becomes small. On the other hand, when the concentration of the substance A to be detected is low, the amount of the labeled secondary antibody CF that binds to the primary antibody C1 increases, and the number of fluorescent substances F on the metal film increases, so that the fluorescence intensity increases. 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 sample cell for detection according to the present embodiment also includes a fluorescent substance F enclosing the fluorescent dye molecule f and one fluorescence enhancing fine particle M as a fluorescent label in advance. Therefore, also in this embodiment, the same effect as the detection sample cell of the first embodiment can be obtained.

(Example of design change of sample cell for detection)
FIG. 15 is a cross-sectional view of a detection sample cell used in a detection method and apparatus that utilizes the enhancement of a photoelectric field by an optical waveguide mode. This detection sample cell has substantially the same configuration as that of the detection sample cell of the first embodiment shown in FIG. 10. However, the optical waveguide layers 58b and 59b are further provided on the metal layers 58a and 59a of the respective sensor units. It differs in that it has. This detection sample cell can be used as a sensor chip in the fourth and fifth embodiments of the detection apparatus and method by appropriately fixing the antibody as described above on the optical waveguide layers 58b and 59b. .

"Detection kit"
The detection kit used in the detection method of the present invention will be described. FIG. 16 is a schematic diagram showing the configuration of the detection kit 60.

  The detection kit 60 according to the present invention includes a detection sample cell 61 and a labeling solution 63 that flows down into the flow path simultaneously with the liquid sample or after the liquid sample flows down. Here, the labeling solution 63 is a labeled secondary antibody BF comprising a secondary antibody B2 (second binding substance) that specifically binds to the antigen A, and a fluorescent substance F modified with the secondary antibody B2. (Fluorescent label binding substance).

  The detection sample cell 61 does not include the labeled secondary antibody adsorption area in which the labeled secondary antibody BF containing the fluorescence enhancing fine particles M is physically adsorbed in the detection sample cell, as described in the second embodiment. Unlike the detection sample cell 50A, the other configuration is substantially the same as that of the detection sample cell 50A.

  In the detection method of the present invention, referring to FIG. 17 for the procedure for performing an assay by the sandwich method as to whether or not an antigen which is a substance to be detected is contained in blood (whole blood) using the detection kit 60 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, blood S (plasma) separated by blood cells by the membrane filter 55 oozes out 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.
step 3: The plasma S gradually flows along the flow path 52 toward the air hole 54b, and the antigen A in the plasma S binds to the primary antibody B1 fixed on the first sensor unit 58.
step 4: The labeling solution 63 containing the labeled secondary antibody BF is injected from the injection port 54a.
step 5: The labeled secondary antibody BF oozes out to 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.
step 6: the labeled secondary antibody BF gradually flows downstream, the labeled secondary antibody BF binds to the antigen A, and the antigen A binds to the primary antibody B1, whereby the antigen A becomes labeled with the primary antibody B1. A so-called sandwich sandwiched between the next antibodies BF is formed. A part of the labeled secondary antibody BF that does not bind to the antigen A binds to the primary antibody B0 fixed on the second sensor unit 59. Furthermore, even if the labeled secondary antibody BF that did not bind to the antigen A or the primary antibody B0 may remain on the sensor part, the subsequent plasma plays a role of washing, floating and non-specific on the plate The adsorbed labeled secondary antibody BF is washed away.

  As described above, the 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 fluorescence enhancement material M is present in the fluorescent substance F in the detection device. By using the effect of increasing the amount of fluorescence and detecting the detection signal from the first sensor unit 58, it is possible to detect the presence and / or concentration of the antigen with high sensitivity. Thereafter, the detection sample cell 61 is moved in the X direction so that the detection signal from the second sensor unit 59 can be detected. Similarly, the fluorescent label binding substance is drawn on the sensor unit, and the second sensor unit 59 The detection signal is detected. The detection signal from the second sensor unit 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 sensor unit 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 to the second sensor unit 59, and a detection signal from the first sensor unit using the fluorescence signal from the second sensor unit 59 as a reference. May be corrected.

An example of a method for modifying a secondary antibody to a fluorescent substance 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 φ150 nm, excitation wavelength 780 nm) prepared in the above-described 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, since the fluorescent substance F containing the fluorescent dye molecule f and one fluorescence enhancing fine particle M is provided in advance as a fluorescent label, the effect is similar to the effect in the detection apparatus and method according to the present invention. In addition, light generated due to excitation of the fluorescent label can be enhanced. As a result, by detecting the substance to be detected using the detection sample kit according to the present embodiment, a stable signal with a good S / N can be detected, and the presence and / or amount of the substance to be detected can be determined. It becomes possible to detect with high accuracy.

(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.

Schematic configuration diagram (SPF) showing a detection apparatus according to the first embodiment of the present invention. Schematic showing the state of fluorescence enhancement effect in the vicinity of the sensor part (Part 1) Schematic showing the state of fluorescence enhancement effect in the vicinity of the sensor part (Part 2) Schematic diagram showing the state of fluorescence enhancement effect in the vicinity of the sensor part (No. 3) Schematic showing the state of fluorescence enhancement effect in the vicinity of the sensor part (part 4) Diagram showing a design change example of the excitation light irradiation optical system 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 The (A) top view and (B) side sectional view showing the sample cell of a 1st embodiment of the present invention. Side sectional view showing a sample cell of a second embodiment of the present invention The figure which shows the assay procedure by the sandwich method using the sample cell of 2nd Embodiment. Side sectional view showing a sample cell of a third embodiment of the present invention The figure which shows the assay procedure by the competition method using the sample cell of 3rd Embodiment. Side sectional view showing an example of sample cell design change The schematic diagram which shows the structure of the kit for detection of one Embodiment of this invention. Diagram showing assay procedure by sandwich method using detection kit 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)
DESCRIPTION OF SYMBOLS 13 Sample holding part 14 Sensor part 16 Translucent material 19 Sensor chip accommodating part 20, 20 'Excitation light irradiation optical system 21 Light source 22 Prism 30 Photodetector 50, 50A, 50B, 61 Sample cell 51 Dielectric plate 52 Flow path 53 Spacer 54 Upper plate 54a Inlet 54b Air hole 55 Membrane filter 57 Labeled secondary antibody adsorption area 58, 59 Sensor unit 60 Detection kit 63 Detection kit labeling solution A Antigen (substance to be detected)
B1, C1 primary antibody (first binding substance)
B2 secondary antibody (second binding substance)
BF, CF labeled secondary antibody (fluorescently labeled binding substance)
C3 secondary antibody (third binding substance)
D Photoelectric field F Fluorescent substance f Fluorescent dye molecule Lo Excitation light Lf Fluorescence Lp Radiation light LS Scattering light M Fluorescence enhancement fine particle Mc Metal coating

Claims (9)

On one surface of the dielectric plate, a sensor chip including a sensor unit having a laminated structure including a metal film adjacent to the dielectric plate is used.
By bringing the sample into contact with the sensor unit, an amount of the fluorescent label binding substance according to the amount of the substance to be detected contained in the sample is bound on the sensor unit,
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 of the fluorescent label-binding substance 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,
As the fluorescent label, using a fluorescent substance comprising a plurality of fluorescent dye molecules and one or more metal fine particles by a light-transmitting material that transmits fluorescence generated from the plurality of fluorescent dye molecules ,
A detection method , wherein plasmons are excited on the metal film by irradiation with the excitation light, and the enhanced photoelectric field is generated by the plasmons . The detection method according to claim 1 , wherein the metal fine particles are metal nanorods. The detection method according to claim 1 or 2 , wherein the metal fine particles have a particle size of 10 to 200 nm. The detection method according to any one of claims 1 to 3 , 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 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 film , thereby causing the other surface opposite to the one surface to the dielectric plate. emitted to the side, the detection method according to claim 1, 3 or, characterized in that detecting the emitted light from the newly induced plasmons. A detection sample cell used as the sensor chip in the detection method according to claims 1 to 5 or,
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 region of the surface ;
A first binding substance immobilized on the sensor unit, the first binding substance for immobilizing the fluorescent label binding substance on the sensor unit;
Any one of the second binding substance that specifically binds to the detected substance and the third binding substance that specifically binds to the first binding substance in competition with the detected substance And a fluorescent label binding substance composed of the fluorescent substance in which the one binding substance is modified, and a fluorescent label binding substance disposed in the flow channel upstream of the sensor unit ,
The detection sample cell, wherein the sensor section has a laminated structure including a metal film adjacent to the dielectric plate. The sample cell for detection according to claim 6 , wherein the laminated structure includes an optical waveguide layer. A detection kit used in the detection method according to claims 1 to 5 or,
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. And a first binding substance fixed on the sensor part, wherein the sensor part is used as the sensor chip having a laminated structure including a metal film adjacent to the dielectric plate. Sample cell for detection, and the liquid A labeling solution that flows into the flow path at the same time as the body sample or after the liquid sample flows, and a second binding substance that specifically binds to the detected substance, and competes with the detected substance. A fluorescently labeled binding substance comprising: a binding substance of any one of the third binding substances that specifically binds to the first binding substance; and the fluorescent substance modified with the one binding substance. A detection kit comprising the labeling solution. The detection kit according to claim 8 , wherein the laminated structure includes an optical waveguide layer.

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