JP2012052917A - Surface plasmon exciting intensified fluorescence measuring device and sensor structure used for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensor structure capable of easily correcting variation in thickness of a metal thin film, and to provide a surface plasmon exciting intensified fluorescence measuring device having the same.SOLUTION: A sensor structure includes a core layer to be a light propagation region and a clad layer laminated on at least either one of an upper surface and a lower surface of the core layer. The sensor structure used for a surface plasmon exciting intensified fluorescence measuring device includes at least a total reflection fluorescence reference region (P) in which a fluorescence dye layer is formed on a surface of the core layer without a metal thin film interposed therebetween, and an electric field enhancing fluorescence reference region (Q) in which the fluorescence dye layer made of the same fluorescence dye with the fluorescence dye layer formed in the region (P) is formed on the surface of the core layer through the metal thin film. The sensor structure includes incident angle adjusting means for adjusting an incident angle of excitation light totally reflected at the region (P) such that the region (Q) is irradiated with the excitation light totally reflected at the region (P) at a predetermined incident angle (θq) for satisfying a total reflection condition.

Description

  The present invention is an SPFS (Surface Plasmon-field enhanced Fluorescence Spectroscopy) measuring device (surface plasmon excitation enhanced fluorescence measuring device) used in the fields of medicine, biotechnology, and the like, and used for this. Sensor structure.

  In SPFS, when the metal thin film formed on the dielectric member is irradiated with excitation light at an angle at which total reflection attenuation (ATR) occurs, the evanescent wave transmitted through the metal thin film is several tens of times due to resonance with the surface plasmon. This is a method of efficiently exciting a fluorescent dye for labeling an analyte (analyte) captured in the vicinity of a metal thin film and measuring the fluorescence signal by using the enhancement of several hundred times. Since such SPFS is extremely sensitive compared to a general fluorescent labeling method, it can be quantified even when only a very small amount of analyte is present in the sample. Basic aspects of a SPFS measurement device (surface plasmon excitation enhanced fluorescence measurement device) are disclosed in Patent Documents 1 and 2, for example.

  By the way, when applying SPFS or the like to clinical examinations, it is necessary to ensure the reliability of the measurement value of the fluorescence signal. For this purpose, the variation (CV) of the measurement value is usually within 10%, and in some cases 1 It is necessary to keep it within 2%. Especially in systems where the absolute value of the signal increases according to the analyte concentration, such as a sandwich system in which a fluorescently modified antibody is immobilized via a specimen-derived analyte on an antibody-immobilized sensor structure, an analyte is generally used. When the concentration decreases, CV deteriorates, leading to a decrease in detection sensitivity and quantitative sensitivity. Therefore, in order to reduce the variation in the measured value of the fluorescent signal, it is necessary to reduce the variation in the electric field enhancement between the sensor structures of the surface plasmon excitation enhanced fluorescence measuring device.

  Here, in considering the influence on the fluorescence signal due to the variation in the electric field enhancement, the variation in the thickness of the metal thin film formed on the sensor structure becomes a problem. However, since it is not practical to manufacture the sensor structure so that the film thickness of the metal thin film is completely the same in all sensor structures, there is no difference in the electric field intensity. In order to minimize the influence of the above, it is conceivable to normalize the electric field enhancement generated in the sensor structure and correct the measurement value based on this. On the other hand, sensor structures used for clinical tests are also required to be inexpensively manufactured. Therefore, the electric field enhancement can be normalized without forming a complicated structure in the sensor structure, and by correcting the measurement value based on this, it is possible to minimize variation in measurement data. It becomes important when applying to.

  Against this background, without introducing a complicated structure to the sensor structure, the influence of variations in the electric field strength derived from the variations in the thickness of the metal thin film that constitutes the sensor structure is eliminated. The present applicant has previously filed an invention relating to a method for correcting measurement data and an assay method using the same, which can improve the measurement accuracy and reliability of SPFS and the like (Patent Document 3, Japanese Patent Application No. 2010-). 181364. Hereinafter referred to as “prior application”).

  The measurement data correction method described in this prior application is such that when excitation light is irradiated under total reflection conditions from the back side of a substrate in which a fluorescent dye layer is formed on a substrate in which a metal thin film is formed on a part of the surface of a dielectric member Based on the relationship between the fluorescence intensity in the area where the metal thin film is not formed and the fluorescence intensity in the area where the metal thin film is formed, the effect on the electric field enhancement due to the variation in the thickness of the metal thin film is estimated. A specific correction method will be described with reference to FIG.

FIG. 10 is a cross-sectional view showing the sensor structure of the prior application.
As shown in FIG. 10, the sensor structure 100 includes a total reflection fluorescent reference region (P) in which only the fluorescent dye layer 116p is formed, and an electric field enhancement in which the fluorescent dye layer 116q is formed through the metal thin film 118. The fluorescence reference region (Q) and the SPFS assay region (A) in which the immobilized ligand-containing layer 120 is formed via the metal thin film 118 are formed on the same transparent dielectric member 112, respectively. The fluorescent dye layer 116p in the region (P) and the fluorescent dye layer 116q in the region (Q) are formed of the same fluorescent dye. A light detection means 130 such as a CCD camera is disposed above the transparent dielectric member 112, and a prism (not shown) is disposed on the lower surface side of the transparent dielectric member 112.

  In addition, the first ligand 122 is fixed to the immobilized ligand-containing layer 120 in the region (A). The first ligand 122 has a property of specifically binding to the analyte 128. By bringing the analyte solution containing the analyte 128 into contact with the immobilized ligand-containing layer 120, as shown in FIG. In addition, the analyte 128 can be bound to the first ligand 122. Further, the analyte 128 is brought into contact with the decorative ligand 125 including the second ligand 124 and the fluorescent molecule 126 bonded to the second ligand 124, as shown in FIG. The fluorescent molecule 126 can be bound to the second ligand 124 via the second ligand 124. Therefore, when this region (A) is irradiated from the lower surface side of the transparent dielectric member 112 with the excitation light 142a at a predetermined incident angle θa, which is a total reflection condition, the fluorescent molecules 126 bound to the analyte 128 emit light. At the same time, the fluorescence is enhanced by the surface plasmon phenomenon. Then, the analyte 128 contained in the sample solution can be quantified by measuring the enhanced fluorescence amount as the fluorescence signal Sa by the light detection means 130.

  In order to correct the influence on the electric field enhancement caused by the variation in the film thickness of the metal thin film between different sensors, the prior application adopts the following method for the fluorescent signal Sa thus obtained.

  First, the region (P) is irradiated with excitation light 142p from the lower surface side of the transparent dielectric member 112 at a predetermined incident angle θp that is a total reflection condition. Then, the amount of fluorescence emitted from the fluorescent dye layer 116p is measured by the light detection means 130 as a total reflection fluorescence signal Sp.

  Similarly, the excitation light 142q is irradiated to the region (Q) from the lower surface side of the transparent dielectric member 112 at a predetermined incident angle θq that is a total reflection condition. The amount of fluorescence emitted from the fluorescent dye layer 116q and enhanced by the surface plasmon phenomenon is measured by the light detection means 130 as an electric field enhanced fluorescence signal Sq. Here, the excitation light 142q is light emitted from the same light source as the excitation light 142p described above, and is light having the same wavelength as the excitation light 142p.

Then, the electric field enhancement intensity R of the sensor structure 100 is calculated from the following formula (1) using the total reflection fluorescence signal Sp and the electric field enhancement fluorescence signal Sq obtained by the above method.
R = Sq / Sp (1)
Further, in the sensor structure 100 ′ (not shown) as a normalization reference having the same configuration as that of the sensor structure 100 shown in FIG. 10, excitation light having the same wavelength as that of the sensor structure 100 described above is emitted. The total reflection fluorescence signal Sp ′ and the electric field enhanced fluorescence signal Sq ′ are measured, and the electric field enhancement intensity R ′ is calculated from the above-described equation (1).

  Then, the metal of the sensor structure 100 shown in FIG. 10 and the sensor structure 100 ′ serving as a standard for normalization is obtained by converting the above-described fluorescence signal Sa into the corrected fluorescence signal S by the following formula (2). The amount of fluorescent signal due to the difference in film thickness is corrected.

S = Sa × (R ′ / R) (2)
In the sensor structure 100 shown in FIG. 10, the region (P), the region (Q), and the region (A) are integrally formed on the same transparent dielectric member 112. The sensor structure 100 is not limited to this. The metal thin film 118 formed in the region (A) and the metal thin film 118 formed in the region (Q) have the same metal and the same film thickness, and both are transparent made of the same material. If formed on the dielectric member 112, a sensor structure in which only the region (P) and the region (Q) are formed and a sensor structure in which only the region (A) is formed are manufactured separately. However, the correction may be performed using these.

Japanese Patent No. 3294605 JP 2006-218169 A Japanese Patent Application No. 2010-181364

  By the way, in the correction method of the above-mentioned prior application, it is necessary to irradiate the total reflection fluorescence reference region (P) and the electric field enhanced fluorescence reference region (Q) with excitation light 142p and 142q at different incident angles θp and θq, respectively. is there. For this reason, for example, when the excitation light 142p and 142q is irradiated to the region (P) and the region (Q) from the same light source, it is necessary to adjust the irradiation direction of the excitation light in the projector serving as the light source.

  However, various optical devices such as a polarizing filter and a beam expander are attached to the projector, and the projector has a complicated configuration. In order to adjust the irradiation direction of the excitation light in such a projector, it is necessary to add driving means such as a motor to the projector, which further complicates the configuration of the projector and increases the size. Further, since high accuracy is required for adjusting the irradiation direction of the excitation light, it is necessary to incorporate a sensor or the like for controlling the irradiation direction in the projector, which further complicates the configuration of the projector and increases the cost. The problem is also considered.

  The present invention has been made in view of such a current situation, and it is possible to easily correct the influence on the electric field enhancement caused by the variation in the film thickness of the metal thin film with a simple structure. An object of the present invention is to provide a surface plasmon excitation enhanced fluorescence measuring apparatus capable of detecting light with high accuracy and a sensor structure used therefor.

The present invention has been invented to solve the above-described problems,
The sensor structure of the present invention comprises:
A core layer serving as a light propagation region, and a cladding layer laminated on at least one of the upper surface and the lower surface of the core layer,
On the surface of the core layer, at least a total reflection fluorescent reference region (P) in which a fluorescent dye layer is formed without using a metal thin film, and a fluorescent dye formed in the region (P) through a metal thin film An electric field-enhanced fluorescent reference region (Q) in which a fluorescent dye layer made of the same fluorescent dye as the layer is formed,
When the excitation light propagating through the core layer irradiates the region (P) at a predetermined incident angle (θp) that is a total reflection condition, the excitation light totally reflected in the region (P) A sensor structure used in a surface plasmon excitation enhanced fluorescence measurement device configured to irradiate the region (Q) at a predetermined incident angle (θq),
The incident angle (θp) of the excitation light when irradiating the region (P) is different from the incident angle (θq) of the excitation light when irradiating the region (Q),
Incident of the excitation light totally reflected in the region (P) so that the excitation light totally reflected in the region (P) irradiates the region (Q) at a predetermined incident angle (θq) that is a total reflection condition. Incident angle adjusting means for adjusting the angle is provided.

  With this configuration, the sensor structure of the present invention includes the incident angle adjusting means for adjusting the incident angle when the excitation light totally reflected in the region (P) irradiates the region (Q). There is no need to adjust the direction of excitation light irradiation. In addition, the total reflection fluorescence signal Sp in the region (P) and the electric field enhanced fluorescence signal Sq in the region (Q) can be measured by irradiating the excitation light only once. Thereby, with a simple sensor structure, it is possible to easily correct the influence on the electric field enhancement due to the variation in the film thickness of the metal thin film, and it becomes possible to detect a desired analyte with high accuracy. .

In the above invention, the sensor structure of the present invention comprises:
The incident angle adjusting means is
It is desirable that it is an inclined surface formed on the surface of the core layer.

  With such a configuration, the incident angle when the excitation light totally reflected in the region (P) irradiates the region (Q) is adjusted by the inclined surface provided on the surface of the core layer, so that excitation is performed in the projector. There is no need to adjust the direction of light irradiation, and the sensor structure of the present invention can be a very simple sensor structure.

In the above invention, the sensor structure of the present invention is
The core layer is configured by bonding a first core having a predetermined refractive index and a second core having a refractive index different from the first core;
It is desirable that the joint surface between the first core and the second core constitutes the incident angle adjusting means.

  With such a configuration, the incident angle when the excitation light totally reflected in the region (P) irradiates the region (Q) by the joint surface between the first core and the second core is adjusted. There is no need to adjust the irradiation direction of the excitation light in the projector, and the sensor structure of the present invention can have a very simple sensor structure.

In the above invention,
Immobilization containing a ligand on the surface of the core layer through a metal thin film having the same thickness and the same metal as the metal thin film formed in the region (Q) without a fluorescent dye layer. It is desirable that the SPFS assay region (A) in which the conjugated ligand-containing layer is formed is formed.

  With this configuration, since the SPFS assay region (A) in which the immobilized ligand-containing layer is formed on the surface of the core layer is formed, the sensor structure according to the present invention has a variation in the film thickness of the metal thin film. Thus, the sensor structure can be used not only for correcting the influence on the electric field enhancement caused by, but also for the quantitative determination of the analyte.

Moreover, the surface plasmon excitation enhanced fluorescence measuring apparatus of the present invention includes the sensor structure described above.
By configuring in this way, the surface plasmon excitation enhanced fluorescence measuring device of the present invention has an incident angle adjusting means for adjusting the incident angle when the excitation light totally reflected in the region (P) irradiates the region (Q). Since the sensor structure is provided, it is not necessary to adjust the irradiation direction of the excitation light in the projector. In addition, the total reflection fluorescence signal Sp in the region (P) and the electric field enhanced fluorescence signal Sq in the region (Q) can be measured by irradiating the excitation light only once. As a result, the surface plasmon excitation enhanced fluorescence measuring apparatus of the present invention can easily correct the influence on the electric field enhancement due to the variation in the film thickness of the metal thin film, and can detect the desired analyte with high accuracy. Can be performed.

  According to the present invention, it is possible to easily correct the influence on the electric field enhancement due to the variation in the film thickness of the metal thin film with a simple sensor structure, thereby performing detection of a desired analyte with high accuracy. It is possible to provide a surface plasmon excitation-enhanced fluorescence measuring apparatus that can be used, and a sensor structure used therefor.

FIG. 1 is a perspective view for explaining a sensor structure according to a first embodiment of the present invention. FIG. 2 is a cross-sectional view for explaining the surface plasmon excitation enhanced fluorescence measuring apparatus of the present invention and the sensor structure of the first embodiment. FIG. 3A is a perspective view for explaining a sensor structure according to a second embodiment of the present invention. FIG. 3B is a perspective view for explaining the sensor structure according to the second embodiment of the present invention, and is a diagram for explaining a case where excitation light is wide light. 4A is a cross-sectional view taken along the line bb in FIG. 3A, and FIG. 4B is a cross-sectional view taken along the line cc in FIG. 3A. FIG. 5 is a perspective view for explaining a modification of the sensor structure according to the second embodiment of the present invention. 6A is a cross-sectional view taken along the line dd in FIG. 5, and FIG. 6B is a cross-sectional view taken along the line ee in FIG. FIG. 7 is a cross-sectional view for explaining a modification of the sensor structure of the present invention. FIG. 8 is a cross-sectional view for explaining a modification of the sensor structure of the present invention. FIG. 9 is a cross-sectional view for explaining a sensor structure according to a third embodiment of the present invention. FIG. 10 is a cross-sectional view showing the sensor structure of the prior application.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
<First Embodiment>
FIG. 1 is a perspective view for explaining a sensor structure according to a first embodiment of the present invention. FIG. 2 is a cross-sectional view for explaining the surface plasmon excitation enhanced fluorescence measuring apparatus of the present invention and the sensor structure of the first embodiment. The sensor structure 10 shown in FIG. 2 is a cross-sectional view taken along the line aa in FIG. In the present specification, the upper surface of the illustrated core layer is referred to as an “upper surface”, and similarly, the lower surface of the illustrated core layer is referred to as a “lower surface”.

  As shown in FIGS. 1 and 2, the sensor structure 10 of the first embodiment includes a core layer 12 and a clad layer 14 laminated on the lower surface 12 l of the core layer 12. On the upper surface 12 u of the core layer 12, a total reflection fluorescence reference region (P) (hereinafter, simply referred to as “region (P)”) in which the fluorescent dye layer 16 p is formed without the metal thin film 18 interposed therebetween; An electric field enhanced fluorescence reference region (Q) (hereinafter sometimes simply referred to as “region (Q)”) in which the fluorescent dye layer 16q is formed via the metal thin film 18 is formed.

  In addition to the sensor structure 10 described above, the surface plasmon excitation enhanced fluorescence measuring apparatus 1 of the present invention includes at least a projector 40 serving as a light source of the excitation light 42 and a total reflection fluorescence signal, as shown in FIG. And a light detection means 30 for measuring Sp and the electric field-enhanced fluorescence signal Sq.

  In the present invention, the core layer 12 is a layer serving as a light propagation region for guiding the excitation light 42 incident from the incident surface 12i of the core layer 12 to the region (P) and the region (Q). The core layer 12 of the present embodiment is formed in a substantially flat plate shape including an upper surface 12u that is a horizontal surface and a lower surface 12l that is inclined so as to descend in a direction away from the incident surface 12i.

The core layer 12 is made of a transparent dielectric member. Here, as the material of the transparent dielectric member constituting the core layer 12, various optically transparent inorganic substances, natural polymers, and synthetic polymers can be used, and the optical stability, manufacturing stability, and optical transparency can be used. From the viewpoint of properties, silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) is desirable.

  In the present invention, the cladding layer 14 is laminated on at least one of the upper surface and the lower surface of the core layer 12. As described above, the clad layer 14 of the present embodiment is laminated on the lower surface 121 of the core layer 12. In addition, the cladding layer 14 is configured so that the excitation light 42 propagating through the core layer 12 is totally reflected at the boundary surface between the core layer 12 and the cladding layer 14 (in this embodiment, the lower surface 12l of the core layer 12). The material is made of a material having a refractive index lower than that of the dielectric member.

Here, the material used for the cladding layer 14 can be basically the same material as that of the dielectric member, except that the refractive index is lower than that of the dielectric member constituting the core layer 12. Examples of plastic materials include polyesters such as polyethylene terephthalate (PET) and polyethylene naphthalate, polyolefins such as polyethylene (PE), polypropylene (PP), polystyrene, and cyclic olefin resins, polyvinyl chloride, polyvinylidene chloride, and the like. Vinyl resin, polyether ether ketone (PEEK), polysulfone (PSF), polyether sulfone (PES), polycarbonate (PC), polyamide, polyimide, acrylic resin, triacetyl cellulose (TAC), etc. can be used. . As the inorganic material, silicon dioxide (SiO ₂ ), titanium dioxide (TiO ₂ ), or the like can be used. In view of chemical stability, production stability and optical transparency, silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) is preferable. From the viewpoint of ease of handling and cost, polyethylene terephthalate, acrylic resin, Triacetyl cellulose is preferred.

  The metal thin film 18 can be made of the same metal as the metal thin film constituting the sensor structure used in a general surface plasmon excitation enhanced fluorescence measuring apparatus. That is, it is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, and more preferably made of gold. About these metals, the form of the alloy may be sufficient and what laminated | stacked the metal may be sufficient. Such a metal species is suitable as a metal for the metal thin film 18 because it is stable against oxidation and has a large electric field enhancement due to surface plasmons. Examples of the method for forming the metal thin film 18 on the surface of the core layer 12 include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like.

  The fluorescent dye layers 16p and 16q are layers composed of fluorescent dyes that emit fluorescence when irradiated with predetermined excitation light. In the sensor structure 10 of the present invention, the fluorescent dye layer 16p in the region (P) and the fluorescent dye layer 16q in the region (Q) are formed from the same fluorescent dye.

  Such a fluorescent dye is not particularly limited as long as the object of the present invention can be achieved. For example, a fluorescent dye of the fluorescein family (manufactured by Integrated DNA Technologies), a fluorescent dye of the polyhalofluorescein family (applied) Biosystems Japan Co., Ltd.), Hexachlorofluorescein family fluorescent dye (Applied Biosystems Japan Co., Ltd.), Coumarin family fluorescent dye (Invitrogen Corporation), Rhodamine family fluorescent dye (GE Health) Care BioScience), cyanine family fluorescent dye, indocarbocyanine family fluorescent dye, oxazine family fluorescent dye, thiazine family fluorescent dye, squara Family fluorescent dyes, chelated lanthanide family fluorescent dyes, BODIPY (registered trademark) family fluorescent dyes (manufactured by Invitrogen), naphthalene sulfonic acid family fluorescent dyes, pyrene family fluorescent dyes, A fluorescent dye of the triphenylmethane family, Alexa Fluor (registered trademark) dye series (manufactured by Invitrogen Corp.) and the like, and further, U.S. Pat. Nos. 6,406,297, 6,221,604, The fluorescent dyes described in US Pat. Nos. 5,994,063, 5,808,044, 5,880,287, 5,556,959, and 5,135,717 are also included. It can be used in the present invention.

  The total reflection fluorescence reference region (P) is a region formed by forming the fluorescent dye layer 16p on the upper surface 12u of the core layer 12 as described above. As shown in FIG. 2, the excitation light 42 propagating through the core layer 12 is irradiated from the inside of the core layer 12 to the region (P) at a predetermined incident angle θp that is a total reflection condition. Then, the fluorescent dye layer 16p emits light and a total reflection fluorescent signal Sp is emitted.

  Further, the electric field enhanced fluorescence reference region (Q) is a region formed by forming the fluorescent dye layer 16p on the upper surface of the core layer 12 via the metal thin film 18, as described above. Then, when the excitation light 42 propagating through the core layer 12 is irradiated from the inside of the core layer 12 to the region (Q) at a predetermined incident angle θq that is a total reflection condition, the fluorescent dye layer 16q , And this fluorescence is enhanced by the surface plasmon phenomenon, and an electric field enhanced fluorescence signal Sq is emitted. Here, the predetermined incident angle θq for the region (Q) and the incident angle θp for the region (P) described above are different incident angles.

  Thus, since the metal thin film 18 is not formed in the region (P) as in the region (Q), the total reflection fluorescence signal Sp is a fluorescence signal in a state where there is no influence of the electric field enhancement due to the surface plasmon phenomenon. It has become. In addition, the electric field enhanced fluorescence signal Sq emitted from the region (Q) is a fluorescence signal enhanced by the surface plasmon phenomenon. Therefore, by measuring the total reflection fluorescence signal Sp and the electric field enhanced fluorescence signal Sq by the above-described light detection means 30, the electric field enhancement intensity R of the fluorescence signal by the metal thin film 18 is calculated by the above-described equation (1). Can be calculated.

  Therefore, a sensor having an SPFS assay region (A) to be described later, in which a metal thin film having the same metal and the same film thickness as the metal thin film 18 in this region (Q) is formed by using the electric field enhancement R described above. In the structure, it is possible to estimate the influence on the electric field enhancement due to the variation in the thickness of the metal thin film.

  In the sensor structure 10 of the present invention, as shown in FIG. 2, the excitation light 42 emitted from the projector 40 enters the core layer 12 from the incident surface 12 i of the core layer 12. (P) is configured to irradiate at a predetermined incident angle θp that is a total reflection condition. In the present embodiment, the excitation light 42 incident from the incident surface 12i directly irradiates the region (P). However, the sensor structure 10 of the present invention is not limited to this, for example, from the incident surface 12i. The incident excitation light 42 may be totally reflected at the boundary surface between the core layer 12 and the cladding layer 14, and the totally reflected excitation light 42 may irradiate the region (P).

  Moreover, in the sensor structure 10 of the present invention, as shown in FIG. 2, the excitation light 42 totally reflected in the region (P) is totally reflected on the lower surface 12l of the core layer 12 so as to satisfy the total reflection condition. The region (Q) is irradiated at an incident angle θq. At this time, as described above, the incident angle θp with respect to the region (P) and the incident angle θq with respect to the region (Q) are different from each other. In this embodiment, the incident angle θq is 2θi rather than θp. It is a big angle. In the sensor structure 10 of the present embodiment, the lower surface 12l of the core layer 12 is inclined by the angle θi correspondingly.

  Therefore, the excitation light 42 totally reflected in the region P irradiates the lower surface 12l of the core layer 12 inclined by θi with respect to the upper surface 12u at an incident angle (θp + θi). Then, the excitation light 42 totally reflected by the lower surface 12l irradiates the region (Q) formed on the upper surface 12u with an incident angle (θp + 2θi), that is, θq.

  Thus, in the sensor structure 10 of the present embodiment, the region (P) is such that the excitation light 42 totally reflected in the region (P) irradiates the region (Q) at the predetermined incident angle θq that satisfies the total reflection condition. ), The lower surface 12l of the core layer 12 is inclined by θi. That is, it is possible to adjust the incident angle when the excitation light 42 totally reflected in the region (P) irradiates the region (Q) with an extremely simple sensor structure in which an inclined surface is provided on the surface of the core layer 12. It can be done.

  Further, in the sensor structure 10 of the present embodiment, the total reflected fluorescence signal Sp of the region (P) and the electric field enhanced fluorescence signal Sq of the region (Q) can be obtained by irradiating the sensor structure 10 once with the excitation light 42. Therefore, it is possible to easily correct the influence of the electric field enhancement caused by the variation in the thickness of the metal thin film.

<Second Embodiment>
FIG. 3A is a perspective view for explaining a sensor structure according to a second embodiment of the present invention. 4A is a cross-sectional view taken along the line bb in FIG. 3A, and FIG. 4B is a cross-sectional view taken along the line cc in FIG. 3A. The sensor structure 10 of the second embodiment has basically the same configuration as the sensor structure 10 of the first embodiment described above, and the same members are denoted by the same reference numerals. Detailed description thereof will be omitted.

  In the sensor structure 10 of the second embodiment, as shown in FIG. 3A and FIG. 4, in addition to the above-described region (P) and region (Q), the region (Q SPFS assay region (A) (hereinafter referred to as “region (A)”) in which the immobilized ligand-containing layer 20 is formed through the same metal thin film 18 having the same metal thickness and the same thickness as ) Is different from the above-described first embodiment.

  The immobilized ligand-containing layer 20 in the region A includes a first ligand 22 having a property of specifically binding to the analyte 28. Accordingly, by bringing the analyte solution containing the analyte 28 into contact with the immobilized ligand-containing layer 20, the analyte 28 can be bound to the first ligand 22 as shown in FIG. Further, by bringing the decorative ligand 25 composed of the second ligand 24 and the fluorescent molecule 26 bonded to the second ligand 24 into contact with the analyte 28, as shown in FIG. The fluorescent molecule 26 can be bound to the second ligand 24 via the second ligand 24. As the fluorescent molecule 26, the same fluorescent dye as the fluorescent dye constituting the fluorescent dye layers 16p and 16q described above can be used, but it is not necessarily the same fluorescent dye.

  When this region (A) is irradiated with excitation light 42 from the inside of the core layer 12 at a predetermined incident angle θa, which is a total reflection condition, the fluorescent molecules 26 bound to the analyte 28 emit light, and the fluorescence is emitted. Enhanced by surface plasmon phenomenon. Then, the analyte 28 contained in the sample solution can be quantified by measuring the enhanced fluorescence amount as the fluorescence signal Sa by the light detection means 30. Note that the predetermined incident angle θa with respect to the region (A) and the predetermined incident angle θq with respect to the region (Q) described above are the same angle.

  As described above, if the sensor structure 10 of the present invention includes the region (A) in addition to the region (P) and the region (Q), the sensor structure 10 of the present invention has the thickness of the metal thin film. The sensor structure 10 can be used not only for correcting the influence on the electric field enhancement caused by the variation but also for actually quantifying the analyte.

  Further, in the sensor structure 10 of the second embodiment, as shown in FIG. 3A, a region (P) is formed on the front side (incident surface 12i side) of the core layer 12, and on the back side thereof. The region (Q) and the region (A) are formed in parallel.

  Therefore, as shown by an arrow X in FIG. 3A, a projector (not shown) that is a light source is moved in parallel with respect to the incident surface 12i of the core layer 12, and as shown in FIG. The total reflection fluorescence signal Sp, the electric field enhancement fluorescence signal Sq of the region (Q), and the fluorescence signal Sa of the region (A) can be easily measured.

  If the excitation light 42 is a wide excitation light 42w as shown in FIG. 3B, the excitation light 42 totally reflected in the region (P) is obtained as shown in FIGS. 4 (a) and 4 (b). In order to simultaneously irradiate the region (Q) and the region (A) at predetermined incident angles θq and θa (in this embodiment, θq = θa) that are the total reflection conditions, the excitation light 42w is applied to the sensor structure 10 once. By simply irradiating, the total reflection fluorescence signal Sp in the region (P), the electric field enhancement fluorescence signal Sq in the region (Q), and the fluorescence signal Sa in the region (A) can be easily measured.

  The formation positions of the region (P), the region (Q), and the region (A) are not limited to the formation positions in the above-described embodiment. For example, the region (P) and the region (A) may be formed in parallel on the front side (incident surface 12i side) of the core layer 12 and the region (Q) may be formed on the back side thereof. However, since the amount of the excitation light irradiated to the region (Q) and the region (A) is significantly reduced due to the total reflection attenuation, the region (Q) and / or the region (A) is formed on the front side of the core layer 12. The region (P) cannot be formed on the back side.

  Further, in the sensor structure 10 of the present invention, in addition to the above-described region (A), as shown in FIGS. 5 and 6, for example, the immobilized ligand-containing layer 20 is formed without using a metal thin film. A total reflection assay region (A ′) (hereinafter may be referred to as “region (A ′)”) may also be formed. Here, FIG. 5 is a perspective view for explaining a modification of the sensor structure according to the second embodiment of the present invention, FIG. 6A is a cross-sectional view taken along the line dd of FIG. 6B is a cross-sectional view taken along the line ee of FIG.

  When this region (A ′) is irradiated with excitation light 42 from the inside of the core layer 12 at a predetermined incident angle θa ′ that is a total reflection condition, the fluorescent molecules 26 combined with the analyte 28 emit light. Then, the analyte 28 contained in the sample solution can be quantified by measuring the enhanced fluorescence amount as a fluorescence signal Sa ′ by the light detection means 30 (not shown). The incident angle θa ′ with respect to the region (A ′) and the predetermined incident angle θp with respect to the region (P) described above are the same angle.

  As described above, if the region (A ′) is formed on the surface of the core layer 12 in addition to the region (A) described above, the sensor structure 10 of the present invention is quantified by the above-described SPFS. In addition, it can be used for the determination of the analyte 28 by the total reflection fluorescence method.

  At this time, if the excitation light 42 is a wide excitation light 42w as shown in FIG. 5, the excitation light 42 totally reflected in the region (P) and the region (A ′) is obtained as shown in FIG. In order to simultaneously irradiate the region (Q) and the region (A) at predetermined incident angles θq and θa (in this embodiment, θq = θa) that are the total reflection conditions, the excitation light 42w is applied to the sensor structure 10 once. By simply irradiating, the total reflection fluorescence signal Sp in the region (P), the electric field-enhanced fluorescence signal Sq in the region (Q), the fluorescence signal Sa in the region (A), and the fluorescence signal Sa ′ in the region (A ′) are measured. can do.

  In the first embodiment and the second embodiment described above, the lower surface 121 of the core layer 12 is an inclined surface that is inclined as a whole, but the sensor structure 10 of the present invention is not limited to this. . For example, as shown in FIG. 7, an inclined portion 12l ′ inclined by an angle θi is formed on a part of the lower surface 12l, whereby the excitation light 42 totally reflected in the above-described region (P) It is also possible to irradiate the region (Q) at a predetermined incident angle θq that is a reflection condition.

  In the first and second embodiments described above, the region (P) and the region (Q) are both formed on the upper surface 12u of the core layer 12, but the sensor structure 10 of the present invention. Is not limited to this. For example, as shown in FIG. 8, it is also possible to form a region (P) on the front side of the upper surface 12u and form a region (Q) on the lower surface 12l on the back side of the region (P). At this time, similarly to the second embodiment described above, the region (A) can be formed in parallel to the region (Q).

  However, when the region (P), the region (Q), and the region (A) are formed on the same side of the upper surface 12u or the lower surface 12l of the core layer 12, the total reflection fluorescence signal Sp and the electric field enhanced fluorescence are generated. Since the signal Sq and the fluorescence signal Sa can be easily measured by one light detection means 30, it is preferable.

<Third Embodiment>
FIG. 9 is a cross-sectional view for explaining a sensor structure according to a third embodiment of the present invention. The sensor structure 10 according to the third embodiment has basically the same configuration as the sensor structure 10 according to the first embodiment and the second embodiment described above, and the same reference numerals denote the same members. The detailed description is omitted.

  In the sensor structure 10 according to the third embodiment, the lower surface 121 of the core layer 12 is not inclined and is formed horizontally, and the core layer 12 includes the first core layer 12a and the second core. It differs from the first embodiment and the second embodiment described above in that the layer 12b is bonded to the bonding surface 13.

  The first core layer 12a and the second core layer 12b are composed of transparent dielectric members having different refractive indexes. Thus, when the excitation light 42 is incident on the joint surface 13 of the two transparent dielectric members having different refractive indexes, the excitation light 42 is refracted at the joint surface 13.

  Therefore, the core layer 12 in which the first core layer 12a having a predetermined refractive index and the second core layer 12b having an appropriate refractive index different from the first core layer 12b are bonded at the bonding surface 13 is provided. As shown in FIG. 9, the excitation light 42 totally reflected in the region (P) is refracted at the joint surface 13 as shown in FIG. 9, and the excitation light 42 has a predetermined incident angle θq that satisfies the total reflection condition (Q ).

  As described above, in the sensor structure 10 of the present embodiment, the excitation light 42 totally reflected in the region (P) is refracted at the bonding surface 13 and irradiates the region (Q) with the predetermined incident angle θq that satisfies the total reflection condition. As described above, the joint surface 13 between the first core layer 12 a and the second core layer 12 b constitutes an incident angle adjusting means in the sensor structure 10. That is, the first core layer 12a having a predetermined refractive index and the second core layer 12b having a refractive index different from that of the first core layer are bonded at the bonding surface 13, which is extremely simple. With the sensor structure, the incident angle when the excitation light 42 totally reflected in the region (P) irradiates the region (Q) can be adjusted.

  Similarly to the above-described embodiment, the sensor structure 10 is irradiated with the excitation light 42 once, and the total reflection fluorescence signal Sp of the region (P) and the electric field enhanced fluorescence signal Sq of the region (Q) are obtained. Since it can be measured, it is possible to easily correct the influence of the electric field enhancement due to the variation in the thickness of the metal thin film.

  In the above description, when various fluorescent signals are measured by single irradiation of excitation light, for example, a condensing member for condensing each fluorescent signal (fluorescent light) is arranged in each region, A light receiving surface of a light detection means such as a CCD camera can be divided and used so as to correspond to each region, and a fluorescence signal can be measured from each divided region. It is also possible to provide light detection means for each region.

  As mentioned above, although preferable embodiment of this invention was described, this invention is not limited to embodiment mentioned above. Various modifications are possible without departing from the spirit of the present invention.

DESCRIPTION OF SYMBOLS 1 Surface plasmon excitation fluorescence measurement apparatus 10 Sensor structure (sensor structure)
12 Core layer 12a First core layer 12b Second core layer 12i Incident surface 12u Upper surface 12l Lower surface 12l 'Inclined portion 13 Joint surface 14 Cladding layers 16p, 16q Fluorescent dye layer 18 Metal thin film 20 Immobilized ligand-containing layer 22 First Ligand 24 Second ligand 25 Decorative ligand 26 Fluorescent molecule 28 Analyte 30 Photodetector 40 Projector 42 Excitation light 100 Sensor structure 112 Transparent dielectric member 116p, 116q Fluorescent dye layer 118 Metal thin film 120 Immobilized ligand-containing layer 122 First ligand 124 Second ligand 125 Decorative ligand 126 Fluorescent molecule 128 Analyte 130 Light detection means 142 Excitation light A SPFS assay region A ′ Total reflection assay region P Total reflection fluorescence reference region Q Electric field enhanced fluorescence reference region Sa Fluorescence signal Sp total reflection fluorescence Gunaru Sq field enhancement fluorescent signal θa, θp, θq incident angle

Claims (5)

A core layer serving as a light propagation region, and a cladding layer laminated on at least one of the upper surface and the lower surface of the core layer,
On the surface of the core layer, at least a total reflection fluorescent reference region (P) in which a fluorescent dye layer is formed without using a metal thin film, and a fluorescent dye formed in the region (P) through a metal thin film An electric field-enhanced fluorescent reference region (Q) in which a fluorescent dye layer made of the same fluorescent dye as the layer is formed,
When the excitation light propagating through the core layer irradiates the region (P) at a predetermined incident angle (θp) that is a total reflection condition, the excitation light totally reflected in the region (P) A sensor structure used in a surface plasmon excitation enhanced fluorescence measurement device configured to irradiate the region (Q) at a predetermined incident angle (θq),
The incident angle (θp) of the excitation light when irradiating the region (P) is different from the incident angle (θq) of the excitation light when irradiating the region (Q),
Incident of the excitation light totally reflected in the region (P) so that the excitation light totally reflected in the region (P) irradiates the region (Q) at a predetermined incident angle (θq) that is a total reflection condition. A sensor structure comprising an incident angle adjusting means for adjusting an angle. The incident angle adjusting means is
The sensor structure according to claim 1, wherein the sensor structure is an inclined surface formed on a surface of the core layer. The core layer is configured by bonding a first core having a predetermined refractive index and a second core having a refractive index different from the first core;
The sensor structure according to claim 1, wherein a joint surface between the first core and the second core constitutes the incident angle adjusting unit.   Immobilization containing a ligand on the surface of the core layer through a metal thin film having the same thickness and the same metal as the metal thin film formed in the region (Q) without a fluorescent dye layer. The sensor structure according to any one of claims 1 to 3, wherein an SPFS assay region (A) in which an activated ligand-containing layer is formed is formed.   A surface plasmon excitation enhanced fluorescence measuring device comprising the sensor structure according to claim 1.