JP2010038624A - Detection method and detector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect fluorescence with high sensitivity by reducing noise light with high efficiency in an evanescent fluorescence detection method using fluorescent coloring matter hard to separate the noise light by a wavelength filter. <P>SOLUTION: In a detection method for detecting an amount of a detection target substance A on the basis of the quantity of light Ld containing signal light Lf caused by the excitation of a fluorescent label F by bonding a fluorescent label bonding substance B<SB>F</SB>, of which the amount corresponds to that of the detection target substance A in a liquid sample S, to a sensor part 14, the amount of the detection target substance A is detected by using a fluorescent label F, which is obtained by including a plurality of fluorescent coloring matter molecules f by a dielectric 16 through which the fluorescence Lf generated from a plurality of the fluorescent coloring matter molecules f is transmitted, as the fluorescent label F, and by detecting the amount of the polarizing component Lc of the light Ld crossing the polarizing direction of exciting light Lo at a right angle. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

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

  Conventionally, a fluorescence detection method has been widely used as a highly sensitive and easy measurement method in biomeasurement and the like. In this fluorescence detection method, the presence of a substance to be detected is detected by irradiating a sample considered to contain a substance to be detected that is excited by light of a specific wavelength to emit fluorescence and then detecting the fluorescence at that time. It is a method to confirm. In addition, when the substance to be detected is not a fluorescent substance, this binding, that is, by detecting the fluorescence in the same manner as described above, by contacting a substance labeled with a fluorescent dye and specifically binding to the substance to be detected, and then detecting fluorescence. The existence of a substance to be detected is also widely confirmed.

  In biomeasurement, for example, in order to detect an antigen that is a detected substance contained in a sample, a primary antibody that specifically binds to the detected substance is fixed on the substrate, and the sample is supplied onto the substrate. By specifically binding the detected substance to the primary antibody, and then adding a secondary antibody to which a fluorescent label is attached that specifically binds to the detected substance and binding the detected substance to the primary antibody. A sandwich method in which a so-called sandwich of primary antibody-substance to be detected-secondary antibody is formed to detect fluorescence from a fluorescent label attached to the secondary antibody, or primary antibody in competition with the target substance A competitive method in which a fluorescently labeled competitive secondary antibody that specifically binds to a primary antibody is competitively bound to a substance to be detected, and fluorescence from the competitive secondary antibody bound to the primary antibody is detected. An assay is made.

  At this time, the fluorescence is excited by evanescent light in order to detect the fluorescence from only the secondary antibody bound to the primary antibody immobilized on the substrate via the substance to be detected or the directly bound competitive secondary antibody. An evanescent fluorescence method has been proposed. In the evanescent and fluorescent methods, excitation light that is totally reflected on the substrate surface is incident from the back surface of the substrate, and the fluorescence is excited by an evanescent wave that exudes to the substrate surface to detect the fluorescence.

  In order to improve sensitivity in the evanescent fluorescence method, Patent Document 1, Non-Patent Document 1, and the like have proposed methods utilizing the effect of electric field enhancement by plasmon resonance. In the surface plasmon enhanced fluorescence method, in order to generate plasmon resonance, a metal layer is provided on the substrate, and excitation light is incident on the interface between the substrate and the metal layer from the back surface of the substrate at an angle greater than the total reflection angle. The surface plasmon is generated in the metal layer by the irradiation of the excitation light, and the S / N is improved by increasing the fluorescence signal by the electric field enhancing action.

  Similarly, in the evanescent fluorescence method, a method using the electric field enhancement effect by the waveguide mode is proposed in Non-Patent Document 2 as having the effect of enhancing the electric field of the sensor unit. 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 substrate, and an angle greater than the total reflection angle from the back surface of the substrate. Excitation light is incident on the optical waveguide layer, an optical waveguide mode is generated in the optical waveguide layer by irradiation of the excitation light, and the fluorescence signal is enhanced by the electric field enhancement effect.

  In Patent Document 2 and Non-Patent Document 3, instead of detecting fluorescence from a fluorescent label excited in an electric field enhanced by surface plasmon, the fluorescence newly induces surface plasmon in the metal layer. A method has been proposed in which the generated radiant light (SPCE: Surface Plasmon-Coupled Emission) is extracted from the prism side.

However, in the signal enhancement method described above, simultaneously with the fluorescent signal to be detected, noise light such as scattered light of excitation light caused by dust or scratches on the sensor part and reflected light of excitation light on the sensor part surface is also enhanced. Will be detected. Therefore, reduction of noise light is an essential issue for further enhancement of sensitivity, particularly improvement of detection limit.
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 "Methods of fluorescence spectroscopy and imaging" pp.14-24, Academic Publishing Center (2006)

  A method using a wavelength filter is well known for separating fluorescence signal light and noise light, but this method is effective when a fluorescent dye having a large Stokes shift is used. Since samples that cause (absorption) are targeted, fluorescent dyes that emit fluorescence in a long wavelength range with a small Stokes shift are often used, and separation is difficult.

  Also known is a method of separating fluorescent signal light and noise light using a polarizing filter. However, since the polarization of the fluorescence signal has a substantially positive correlation with the polarization of the excitation light (Non-Patent Document 4), this method causes the rotation of the fluorescence molecules due to Brownian motion and the energy transfer between adjacent fluorescence molecules. It can only be applied to cases where such a resolution occurs. In the fluorescence detection method using near-field light such as the above evanescent fluorescence method, since the fluorescent dye is fixed to the sensing surface, rotation due to Brownian motion is difficult to occur, and when the detection limit is further improved, the target substance itself is not detected. Therefore, the fluorescent molecules become sparse and energy transfer between the fluorescent molecules hardly occurs. In Non-Patent Document 3, it has been confirmed that in fluorescence detection using surface plasmon resonance, the polarization of the fluorescence signal is strongly biased to the polarization of excitation light, that is, p-polarization.

  The present invention has been made in view of the above circumstances, and in a fluorescence detection method using near-field light using a fluorescent dye having a small Stokes shift and difficult to separate noise light by a wavelength filter, noise light is reduced with high efficiency. It is an object of the present invention to provide a fluorescence detection method and apparatus capable of highly sensitive detection.

The detection method of the present invention prepares a sensor chip comprising a dielectric plate and a sensor unit in which at least a metal layer is provided on one surface of the dielectric plate,
By bringing a liquid 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 in the liquid sample is bound on the sensor unit,
By irradiating the sensor unit with linearly polarized excitation light, an enhanced photoelectric field is generated on the sensor unit, the fluorescence label of the fluorescent label binding substance is excited in the enhanced photoelectric field, and the fluorescence label In a detection method for detecting the amount of the substance to be detected based on the amount of light including signal light generated due to excitation,
As the fluorescent label, using a fluorescent substance comprising a plurality of fluorescent dye molecules included by a dielectric that transmits fluorescence generated from the plurality of fluorescent dye molecules,
The amount of the substance to be detected is detected by detecting the amount of the polarization component of the light orthogonal to the polarization direction of the excitation light.

  Here, the “fluorescent label binding substance” is a fluorescently labeled binding substance that binds on the sensor unit by an amount corresponding to the amount of the target substance. For example, when performing an assay by the sandwich method, When the assay is performed by a competitive method, the binding substance specifically binds to the substance and the fluorescent label.

  In the present specification, the fluorescent dye molecules include organic dyes and inorganic dyes such as quantum dots. The excitation light incident on the sensor unit may be excitation light that is linearly polarized immediately after being emitted from the excitation light irradiation optical system, and the polarization state immediately before entering the sensor unit may be linear. It may be excitation light that is polarized light.

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

Here, the “photoelectric field” refers to an electric field caused by evanescent light or near-field light generated by irradiation with excitation light.
Further, “to generate an enhanced photoelectric field” means that an enhanced photoelectric field is formed by enhancing the photoelectric field, and a method for enhancing the photoelectric field is based on plasmon resonance. It may be due to excitation of an optical waveguide mode.

Further, as a method of “detecting the amount of the substance to be detected based on the amount of signal light generated due to the excitation of the fluorescent label of the fluorescent label-binding substance”, a method for directly detecting fluorescence from the fluorescent label Or may be detected indirectly.
Specifically, the following modes (1) to (4) are mentioned.

(1) The plasmon is excited on the metal layer by irradiation of the excitation light, the enhanced photoelectric field is generated by the plasmon, and the fluorescence label is generated by the excitation as the light generated due to the excitation of the fluorescence label. The amount of the substance to be detected is detected by detecting the fluorescence generated from.
(2) The plasmon is excited on the metal layer by irradiation of the excitation light, the enhanced photoelectric field is generated by the plasmon, and the fluorescence label is generated by the excitation as the light generated due to the excitation of the fluorescence label. The amount of the substance to be detected is detected by detecting the radiated light emitted from the other surface of the dielectric plate by newly inducing the plasmon in the metal layer by the fluorescence generated from.
(3) The sensor chip having an optical waveguide layer on the metal layer is used to excite an optical waveguide mode in the optical waveguide layer by irradiation of the excitation light, and the enhanced photoelectric by the optical waveguide mode. An amount of the substance to be detected is detected by generating a field and detecting fluorescence generated from the fluorescent label by the excitation as the light generated due to the excitation of the fluorescent label.
(4) The sensor chip is provided with an optical waveguide layer on the metal layer, the optical waveguide mode is excited in the optical waveguide layer by the irradiation of the excitation light, and the enhanced photoelectric is generated by the optical waveguide mode. As the light generated due to excitation of the fluorescent label, a fluorescence generated from the fluorescent label due to the excitation newly induces a plasmon in the metal layer, thereby generating the other surface of the dielectric plate. The amount of the substance to be detected is detected by detecting the emitted light emitted from the sensor.

  In (1) and (2), the metal layer is a metal film, p-polarized 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, and the surface of the metal film is exposed. It may be one that excites plasmons, and the metal layer has a metal fine structure having irregularities with a period smaller than the wavelength of excitation light on the surface, or a plurality of metal nanorods having a size smaller than the wavelength of excitation light And the local plasmon may be excited in the metal microstructure or the metal nanorod by irradiation with excitation light.

The detection device of the present invention is a detection device used in the detection method of the present invention described above,
An accommodating portion for accommodating the sensor chip;
An excitation light irradiation optical system including an excitation light source that irradiates the excitation light to the sensor unit;
In the enhanced photoelectric field generated on the sensor unit by the irradiation of the excitation light, a polarization component orthogonal to the polarization direction of the excitation light is separated from light including signal light generated due to excitation of the fluorescent label. Polarized light separating means to be taken out,
And a light detecting means for detecting the amount of the separated polarization component.

  The detection apparatus of the present invention further includes a photodetector that detects an intensity of reflected light of the excitation light, a polarization adjustment element that controls a polarization state of the excitation light emitted from the excitation light source, and the detected It is preferable to have control means for operating the polarization adjusting element so that the intensity of the p-polarized component of the excitation light incident on the sensor unit is maximized according to the intensity.

  According to the detection method and apparatus of the present invention, a plurality of fluorescent dye molecules are used as a fluorescent label of a fluorescent label binding substance that is bound on the sensor unit according to the amount of the target substance contained in the sample. By using a fluorescent substance that is included by a dielectric that transmits fluorescence from the dye molecule, a fluorescence signal having a polarization component orthogonal to the polarization direction of the excitation light can be generated, so noise light included in the detection light And the fluorescence signal are polarized and separated, and noise light can be reduced with high efficiency.

  According to the present invention, in the fluorescence detection method using near-field light, which has been difficult to separate noise light using polarized light, polarization of noise light can be separated, so that the Stokes shift is small and the wavelength filter is reduced. Even when using fluorescent dyes where it is difficult to separate noise light, it is possible to reduce noise light with high efficiency, detect stable signals with good S / N, and accurately detect the presence and / or amount of the substance to be detected. Can be detected well.

"Detection method and apparatus"
The detection method and apparatus of the present invention improves the S / N ratio by separating noise light and signal light by polarization separation in fluorescence detection using near-field light such as evanescent fluorescence, and thus more sensitive fluorescence. The detection and the detection limit are improved.

  As described in the section of “Background Technology”, in fluorescence detection using near-field light such as evanescent fluorescence, the fluorescent label is fixed to the sensing surface. Rotation of the fluorescent dye molecules due to Brownian motion is unlikely to occur, and when the detection limit is further improved, the substance to be detected itself is reduced, so that energy transfer between fluorescent molecules is also unlikely to occur. Therefore, since the polarization of the fluorescence signal of the excitation light has a substantially positive correlation with the polarization of the excitation light, it has not been possible to improve the S / N ratio by separating noise light and signal light by polarization separation.

  The present inventor succeeded in separating noise light with high efficiency by polarization separation by using a fluorescent label having a function of depolarizing polarization in a fluorescence detection method using near-field light. Hereinafter, embodiments of the present invention will be described with reference to the drawings. For convenience of explanation in each figure, the dimensions of each part are different from the actual ones.

The detection method of the present invention is a fluorescence detection method using near-field light such as evanescent fluorescence. For example, as shown in FIG. 1, a dielectric plate 11 and at least a metal layer 12 provided on one surface of the plate 11 are provided. The amount corresponding to the amount of the substance A to be detected contained in the liquid sample S on the sensor unit 14 by bringing the liquid sample S into contact with the sensor unit 14 using the sensor chip 10 including the sensor unit 14 included. In this enhanced photoelectric field D, the fluorescence label binding substance _BF is bound and the sensor unit 14 is irradiated with linearly polarized excitation light Lo, thereby generating an enhanced photoelectric field D on the sensor unit 14. exciting the fluorescent labels F of the fluorescent label binding substance B _F, based on the amount of light Ld including the signal light Lf generated due to the excitation of the fluorescent labels F, a detection method for detecting the amount of the detection target substance a Fluorescent As the light F, a fluorescent material F including a plurality of fluorescent dye molecules f including a dielectric that transmits the fluorescence from the fluorescent dye molecules f is used, and the polarization of the light Ld is orthogonal to the polarization direction of the excitation light Lo. The amount of the substance A to be detected is detected by detecting the amount of the component Lc.

Incidentally, the sensor unit 14, after binding the fluorescent label binding substance B _F, in the liquid sample S, a fluorescent label binding substance B _F the sensor portion staying or non-specific adsorption without binding to the sensor unit 14 14 is removed from above, and then the amount of the substance A to be detected is preferably detected. Here, binding to the sensor unit 14 means specifically binding to the sensor unit 14, specifically, the substance B _{1 to} which the fluorescent label binding substance _BF specifically binds. It is provided in the sensor part 14 as a fixed layer, and the form couple | bonded with this fixed layer is mentioned.

  Detection devices 1 to 6 according to an embodiment of the present invention described below are for carrying out the above-described detection method. The detection devices 1 to 6 include an accommodating portion 13 that accommodates the sensor chip 10 and excitation light Lo on the sensor portion 14. Light including signal light Lf generated due to excitation of the fluorescent label f in the excitation light irradiation optical system 20 including the excitation light source 21 to be irradiated and the enhanced photoelectric field D generated on the sensor unit 14 by irradiation of the excitation light Lo. A polarization separation unit 40 that separates and extracts a polarization component Lc orthogonal to the polarization direction of the excitation light Lo from Ld and a light detection unit 30 that detects the amount of the separated polarization component Lc are provided.

If it is preferable excitation light Lo incident on the sensor unit 14 is p-polarized light, a photodetector 50 for detecting the intensity of the reflected light L _R of the excitation light Lo, the excitation light emitted from excitation light source 21 The polarization adjusting element 23 that controls the polarization state and the polarization adjusting element 23 so that the intensity of the p-polarized component of the excitation light Lo incident on the sensor unit 14 is maximized according to the intensity detected by the photodetector 50. It is good also as a structure provided with the control means 60 which operates (refer FIG. 4).

As shown in FIG. 2, the fluorescent label F includes a plurality of fluorescent dye molecules f, and a dielectric 16 that transmits the fluorescent Lf excited by the evanescent light L _E (photoelectric field D) from the plurality of fluorescent dye molecules f. It is a fluorescent substance comprising. If a fluorescent substance F containing a plurality of fluorescent dye molecules f is used, the amount of fluorescent Lf can be increased, and the fluorescent dye molecules f are encapsulated by the dielectric 16 that transmits the fluorescent Lf. Metal quenching that occurs when the molecule f approaches the metal layer 12 can be prevented, and the polarization of the fluorescence Lf can be randomized.

The quenching that occurs when the fluorescent dye molecules are too close to the metal layer is associated with the energy transfer to the metal, and the degree of this energy transfer is the cube of the distance if the metal is a plane with a semi-infinite thickness. When the metal is an infinitely thin flat plate, it is inversely proportional to the fourth power of the distance, and when the metal is a fine particle, it is decreased inversely proportional to the sixth power of the distance. Therefore, it is desirable to ensure the distance between the metal layer 12 and the fluorescent dye molecule f at least several nm or more, more preferably 10 nm or more.
Conventionally, as a method for preventing metal quenching, a self-assembled film (SAM) is formed on the metal layer 12, and a carboxymethyl dextran (CMD) film is formed to increase the distance between the metal film and the fluorescent dye molecule. It has been known. However, providing a film for preventing such metal quenching on the metal film complicates the sensor chip manufacturing process and is very laborious. On the other hand, if the fluorescent substance F is used, the metal layer 12 can be kept at a predetermined distance or more, so that the metal can be effectively effectively provided by a very simple method without providing a film for preventing metal quenching as described above. Quenching can be prevented.

Further, since the fluorescent label F is fixed on the surface of the sensor unit 14, it does not easily rotate itself, but as shown in FIG. 2, the evanescent wave L generated on the surface of the sensor unit 14 by the excitation light Lo as shown in FIG. 2. When _{E (optical} field D) is incident on the fluorescent labels F, it is excited by the evanescent wave L _E and its optical field D, enhanced fluorescence Lf is reflected and multiple scattering inside the dielectric 16 of the fluorescent labels F Is done. Further, as shown in the figure, since there are a plurality of fluorescent labels F, energy transfer between the fluorescent dye molecules f is likely to occur. Accordingly, the polarization is randomized by reflection and multiple scattering in the fluorescent label F, and energy transfer between the fluorescent dye molecules f, and the polarization of the excitation light Lo is eliminated.

  This depolarization effect increases as the density of the fluorescent dye molecules f in the fluorescent label F increases, the anisotropy of the fluorescent dye molecules f increases, and the birefringence of the dielectric 16 increases. The higher the density of the fluorescent dye molecule f, the larger the interaction of the fluorescent dye molecule and the effect of multiple scattering, and the stronger the anisotropy of the fluorescent dye molecule f, the more the direction in which the dipole in the fluorescent dye molecule is induced. Since they are present in close proximity to each other, the polarization is more randomized. If the birefringence of the dielectric 16 itself is high, the polarization is disturbed by the birefringence and the polarization is eliminated. As described above, by using the fluorescent label F as a label, the polarization of the fluorescence Lf that becomes signal light can be randomized.

Not only polarization of the signal light, the polarization of the evanescent light L _E incident on the fluorescent labels F are also randomized. However, the noise light in which is included in front of the light Ld where the signal light and noise light is separated, the fluorescence-labeled ratio of F in incident on the evanescent light L _E is small, scratches and the surface of the sensor portion 14, dust suspended with or near the surface on the surface, also the scattered light component of the evanescent light L _E caused by the substance B ₁ such that the detection target substance a specifically bind fixed on the sensor section 14 is almost Therefore, it is considered that most of the polarization state of the noise light is the same as that of the excitation light Lo.

  Accordingly, the noise light intensity included in the component Lc of the light Ld orthogonal to the polarization direction of the excitation light Lo extracted from the light Ld via the polarization separation means 40 (hereinafter referred to as detection light Lc) is the noise light intensity of the light Ld. For example, it is reduced to about 1/50 (see the examples described later).

  The intensity of the signal light Lf detected through the polarization separation means 40 becomes small because it becomes only a component orthogonal to the polarization direction of the excitation light Lo as in the case of noise light, but unlike the noise light, most of the polarization of the signal light is reduced. Is randomized, for example, about one third of the signal light intensity of the light Ld (see the examples described later). Even if the signal light intensity in the detection light Lc is 1/3, if the noise light intensity is 1/50, the S / N is improved by one digit. Therefore, high sensitivity detection with high S / N and improvement in detection limit can be realized.

As described above, the fluorescent labels F is a plurality of fluorescent dye molecules f, and encompassed by a dielectric 16 that transmits fluorescence Lf to be excited from the fluorescent dye molecules f the plurality of the evanescent light L _{E (optical} field D) (FIG. 2).

The dielectric 16 can be encapsulated fluorescent dye molecules f, and is not particularly limited as long as it can transmit the evanescent light L _{E (optical} field D) and fluorescence Lf, and the like polystyrene or SiO ₂ is. From the viewpoint of polarization randomization, the dielectric 16 itself preferably has birefringence. Polystyrene is known as a polymer material having a large birefringence. Further, in order to increase the birefringence, the dielectric 16 includes a highly anisotropic inorganic substance or organic substance such as a needle-like crystal (strontium carbonate, etc.) in the matrix such as polystyrene or SiO ₂ described above. You may use what was made to do. It is preferable that the anisotropic substance does not cause a scattered light that greatly reduces the S / N, and therefore it is preferable that the anisotropic substance has a size and density that does not generate the scattered light.

  The fluorescent dye molecule f is not particularly limited, and includes organic dyes and inorganic dyes such as quantum dots. Since the sample that generates autofluorescence (absorption) is targeted for biomeasurement use, it is preferable to use a fluorescent dye / fluorescent dye molecule that emits fluorescence in a long wavelength range with a small Stokes shift. Examples of fluorescent dyes that can be excited by infrared light include Hayashibara Biochemical Research Institute NK-529 (excitation wavelength: 640 nm), NK-1836 (excitation wavelength: 650 nm), NK-2014 (excitation wavelength: 780 nm), DY- manufactured by Dyomics. 785-L (excitation wavelength to 760 nm), DY-785-L (excitation wavelength to 770 nm), and the like.

  The particle size of the fluorescent label F is not particularly limited, but is preferably 70 nm to 900 nm, more preferably 130 nm to 500 nm from the viewpoint of the amount of fluorescence and the closest packing density on the sensor part and the disturbance of the surface plasmon. Particularly preferred. In the present specification, the particle size of the fluorescent label F is defined by the average length of the maximum width and the minimum width in the case of a substantially spherical particle, and in the case of a non-spherical particle. To do.

  The fluorescent label (fluorescent substance) F may be prepared by impregnating dielectric particles such as polystyrene with the above-described fluorescent dye, and commercially available fluorescent beads are used as long as they can be excited at a desired wavelength. May be. For example, when using an LD light source (product number DL-3147-160F, DL-3357-165, wavelength 655 nm) manufactured by StockerYale, etc., fluorescent beads manufactured by Bangs Laboratories (product number FC03F / 8632), diameter 510 nm, excitation wavelength 660 nm, Fluorescence wavelength 690 nm), Molecular Probes fluorescent beads (product number F8807, diameter 200 nm, excitation wavelength 660 nm, fluorescence wavelength 680 nm), StockerYale LD light source (product numbers DL-3148-023, DL-3038-011, wavelength 635 nm), etc. Can be used with commercially available fluorescent beads such as Molecular Probes (Part No. F8816, diameter 1000 nm, excitation wavelength 625 nm, fluorescence wavelength 645 nm). wear. Since the particle surface of the fluorescent beads exemplified above is modified with a carboxyl group, the secondary antibody can be bound by amine coupling.

The fluorescently labeled binding substance _BF is a fluorescently labeled binding substance that binds on the sensor unit 14 by an amount corresponding to the amount of the target substance A. As shown in FIG. 1, when performing an assay by the sandwich method, Is composed of a binding substance that specifically binds to the substance to be detected and the above-mentioned fluorescent substance F. When assaying by the competitive method described later, the binding substance that competes with the substance to be detected and fluorescence It consists of the substance F. More specifically, when a sensor chip 10 is used in which the first binding substance B ₁ that specifically binds to the substance A to be detected is fixed as a fixed layer to the sensor unit 14, the sandwich method uses a fluorescence. As the label binding substance _BF , a substance composed of a second binding substance B ₂ that specifically binds to the substance A to be detected and a fluorescent substance F modified with this binding substance B ₂ is used. As the label binding substance, a substance composed of a third binding substance that specifically binds to the first binding substance in competition with the substance to be detected A and a fluorescent substance F in which the binding substance is modified is used. When the substance A to be detected is an antigen, a so-called primary antibody may be used as the first binding substance B _{1 and} a so-called labeled secondary antibody may be used as the fluorescent label binding substance.

  The detection method of the present invention generates an enhanced photoelectric field on the sensor unit by irradiation with excitation light, and detects light generated by excitation of a fluorescent label in the enhanced photoelectric field. The method of enhancing the power may be by surface plasmon resonance or localized plasmon resonance, or by excitation of an optical waveguide mode. Further, the fluorescence generated from the fluorescent label may be detected directly or indirectly. Specific embodiments will be described in the following embodiments.

<First Embodiment>
A detection method and apparatus according to the first embodiment will be described with reference to FIG. FIG. 1 is an overall view showing a schematic configuration of a detection apparatus according to the first embodiment. The detection method and apparatus of the present embodiment is a fluorescence detection method and apparatus that enhances a photoelectric field by surface plasmon resonance and detects fluorescence excited in the enhanced photoelectric field.

In the present fluorescence detection method of the present embodiment, a sensor chip 10 including a sensor part 14 in which a metal film is provided as at least a metal layer 12 in a predetermined region on one surface of the dielectric plate 11 is used. Is provided with a sample holding part (accommodating part) 13 for holding a liquid sample S, and uses a box-shaped sample cell with the sensor chip 10 and an accommodating part 13 capable of holding a liquid sample.
The metal film 12 can be formed by a known vapor deposition method by forming a mask having an opening in a predetermined region on one surface of the plate 11. The thickness of the metal film 12 is desirably 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. For example, when laser light having a center wavelength of 655 nm is used as excitation light and a gold (Au) film is used as the metal film, the thickness of the metal film is preferably 50 nm ± 20 nm. More preferably, it is 47 nm ± 10 nm. The metal film is preferably composed mainly of at least one metal selected from the group consisting of Au, Ag, Cu, Al, Pt, Ni, Ti, and alloys thereof. Here, the “main component” is defined as a component having a content of 90% by mass or more.

  The constituent material of the dielectric plate 11 is not particularly limited as long as it has translucency with respect to the excitation light Lo, and glass or the like is preferable. In this specification, having translucency is defined as having a transmittance of 80% or more for light of a predetermined wavelength.

  The fluorescence detection device 1 is configured to receive a linearly polarized excitation light Lo at the interface between a housing part 13 that houses the sensor chip 10 and the dielectric plate 11 and the metal film 12 of the sensor chip 10 housed in the housing part 13. The excitation light irradiation optical system 20 that is incident from the other surface side opposite to the metal film formation surface of the sensor chip 10 at the above incident angle, and the enhanced photoelectric field generated on the sensor unit 12 by irradiation of the excitation light Lo. The light for detecting the polarization component Lc, and the polarization separation means 40 for separating and extracting the polarization component Lc orthogonal to the polarization direction of the excitation light Lo from the light Ld including the signal light generated due to the excitation of the fluorescent label F in D Detection means 30.

    In the present embodiment, the excitation light Lo is incident on the interface between the dielectric plate 11 and the metal film 12 with p-polarization so as to induce surface plasmons. The excitation light irradiation optical system 20 includes an excitation light source 21 composed of a semiconductor laser (LD) or the like that outputs excitation light Lo, a polarization adjusting element 23 that makes the excitation light Lo linearly polarized light (p-polarized light), and a dielectric plate 11. And a prism 22 arranged so that one surface is in contact therewith. 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 polarization adjusting element 23 is a polarizer that extracts only p-polarized light. The constituent material of the prism 22 is not particularly limited, and glass, polymethyl methacrylate (PMMA), polycarbonate (PC), amorphous polyolefin (APO) containing cycloolefin, and the like are preferable.
In the present embodiment, the prism 22 and the dielectric plate 11 are in contact via a refractive index matching oil. The excitation light source 21 is arranged so that the excitation light Lo is incident on the sample contact surface 10a 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. ing. Further, a light guide member may be disposed between the light source 21 and the prism 22 as necessary. Further, the prism 22 and the dielectric plate 11 may be integrally formed.

  The accommodating portion 13 is configured such that when the sensor chip 10 is accommodated, the sensor portion 14 of the sensor chip is disposed on the prism 22 so that the light detection means 30 can detect fluorescence. As a constituent material of the accommodating part 13, if it has translucency with respect to excitation light Lo and signal light (fluorescence) Lf, it will not restrict | limit, Glass etc. are mentioned. The cell (sensor chip 10) in the accommodating part 13 is replaceable.

The fluorescence (signal light) Lf is detected by the light detection means 30 from the surface side (upper side in the drawing) of the housing portion 13 opposite to the prism 22. The detection light Lc detected by the light detection means 30 is obtained by extracting the polarization component orthogonal to the polarization direction of the excitation light Lo, that is, the s-polarization component, from the light Ld detected from the accommodating portion 13 by the polarization separation means 40. is there. The polarization separation unit 40 is a polarization filter arranged in a crossed Nicols state with the polarization adjustment unit 23. As the light detection means 30, a light detector such as a CCD, PD (photodiode), photomultiplier, c-MOS can be used as appropriate. For example, LAS-1000 plus (trade name) manufactured by FUJIFILM Corporation. Can be suitably used. Further, the light detection means 30 can be used in combination with a spectral means such as a filter other than a polarizing filter or a spectroscope depending on detection conditions.

Below, the fluorescence detection method of this embodiment using the fluorescence detection apparatus 1 is demonstrated.
Here, as an example, a case where the antigen A is detected as the substance to be measured contained in the sample S will be described.

A sensor chip 10 is prepared in which a primary antibody B ₁ is modified as a fixed layer on the metal film 12 of the sensor chip 10 as a first binding substance that specifically binds to the antigen A.
First, the liquid sample S to be inspected is caused to flow through the storage unit 13, and the sample S is brought into contact with the metal film 12 of the sensor chip 10. Then Similarly flow a solution containing the second binding agent is a secondary antibody B ₂ and the fluorescent labels F consisting of a fluorescent label binding substance (labeled secondary antibody) B _F to bind to antigen A specifically. In this case, as the secondary antibody B ₂ of the primary antibody B ₁ and the fluorescent label binding substance B _F is the surface modified metal film 12, those that bind to different sites from each other with respect to antigen A to be detected Use. The fluorescent label F is a fluorescent substance F that encloses fluorescent dye molecules f in dielectric particles.

If antigen A is present in sample S, antigen A specifically binds to primary antibody B ₁ , and further, secondary antibody B ₂ of fluorescently labeled binding substance _BF binds to antigen A, and primary antibody B1- antigen A-2 primary antibody _{B 2} conjugate (hereinafter, referred to as a sandwich conjugate.) are formed.

Thereafter, in order to separate the sandwich conjugate from the unreacted fluorescently labeled binding substance _BF , a buffer is passed to eliminate the unreacted fluorescently labeled binding substance.

The timing of labeling the target substance (antigen A) is not particularly limited, and before the target substance (antigen A) is bound to the first binding substance (primary antibody B ₁ ), the sample is labeled with a fluorescent label F. May be added.

Thereafter, 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 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 waves ooze and surface plasmons are excited in the metal film 12 by the evanescent waves. Optical field that occurs on the metal layer 12 by the incident excitation light Lo (electric field caused by the evanescent wave L _E) is enhanced by the surface plasmon, optical field enhancement region D is formed on the metal layer. In the electric field enhancement region D, particularly in the vicinity of the surface of the metal film 12, the fluorescent label F is excited (substantially, the fluorescent dye molecule f in the fluorescent material is excited) to generate fluorescence Lf. The fluorescence is enhanced by the effect of enhancement of the photoelectric field by the surface plasmon.

  By detecting this fluorescence Lf by the light detection means 30, it is possible to detect the presence and / or amount of the target substance bound to the fluorescent label binding substance. The light Ld present on the surface opposite to the prism 22 of the accommodating portion 13 is not only the fluorescence Lf that is signal light, but also the scattered light caused by scratches and dust on the sensor portion 14, the primary antibody B1, etc. Such noise light is included. On the other hand, in the present embodiment, as described above, since the polarization state is different between the signal light and the noise light, the noise light is increased by detecting the detection light Lc by the light detection means 30 via the polarization separation means 40. It is possible to detect fluorescence with high S / N which is cut efficiently.

Further, the step of forming a sandwich bonded product on the sensor unit 14 and separating the sandwich bonded product from the unreacted fluorescent label binding substance _BF is performed before the sensor chip 10 is set in the housing unit 13 of the detection device 1. Or may be performed after setting.

According to the detection method and apparatus of the above-described embodiment, a plurality of fluorescent lights are used as the fluorescent label of the fluorescent label binding substance _BF that is bound on the sensor unit 14 according to the amount of the target substance A contained in the sample S. By using the fluorescent substance F that includes the dye molecule f with a dielectric that transmits the fluorescence from the fluorescent dye molecule f, a fluorescence signal Lf having a polarization component orthogonal to the polarization direction of the excitation light Lo can be generated. Therefore, the noise light and the fluorescence signal included in the light Ld can be polarized and separated, and the noise light can be reduced with high efficiency.

  According to the present invention, in the fluorescence detection method using near-field light, which has been difficult to separate noise light using polarized light, polarization of noise light can be separated, so that the Stokes shift is small and the wavelength filter is reduced. Even when a fluorescent dye f that is difficult to separate by noise light is used, it is possible to reduce noise light with high efficiency and detect a stable signal with good S / N, and the presence and / or amount of the substance A to be detected. Can be detected with high accuracy.

<Design change example of the first embodiment>
In each of the embodiments described above, parallel light incident on the interface at a predetermined angle θ is incident as the excitation light Lo, but the excitation light is centered on the angle θ as schematically shown in FIG. Alternatively, a fan beam (focused light) having an angular width Δθ 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. 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.

  When the dielectric prism 22 or the dielectric plate 11 is made of, for example, a resin having a large birefringence, the excitation light Lo is incident on the dielectric prism 22 and reaches the metal film 20. In addition, there is a case where a phase difference δ is generated between the p-polarized light and the s-polarized light, for example, changing to a clockwise elliptically polarized state.

  At this time, the p-polarized component of the excitation light Li can contribute to surface plasmon excitation, but the s-polarized component generated by the rotation of the polarization state cannot excite the surface plasmon. That is, there arises a problem that the amount of excitation light that can excite surface plasmons is substantially reduced.

In such a case, as the detection device 2 shown in FIG. 4, a light detector 50 for detecting the intensity of the reflected light L _R of the excitation light Lo, depending on the detected intensity in the light detector 50, Excitation light that reaches the metal film 12 is configured by including a control unit 60 that operates the polarization adjusting element 23 so that the intensity of the p-polarized component of the excitation light Lo incident on the sensor unit 14 is maximized. Is changed to linearly polarized light having only p-polarized light, and a decrease in the amount of excitation light can be prevented.

  In the detection device 2, the excitation light Lo is incident on the dielectric prism 22 after the polarization state of the excitation light Lo is previously changed to a counterclockwise elliptical polarization state having a phase difference opposite to the above phase difference. For example, in the case where a right-handed elliptical polarization state having a phase difference δ is caused in the dielectric prism 22 as described above due to the influence of birefringence by the dielectric prism 22, before entering the dielectric prism 22. The polarization state of the excitation light Lo is adjusted in advance so that it becomes a counterclockwise elliptical polarization state with a phase difference of −δ. Thus, by canceling out the phase difference δ generated in the dielectric prism 6 with the phase difference −δ given in advance, the polarization state when the excitation light Lo reaches the interface 20a is a straight line having only p-polarized light. By setting the polarization state, the energy of the excitation light can be effectively used for the excitation of the surface plasmon.

  In this case, the polarization adjusting element 23 is composed of, for example, a λ / 2 plate and a λ / 4 plate, and the inclination of the polarization axis is adjusted by the λ / 2 plate and the spread state of the polarization state is adjusted by the λ / 4 plate. To do. The polarization adjusting element 23 is not limited to these, and a Pockels cell or the like may be used.

Polarizers H is, if it is possible to extract a specific polarized component of the reflected light L _R, can be used particularly restricted without polarizing prism and the polarization plate or the like.

  The automatic polarization adjusting mechanism 60 is incident on the dielectric prism 22 so that the intensity of the p-polarized light of the excitation light Lo is maximized when incident on the metal film 12 according to the detection signal from the photodetector 50. The polarization adjusting element 23 is operated in order to adjust the polarization state of the excitation light Lo before starting. The automatic polarization adjusting mechanism 60 includes a personal computer (PC) or the like.

Then, the excitation light Lo is incident so that the total reflection condition is satisfied at the interface between the dielectric plate 11 and the metal film 12, and the reflection angle at the interface is changed so as to excite the surface plasmon in the metal film 12, monitored by the photodetector 50 reflected light L _R through a polarizer H, seeking angular spectrum of the reflected light L _R, the absorption peak appearing in the spectrum (dark line), of the sample S in contact with the metal film 12 Measure physical properties such as refractive index or its change. In this case, the optical detector 50, while the s-polarized component of the reflected light L _R extracted by the polarizer H, and feeds back the detection signal to the automatic polarization adjusting mechanism 60, also, an automatic polarization adjusting mechanism 60 is the s-polarized light The polarization adjustment element 23 is operated so as to minimize the intensity of the excitation light Lo, and the polarization state of the excitation light Lo is adjusted. Note when the dark line measurements, only p-polarized light component of the reflected light L _R or remove polarizer is adjusted so as to transmit.

In the above method, the excitation light Lo is reflected on the metal film 12, when the reflected light L _R is propagated through the dielectric prism 22, it is affected by the birefringence due to the dielectric prism 22, which is excitation light This can be avoided by adjusting the irradiation point of Lo and shortening the distance that the reflected light _LR is reflected and propagates through the dielectric prism 22 as much as possible.

The configuration provided with the automatic polarization adjustment mechanism described above can also be applied to the third to fifth embodiments described below. Like the first embodiment in any case, since by detecting the polarized components of the reflected light L _R of the excitation light is to correct the polarization state of the pumping light Lo by automatic polarization adjusting mechanism, the third to fifth Description of the embodiment applied to the embodiment is omitted.

<Second Embodiment>
A detection method and apparatus according to the second embodiment will be described with reference to FIG. FIG. 5 is an overall view showing a schematic configuration of the detection apparatus according to the second embodiment. The detection method and apparatus of the present embodiment is a fluorescence detection method and 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 3 shown in FIG. 5 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 irradiated with excitation light to generate a so-called localized plasmon, and has a metal fine structure having a concavo-convex structure smaller than the wavelength of the excitation light Lo on the surface. A structure or a plurality of metal nanorods having a size smaller than the wavelength of the excitation light Lo is provided. When the metal layer 12 ′ that generates such localized plasmons is provided, it is not necessary to make the excitation light incident on the interface between the metal layer 12 ′ and the dielectric plate 11 so as to be totally reflected. The excitation light irradiation optical system 20 ′ is configured to irradiate the excitation light Lo from above the dielectric plate 11.

  The excitation light irradiation optical system 20 ′ includes a light source 21 composed of a semiconductor laser (LD) or the like that outputs excitation light Lo, a polarization adjusting element 23 that makes the excitation light Lo linearly polarized light (p-polarized light), and reflects the excitation light Lo. And a half mirror 23 for guiding light 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. 6A to 6C in perspective views.
A sensor chip 10A shown in FIG. 6A includes a dielectric plate 11 and a metal microstructure 73 composed of a plurality of metal particles 73a fixed in an array on a predetermined region of the plate 11. The arrangement pattern of the metal particles 73a can be designed as appropriate, but is preferably substantially regular. In such a configuration, the average diameter and pitch of the metal particles 73a are designed to be smaller than the wavelength of the excitation light Lo.

  A sensor chip 10B shown in FIG. 6B is composed of a dielectric plate 11 and a metal microstructure 74 which is provided in a predetermined region on the plate and is composed of a metal pattern layer in which fine metal wires 74a are patterned in a lattice pattern. Has been. The pattern of the metal pattern layer can be designed as appropriate and is preferably substantially regular. In such a configuration, the average line width and pitch of the fine metal wires 74a are designed to be smaller than the wavelength of the excitation light Lo.

A sensor chip 10C shown in FIG. 6C 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. This metal microstructure 75 is obtained by anodizing a part of a metal body (Al, etc.) to form a metal oxide body (Al ₂ O ₃ etc.), and a plurality of fine metal oxide bodies 77 formed in the process of anodization. Each metal 75a can be grown in the hole 77a by plating or the like.
In the example shown in FIG. 6C, 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 3 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.
With the sensor chip 10 ′ set in the housing portion 13, 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 ′. A photoelectric field (electric field caused by near-field light) generated on the metal layer 12 ′ by the incidence of the excitation light Lo is enhanced by this localized plasmon, and a photoelectric field enhancement region D is formed on the metal layer. In the electric field enhancement region D, particularly in the vicinity of the surface of the metal film 12 ′, the fluorescent label F is excited (substantially, the fluorescent dye molecule f in the fluorescent material is excited) to generate fluorescence Lf. At this time, the fluorescence is enhanced by the effect of enhancement of the photoelectric field by the localized plasmons. By detecting this fluorescence Lf by the light detection means 30, it is possible to detect the presence and / or amount of the target substance bound to the fluorescent label binding substance.

  Also in the present embodiment, as in the first embodiment, a fluorescent substance F including a plurality of fluorescent dye molecules f by a dielectric that transmits fluorescence from the fluorescent dye molecules f is used, and the polarization separating means 40 is provided. Thus, since the detection light Lc of the polarization component orthogonal to the polarization direction of the excitation light Lo is detected, the same effect as in the first embodiment can be obtained.

<Third Embodiment>
A detection method and apparatus according to the third embodiment will be described with reference to FIG. FIG. 7 is an overall view showing a schematic configuration of the detection apparatus of the third embodiment. The detection method and apparatus of the present embodiment enhances an electric field by surface plasmon resonance, and fluorescence excited in the enhanced electric field newly induces plasmon in the metal layer, thereby forming a metal layer forming surface of the dielectric plate. A radiation detection method and apparatus for detecting radiation emitted from a newly induced plasmon emitted from the opposite surface side.

  The emitted light detection device 4 shown in FIG. 7 is different from the fluorescence detection device of the first embodiment in the arrangement of the photodetectors. In the present radiation detection device 4, the light detection means 30 is opposite to the metal layer forming surface of the dielectric plate 11 by the fluorescence excited by the enhanced electric field newly inducing the surface plasmon in the metal layer 12. It arrange | positions so that the emitted light Lp from the newly induced plasmon radiated | emitted from the surface side (lower side of illustration) may be detected.

A radiation light detection method of the present embodiment using the radiation light detection device 4 will be described.
In the state where the sensor chip 10 is set in the housing part 13, the excitation light Lo is irradiated by the excitation light irradiation optical system 20 as in the first embodiment. The excitation light Lo is incident on the interface between the dielectric plate 11 and the metal film 12 by the excitation light irradiation optical system 20 at a specific incident angle that is equal to or greater than the total reflection angle, thereby entering the sample S on the metal film 12. Evanescent waves ooze and surface plasmons are excited in the metal film 12 by the evanescent waves. A photoelectric field (electric field caused by evanescent waves) generated on the metal layer by the incidence of the excitation light Lo is enhanced by this surface plasmon, and a photoelectric field enhancement region D is formed on the metal layer. In the electric field enhancement region D, particularly in the vicinity of the surface of the metal film 12, the fluorescent label F is excited (substantially, the fluorescent dye molecule f in the fluorescent material is excited) to generate fluorescence Lf. At this time, the fluorescence is enhanced by the effect of enhancing the photoelectric field by the surface plasmon. In this embodiment, the fluorescence Lf generated on the metal film 12 induces a new surface plasmon on the metal film 12, and the surface plasmon emits at a specific angle from the side opposite to the metal film formation surface of the sensor chip 10. Light Lp is emitted. By detecting this emitted light Lp by the light detection means 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.

  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.

  On the surface of the dielectric plate 11 opposite to the surface on which the metal layer is formed, there is light Ld containing the radiated light Lp and noise light. Also in the present embodiment, as in the first embodiment, a fluorescent substance F including a plurality of fluorescent dye molecules f by a dielectric that transmits fluorescence from the fluorescent dye molecules f is used, and the polarization separating means 40 is provided. Thus, the detection light Lc of the polarization component orthogonal to the polarization direction of the excitation light Lo of the light Ld is detected, so that the same effect as in 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. 8 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 fluorescence detection method and apparatus for detecting fluorescence excited in an enhanced photoelectric field.

  The configuration of the fluorescence detection device 5 shown in FIG. 8 is the same as the configuration of the fluorescence detection device of the first embodiment, but the sensor chip used is different, and the mechanism of photoelectric field enhancement is different due to the difference in this 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 5 will be described.
In the state where the sensor chip 10 ″ is set in the housing portion 13, the excitation light Lo is irradiated by the excitation light irradiation optical system 20 in the same manner as in the first embodiment. The excitation light Lo is radiated by the excitation light irradiation optical system 20. By being incident on the interface between the plate 11 and the metal layer 12a at a specific incident angle equal to or greater than the total reflection angle, an evanescent wave oozes out on the metal layer 12a, and the evanescent wave is guided through the optical waveguide layer 12b. The optical waveguide mode is excited by coupling with the mode, and the photoelectric field (electric field caused by the evanescent wave) generated on the optical waveguide layer by the incidence of the excitation light is enhanced by this optical waveguide mode, and on the optical waveguide layer. In the electric field enhancement region D, particularly in the vicinity of the surface of the metal film 12, the fluorescent label F is excited (substantially, the fluorescent dye molecule f in the fluorescent material is excited). At this time, the fluorescence is enhanced by the effect of the enhancement of the photoelectric field by the optical waveguide mode, and the fluorescence label binding substance is detected by detecting the fluorescence Lf by the light detection means 30. It is possible to detect the presence and / or amount of the substance to be detected bound to.

  Also in the present embodiment, as in the first embodiment, a fluorescent substance F including a plurality of fluorescent dye molecules f by a dielectric that transmits fluorescence from the fluorescent dye molecules f is used, and the polarization separating means 40 is provided. Thus, since the detection light Lc of the polarization component orthogonal to the polarization direction of the excitation light Lo is detected, the same effect as that of the first embodiment can be obtained.

  Furthermore, the light-enhanced electric field distribution 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 light-enhanced electric field distribution generated by the surface plasmon. When a fluorescent substance having a large diameter encapsulating the fluorescent dye molecule is used, a larger fluorescence amount increasing effect can be obtained than when the photoelectric field enhancement by surface plasmon is used.

<Fifth Embodiment>
A detection method and apparatus according to a fifth embodiment will be described with reference to FIG. FIG. 9 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 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. The fluorescence excited in the enhanced photoelectric field induces a new plasmon in the metal layer, which is emitted from the surface opposite to the metal layer forming surface of the dielectric plate, from the newly induced plasmon. A radiation detection method and apparatus for detecting radiation.

  The synchrotron radiation detection device 6 of this embodiment shown in FIG. 9 has the same configuration as that of the synchrotron radiation detection device of the third embodiment, and the sensor chip used in the detection method of this embodiment is the same as that of the fourth embodiment. This is the same as the sensor chip used in the fluorescence detection method.

  The fluorescence detection method of this embodiment using the synchrotron radiation detection device 6 will be described.

  In the state where the sensor chip 10 ″ is set in the housing portion 13, the excitation light Lo is irradiated by the excitation light irradiation optical system 20 in the same manner as in the first embodiment. The excitation light Lo is radiated by the excitation light irradiation optical system 20. By being incident on the interface between the plate 11 and the metal layer 12a at a specific incident angle equal to or greater than the total reflection angle, an evanescent wave oozes out on the metal layer 12a, and the evanescent wave is guided through the optical waveguide layer 12b. The optical waveguide mode is excited by coupling with the mode, and the photoelectric field generated on the optical waveguide layer by the incidence of the excitation light (the electric field due to the evanescent wave) is enhanced by this optical waveguide mode, and on the optical waveguide layer. In the electric field enhancement region D, particularly in the vicinity of the surface of the metal film 12, the fluorescent label F is excited (substantially, the fluorescent dye molecule f in the fluorescent material is excited). In this case, the fluorescence Lf is generated, and the fluorescence is enhanced by the effect of enhancement of the photoelectric field by the optical waveguide mode, and the fluorescence Lf generated on the optical waveguide layer 12b is applied to the surface plasmon on the metal film 12. Then, the surface plasmon emits the radiated light Lp at a specific angle from the side opposite to the metal film forming surface of the sensor chip 10. By detecting the radiated light Lp by the light detection means 30, The presence and / or amount of the substance to be detected labeled with the fluorescent label F can be detected.

  Also in the present embodiment, as in the first embodiment, the polarization separating unit 40 uses a fluorescent material F including a plurality of fluorescent dye molecules f including a dielectric 16 that transmits fluorescence from the fluorescent dye molecules f. Since the detection light Lc of the polarization component orthogonal to the polarization direction of the excitation light Lo is detected via the above, the same effect as in 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 third embodiment can be obtained.
Furthermore, since the electric field enhancement by excitation of the optical waveguide mode is used, the effect of increasing the amount of fluorescence can be obtained as in the fourth embodiment.

  As the fluorescent label, as shown in FIG. 10, (F ′) may be used in which the surface of the dielectric 16 is further provided with a metal film 19 having a thickness that transmits fluorescence. The metal coating 19 may cover the entire surface of the dielectric 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 19, the same metal material as that of the above-described metal layer can be used.

  When the metal film 19 is provided on the surface of the fluorescent material, the surface plasmon or local plasmon generated on the metal layers 12 and 12 ′ of the sensor chip 10, 10 ′ is a whispering gallery of the metal film 19 of the fluorescent material F ′. By coupling to the mode, the fluorescent dye molecule f in the fluorescent substance F ′ can be excited with higher efficiency. The whispering gallery mode is an electromagnetic wave mode that localizes and circulates on the surface of a microsphere such as a fluorescent material having a diameter of about 5300 nm or less used here.

An example of a metal coating method on a fluorescent material is given.
First, a quenching-resistant fluorescent material is prepared by the above-described procedure, and polyethyleneimine (PEI) (Epomin, Nippon Shokubai Co., Ltd.) is modified on the surface.
Next, Pd nanoparticles having an average particle diameter of 15 nm (average particle diameter of 19 nm, Tokuru Head Office) are adsorbed on the PEI on the particle surface.
By immersing polystyrene particles adsorbed with Pd nanoparticles in an electroless gold plating solution (HAuCl ₄ , Kojima Chemical Co., Ltd.), a gold film is formed on the polystyrene particle surface using electroless plating using Pd nanoparticles as a catalyst. Make it.

Embodiments according to the present invention will be described.
Example 1
As fluorescent labels, fluorescent particles manufactured by Bangs Laboratories (product number FC03F / 8863), diameter 510 nm, excitation wavelength 660 nm, fluorescence wavelength 690 nm) are prepared, and a secondary antibody comprising an anti-hCG monoclonal antibody is applied to the particle surface by amine coupling. A labeled secondary antibody (fluorescent label binding substance) was prepared by binding.

  Next, a sample cell having a gold film on which a primary antibody composed of an anti-hCG monoclonal antibody having a different epitope from that of the secondary antibody was fixed as a fixed layer on the surface was prepared. In physiological saline (containing NaCl, 150 mM (mmol / L)), a sample solution containing a labeled secondary antibody and hCG as a substance to be detected is prepared, the sample solution is allowed to flow through the sample cell, and an antigen-antibody reaction is performed. A sandwich conjugate was formed on the fixed layer, and unreacted labeled secondary antibody was washed away with a buffer.

  Next, a sample cell is set in a detection device having the same configuration as the detection device shown in FIG. 1, and excitation light obtained by extracting only p-polarized light from the laser light is completely applied to the gold film using a dielectric prism. Incident light is reflected so that fluorescence is excited in the fluorescent substance in the sample cell, and signal light (fluorescence) and scattered light (noise light) are measured from the surface of the sample cell opposite to the dielectric prism. It was.

  As the light source, an LD light source (product number DL-3147-160F, DL-3357-165, wavelength 655 nm) manufactured by Stocker Yale is used, and the wavelength is used for separation and measurement of scattered light (˜655 nm) and signal light (˜690 nm). In each case, a p-polarized light component and an s-polarized light component were taken out using a selection filter and detected by LAS-1000plus manufactured by FUJIFILM Corporation, and their intensities were compared.

  As a result, in the signal light (fluorescence), the intensity ratio of the p-polarized component to the s-polarized component is 2: 1, and the intensity ratio of the p-polarized component to the s-polarized component of the scattered light (noise light) is 50: 1. It was. By detecting the s-polarized component, the signal light intensity is reduced to 1/3, but the scattered light component is reduced to 1/50. Therefore, by using the fluorescent substance F containing fluorescent dye molecules with polystyrene as the fluorescent label and detecting the s-polarized component, the ratio of noise light is reduced by about an order of magnitude and high-sensitivity detection with high S / N. Was confirmed to be possible.

  Moreover, the pattern of each polarization component of the signal light imaged with LAS-1000plus (exposure time 1 second) is shown in FIGS. 11A and 11B, and the pattern of scattered light imaged with LAS-1000plus (exposure time 0.01 seconds) is also used. The pattern of each polarization component is shown in FIGS. 12A and 12B. In FIG. 12A, the hatched portion in the middle part is slightly saturated and is saturated. From FIG. 11 and FIG. 12, the improvement of S / N could be confirmed.

  The surface plasmon sensor of the present invention can be preferably used as a biosensor or the like.

1 is a schematic configuration diagram showing a fluorescence detection device according to a first embodiment of the present invention. Schematic diagram showing the cross section of a fluorescent label including a plurality of fluorescent dye molecules by a dielectric and the state of multiple reflection of light in the fluorescent label Diagram showing a design change example of the excitation light irradiation optical system The figure which shows the example of a design change of 1st Embodiment provided with the polarization adjusting element and its control means The schematic block diagram which shows the fluorescence detection apparatus by 2nd Embodiment of this 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 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 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 which shows the detection apparatus by 3rd Embodiment of this invention. The schematic block diagram which shows the detection apparatus by 4th Embodiment of this invention. The schematic block diagram which shows the detection apparatus by 5th Embodiment of this invention. Schematic showing a fluorescent substance with a metal coating Intensity pattern of p-polarized component of signal light of Example 1 Intensity pattern of the s-polarized component of the signal light of Example 1 Intensity pattern of the p-polarized component of the scattered light of Example 1 Intensity pattern of the s-polarized component of the scattered light of Example 1

Explanation of symbols

1, 2, 3, 4, 5, 6 Detector 10, 10 ', 10 "Sensor chip 11 Dielectric plate 12 Metal layer (metal film)
12 'metal layer (metal microstructure)
16 Dielectric 19 Metal coating 20, 20 ′ Excitation light irradiation optical system 21 Excitation light source 23 Polarization adjusting element 30 Photodetection means 40 Polarization separation means 50 Photodetector A Antigen (substance to be detected)
B ₁ primary antibody (first binding substance)
B ₂ secondary antibody (second binding substance)
_BF labeled secondary antibody (fluorescently labeled binding substance)
F Fluorescent substance (fluorescent label)
f Fluorescent dye molecule Lo Excitation light Lf Fluorescence Lp Radiation light Ld Light including signal light Lc Polarization component of light including signal light (detection light) orthogonal to the polarization direction of excitation light
Reflected light of _LR excitation light

Claims (8)

Preparing a sensor chip comprising a dielectric plate and a sensor portion in which at least a metal layer is provided on one surface of the dielectric plate;
By bringing a liquid 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 in the liquid sample is bound on the sensor unit,
By irradiating the sensor unit with linearly polarized excitation light, an enhanced photoelectric field is generated on the sensor unit, the fluorescence label of the fluorescent label binding substance is excited in the enhanced photoelectric field, and the fluorescence label In a detection method for detecting the amount of the substance to be detected based on the amount of light including signal light generated due to excitation,
As the fluorescent label, using a fluorescent substance comprising a plurality of fluorescent dye molecules included by a dielectric that transmits fluorescence generated from the plurality of fluorescent dye molecules,
A detection method comprising: detecting an amount of the substance to be detected by detecting an amount of a polarization component of the light orthogonal to a polarization direction of the excitation light. Excitation of plasmons in the metal layer by irradiation of the excitation light, causing the enhanced photoelectric field by the plasmons,
The detection method according to claim 1, wherein the amount of the detection target substance is detected by detecting fluorescence generated from the fluorescent label by the excitation as the light generated due to the excitation of the fluorescent label. . Excitation of plasmons in the metal layer by irradiation of the excitation light, causing the enhanced photoelectric field by the plasmons,
As the light generated due to the excitation of the fluorescent label, the fluorescence generated from the fluorescent label by the excitation induces a new plasmon in the metal layer, thereby radiating from the other surface of the dielectric plate. The detection method according to claim 1, wherein the amount of the substance to be detected is detected by detecting light.   The detection method according to claim 2 or 3, wherein the linearly polarized light is p-polarized light, and the plasmon is a surface plasmon. As the sensor chip, one having an optical waveguide layer on the metal layer,
Excitation of an optical waveguide mode in the optical waveguide layer by irradiation of the excitation light, causing the enhanced photoelectric field by the optical waveguide mode,
The detection method according to claim 1, wherein the amount of the detection target substance is detected by detecting fluorescence generated from the fluorescent label by the excitation as the light generated due to the excitation of the fluorescent label. . As the sensor chip, one having an optical waveguide layer on the metal layer,
Excitation of an optical waveguide mode in the optical waveguide layer by irradiation of the excitation light, causing the enhanced photoelectric field by the optical waveguide mode,
As the light generated due to the excitation of the fluorescent label, the fluorescence generated from the fluorescent label by the excitation induces a new plasmon in the metal layer, thereby radiating from the other surface of the dielectric plate. The detection method according to claim 1, wherein the amount of the substance to be detected is detected by detecting light. A detection device used in the detection method according to claim 1,
An accommodating portion for accommodating the sensor chip;
An excitation light irradiation optical system including an excitation light source that irradiates the excitation light to the sensor unit;
In the enhanced photoelectric field generated on the sensor unit by the irradiation of the excitation light, a polarization component orthogonal to the polarization direction of the excitation light is separated from light including signal light generated due to excitation of the fluorescent label. Polarized light separating means to be taken out,
And a light detecting means for detecting the amount of the separated polarization component.   A light detector that detects the intensity of reflected light of the excitation light, a polarization adjustment element that controls the polarization state of the excitation light emitted from the excitation light source, and a sensor unit according to the detected intensity. The detection apparatus according to claim 7, further comprising a control unit that operates the polarization adjustment element so that the intensity of the p-polarized component of the incident excitation light is maximized.