JP6114097B2 - Surface plasmon enhanced fluorescence measurement device - Google Patents

Description

  The present invention relates to a surface plasmon enhanced fluorescence measuring apparatus using a surface plasmon enhanced fluorescence method.

  Conventionally, detection and analysis of biomolecules using surface plasmon-enhanced fluorescence spectroscopy (SPFS: Surface Plasmon-field enhanced Fluorescence Spectroscopy) have been performed. Specifically, by irradiating light (excitation light) at a predetermined incident angle to a sensor chip in which a metal film is formed on a prism or diffraction grating, total reflection on the prism surface or light from the diffraction grating The electric field of the evanescent wave is enhanced by causing the evanescent wave (light wave) generated by this diffraction to resonate with the surface plasmon generated on the surface of the metal film (surface plasmon resonance). This enhanced electric field excites a fluorescent dye that labels the analyte (biomolecule) placed in the vicinity of the metal film (on the sensor chip), generates fluorescence from the fluorescent dye, and detects this fluorescence. This is to detect and analyze biomolecules.

For example, in Patent Document 1, a sample containing a fluorescent dye is irradiated with excitation light, and fluorescence emitted from the fluorescent dye is imaged on a two-dimensional detector such as a CCD camera to obtain a fluorescent image. A fluorescence image acquisition device is described. In the optical path from the fluorescent dye to the two-dimensional detector, a multilayer interference filter and a color filter are arranged. The multilayer interference filter transmits light in a wavelength band including the fluorescent wavelength band, and blocks light of other wavelengths (especially the wavelength band of excitation light) with a transmittance of about 1 × 10 ^−6. It is configured. The color filter transmits light in the fluorescent wavelength band and longer wavelengths, and transmits light having a wavelength shorter than the fluorescent wavelength band (excitation light side) with a transmittance of about 1 × 10 ^−3. It is configured to block. The double light shielding by the multilayer interference filter and the color filter prevents light having a wavelength other than fluorescence (particularly excitation light) from entering the two-dimensional detector and becoming noise.

International Publication WO2010 / 032306

  However, in the apparatus of Patent Document 1, when the excitation light and fluorescence wavelength bands are close, the intensity (light quantity) of the light in the fluorescence wavelength band (transmission range of the multilayer interference filter and the color filter) of the excitation light becomes high. End up. In addition, unlike the multilayer interference filter, the color filter has a gradual transmittance gradient in the transient region from the transmission region to the light shielding region. Therefore, if the excitation light and fluorescence wavelength bands are close, the color filter transient region The intensity of the excitation light that passes through increases. Therefore, if the excitation light and fluorescence wavelength bands are close, the multilayer interference filter and the color filter can remove the excitation light and the noise component on the short wavelength side, but the noise component on the long wavelength side is sufficient. Therefore, a clear fluorescent image cannot be obtained.

  The present invention has been made in view of the above problems, and provides a surface plasmon enhanced fluorescence measuring apparatus capable of reliably detecting fluorescence by removing noise even when excitation light and fluorescence wavelength bands are close to each other. The purpose is to do.

  The surface plasmon enhanced fluorescence measuring apparatus according to the present invention generates fluorescence from a fluorescent substance in the vicinity of the metal film by generating surface plasmon resonance on the metal film formed on the substrate based on light irradiation to the substrate. A surface plasmon-enhanced fluorescence measurement device that generates and measures the fluorescence, a photodetector that detects light including fluorescence emitted from the fluorescent substance in photon units, and a detection from the substrate to the photodetector A fluorescent filter that is disposed in the optical path and transmits only light in the fluorescent wavelength band, and a fluorescent filter that is disposed in the detection optical path from the fluorescent filter to the photodetector, and reduces light of all wavelengths at a predetermined rate. And a neutral density filter that allows the light to pass therethrough.

According to the above configuration, most of the light other than the fluorescence wavelength band is removed by the fluorescent filter from the light including the fluorescence emitted from the fluorescent material and the irradiation light (excitation light) that has passed or reflected through the substrate. After that, the light of all wavelengths is attenuated at a predetermined ratio by the neutral density filter, and then enters the photodetector.
When the wavelength band of fluorescence and excitation light is close, the excitation light cannot be removed sufficiently only with a filter that allows only light in the wavelength band of fluorescence to pass through, but excitation can be achieved by combining a fluorescence filter and a neutral density filter. Light can be removed sufficiently. Further, the neutral density filter tends to have higher transmittance as the wavelength is longer, and the fluorescence is higher in wavelength than the excitation light, so the SN ratio of fluorescence measurement is higher. In addition, the color filter can attenuate only the light on the short wavelength side from the peak wavelength of the excitation light, but the neutral density filter can attenuate the light on the short wavelength side and the long wavelength side of the peak wavelength of the excitation light. Noise can be removed more than when used.
In addition, although the neutral density filter also attenuates fluorescence, the photodetector can detect light in units of photons, and therefore can detect even attenuated fluorescence.
Therefore, even when the wavelength bands of the fluorescence and the excitation light are close, it is possible to reliably detect the fluorescence by removing the excitation light and its short wavelength side noise and long wavelength side noise.

  Further, the surface plasmon enhanced fluorescence measuring device of the present invention is arranged in a light source that emits the light, and in an irradiation light path from the light source to the substrate, and has a wavelength band that generates an evanescent wave that generates the surface plasmon resonance. You may have the excitation filter which lets only light pass.

  According to said structure, since the light of the wavelength used as noise can be removed with an excitation filter in an irradiation optical path, noise can be reduced more.

  Further, in the surface plasmon enhanced fluorescence measuring apparatus according to the present invention, the metal film is formed on a diffraction grating formed on the surface of the substrate, and the light source irradiates the diffraction grating of the substrate with light. It may be set to do.

  According to the above configuration, the incident angle of light with respect to the metal film is reduced to about 10 to 35 degrees as compared with the case where the substrate is a prism and the light is irradiated so as to be totally reflected at the interface between the prism and the metal film. Therefore, the apparatus can be miniaturized with respect to the surface direction of the metal film.

  Further, in the surface plasmon enhanced fluorescence measuring apparatus of the present invention, the detection optical path is provided on the diffraction grating side of the substrate, and the irradiation optical path is provided on the side of the substrate where the diffraction grating is not formed. May be.

  According to the above configuration, since the irradiation optical path and the detection optical path are arranged on both sides of the substrate, the configuration of the apparatus can be simplified and the apparatus can be made smaller than the configuration in which the illumination optical path and the detection optical path are arranged on one side of the substrate. Can be

  In the surface plasmon enhanced fluorescence measuring apparatus of the present invention, the optical system of the irradiation light may be formed of a polarization maintaining fiber.

According to the above configuration, since the optical system of the irradiation light is formed of the polarization maintaining fiber, the polarization state in the optical fiber can be maintained even when stress is applied to the polarization maintaining fiber, so that the excitation by fluctuation of the polarization state Power fluctuation can be suppressed and measurement can be performed stably.
Further, when the optical system of the irradiation light is formed of a polarization maintaining fiber, a light source with an optical fiber output can be used. When a light source having an optical fiber output is used, the light emitting portion and the laser oscillating portion can be separated. Therefore, since the laser oscillation unit and the control unit can be arranged in a space in the apparatus, the layout inside the apparatus can be designed relatively freely, and the apparatus can be miniaturized.
Further, when a polarization maintaining fiber is connected to the light source by using a light source with an optical fiber output, the incident optical system does not shift when the light source is replaced, so that realignment is not necessary when the light source is replaced.

  In the present invention, even when the wavelength bands of fluorescence and excitation light are close, noise can be reliably detected by removing noise.

It is a figure which shows schematic structure of the surface plasmon enhanced fluorescence measuring apparatus which concerns on embodiment. It is an expanded sectional view of the sensor chip in FIG. (A) is a schematic diagram of a spectrum of light emitted from a light source, (b) is a schematic diagram of a spectrum of light emitted to a sensor chip, and (c) is a diagram of light incident on a fluorescent filter. It is a schematic diagram of a spectrum, (d) is a schematic diagram of the spectrum of the light which injects into a neutral density filter, (e) is a schematic diagram of the spectrum of the light which injects into a photodetector. It is a graph which shows the attenuation factor of a neutral density filter and a color filter. It is a graph which shows the relationship between the intensity | strength of the excitation light attenuate | damped using each of a neutral density filter and a color filter, and an incident angle.

Embodiments of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, the surface plasmon enhanced fluorescence measurement device 1 of the present embodiment irradiates light to the sensor chip 20 on which a sample 30 (see FIG. 2) containing a fluorescent dye (fluorescent substance) 35 is placed. Thus, it is an apparatus that generates fluorescence from the fluorescent dye 35 by surface plasmon resonance and measures the fluorescence.

  The surface plasmon enhanced fluorescence measurement apparatus 1 includes a light source 2, a collimator lens 3, an excitation filter 4, a polarizing plate 5, and a fluorescence emitted from a fluorescent dye 35 disposed in an irradiation light path from the light source 2 to the sensor chip 20. And a collimator lens 7, a right angle mirror 8, a diaphragm 9, a fluorescent filter 10, a condensing lens 11, and a dimming element disposed in the detection optical path from the sensor chip 20 to the photodetector 14. A filter 12 and a diaphragm 13 are provided.

  As shown in FIG. 2, the sensor chip 20 includes a substrate 21, and a first adhesive film 22, a metal film 23, a second adhesive film 24, and an antibody binding film 25 stacked on the substrate 21. . The sample 30 is held on the antibody binding film 25.

  The sample 30 includes a biomolecule 32 to be analyzed (for example, BDNF (brain-derived neurotrophic factor) which is a biomarker for depression) in a state of being bound to the fluorescent dye 35. Examples of the fluorescent dye 35 include Cy5, Alexa Fluor 647, Dylight 647, Hilite Fluor 647, and the like.

  An example of a procedure for holding the sample 30 on the sensor chip 20 will be described. First, a solution containing an antibody 31 capable of binding to a biomolecule (antigen) 32 is dropped on the surface of the antibody binding film 25 of the sensor chip 20 to bind the antibody 31 to the antibody binding film 25. Then, after rinsing (washing) with a phosphate buffer, a solution containing the biomolecule 32 is dropped, and the biomolecule 32 is bound to the antibody 31 bound to the antibody binding membrane 25. Then, after rinsing with a phosphate buffer, a solution containing an antibody (primary antibody) 33 that can bind to the biomolecule 32 is dropped to bind the antibody 33 to the biomolecule 32 on the sensor chip 20. Then, after rinsing with a phosphate buffer, a solution containing an antibody (secondary antibody) 34 that can be bound to the antibody 33 and previously bound to the fluorescent dye 35 is dropped, and the antibody on the sensor chip 20 The antibody 34 is bound to 33.

  The configuration of the sample 30 held on the antibody binding film 25 may be other than the above as long as it has a fluorescent substance. For example, instead of the antibodies 33 and 34, an antibody that can be bound to the biomolecule 32 and bound to the fluorescent dye 35 may be used. Further, the fluorescent substance may be a fluorescent protein.

  The substrate 21 is made of a transparent material, for example, a synthetic resin such as cycloolefin polymer, polymethyl methacrylate (PMMA), or polycarbonate, or glass. A diffraction grating 21 a is formed on the surface of the substrate 21. The diffraction grating 21a has a configuration in which rectangular grooves extending linearly are arranged at equal intervals. Note that the pattern of the diffraction grating 21a and the shape of the groove are not limited to the above.

  The first adhesive film 22 is for bonding the substrate 21 and the metal film 23. If the substrate 21 and the metal film 23 can be directly fixed, the first adhesive film 22 may be omitted.

  The metal constituting the metal film 23 is preferably, for example, a transition metal such as gold, silver, copper, platinum, nickel, and particularly preferably silver, but may be any other metal as long as it can generate surface plasmon resonance. . When the metal film 23 is silver, the first adhesive film 22 is formed of chromium, titanium, or the like, for example.

  The second adhesive film 24 is for bonding the metal film 23 and the antibody binding film 25. The second adhesive film 24 may be omitted as long as the metal film 23 and the antibody binding film 25 can be directly adhered. When the metal film 23 is silver, the second adhesive film 24 is formed of, for example, chromium, titanium, or the like. Further, since silver is an unstable metal, when the metal film 23 is silver, the second adhesive film 24 also plays a role of preventing deterioration of the silver due to the phosphate buffer solution.

  The antibody binding film 25 is made of zinc oxide (ZnO) and is a transparent thin film. If the distance between the fluorescent dye 35 and the metal film 23 is short, the excited fluorescence energy moves to the metal film 23 and is quenched, so the antibody binding film 25 is set to a thickness that can prevent fluorescence quenching. ing. That is, the antibody binding film 25 has a role of binding the antibody 31 and a role of preventing fluorescence quenching with a predetermined interval between the fluorescent dye 35 and the metal film 23.

  As the light source 2, for example, a gas laser such as a semiconductor laser (LD) or a He—Ne laser, a fiber laser, or the like is used. The light intensity at the peak wavelength of the light (excitation light) La emitted from the light source 2 is, for example, 0.01 to 100 mW. The light source 2 has a circuit for driving and a temperature control mechanism such as a Peltier element to obtain a stable output / oscillation wavelength, and is an optical fiber output type light source.

  A polarization maintaining fiber 6 is disposed between the light source 2 and the collimator lens 3, and the excitation light La emitted from the light source 2 is transmitted to the collimator lens 3 while maintaining the polarization state. The polarization maintaining fiber 6 is connected to the light source 2 and the collimator lens 3 by a connector.

  The excitation filter 4 is formed by a multilayer interference filter. As shown in FIGS. 3A and 3B, the excitation filter 4 emits light in the wavelength band W1 that generates an evanescent wave that generates surface plasmon resonance, among the excitation light La emitted from the light source 2. It is configured to transmit and block (reflect) light of other wavelengths with an attenuation factor of 40 to 60 dB, for example. The wavelength band W1 of the transmission region of the excitation filter 4 is determined according to the absorption spectrum of the fluorescent dye 35. 3A and 3B schematically show the spectrum of the excitation light La emitted from the light source 2 and the spectrum of the excitation light La that has passed through the excitation filter 4. The wavelength band W1 of the transmission region of the excitation filter 4 is, for example, 5 to 20 nm.

  The polarizing plate 5 is for changing the excitation light La to P-polarized light. In the surface plasmon excitation enhanced fluorescence method, the polarization state of the excitation light must be P-polarized with respect to the sensor chip 20. S-polarized light does not contribute to the electric field enhancement and becomes a measurement noise component. A polarizing plate 5 having a high extinction ratio is used.

  After the excitation light La emitted from the light source 2 is converted into parallel light by the collimator lens 3, light other than the predetermined wavelength band W <b> 1 is removed by the excitation filter 4, and then P-polarized by the polarizing plate 5. The sensor chip 20 is irradiated at a predetermined incident angle from the substrate 21 side. The excitation light La diffracts the diffraction grating 21a to generate an evanescent wave, and the surface generated on the surface of the metal film 23 (boundary surface with the first adhesive film 22) by irradiation of the evanescent wave and the excitation light La. Resonance with plasmons enhances the electric field of the evanescent wave. This enhanced electric field excites the fluorescent dye 35 and the fluorescent Lb is emitted from the fluorescent dye 35. A part of the excitation light La irradiated to the sensor chip 20 passes through the sensor chip 20.

  Although not shown, the light source 2, the collimator lens 3, the excitation filter 4, and the polarizing plate 5 rotate integrally around the sensor chip 20 so that the incident angle of the excitation light La to the sensor chip 20 can be arbitrarily changed. It is configured to be movable. The incident angle of the excitation light La capable of generating surface plasmon resonance varies depending on the wavelength of the excitation light La, the structure of the diffraction grating 21a, the configuration (material and film thickness) of the sensor chip 20, the concentration of the sample 30, and the like. Irradiation of the excitation light La to the sensor chip 20 may be performed with a fixed incident angle, but is preferably performed while changing the incident angle (angle scanning).

  When laser angle scanning is performed, a strong stress is applied to the polarization maintaining fiber 6. When a single mode fiber is used instead of the polarization maintaining fiber 6, the polarization state fluctuates in the optical fiber due to stress, and the excitation power varies greatly. However, the polarization maintaining fiber 6 can maintain the polarization state. Therefore, fluctuations in excitation power can be suppressed to within ± 0.5%, and stable measurement can be performed.

  As shown in FIG. 3C, the wavelength band W2 of the fluorescence Lb emitted from the fluorescent dye 35 appears on the longer wavelength side than the wavelength band (W1) of the excitation light La that causes surface plasmon resonance. The wavelength band W2 of the fluorescence Lb varies depending on the wavelength band of the excitation light La and the fluorescent dye. For example, when the excitation light La has a peak wavelength of 649 nm and a full width at half maximum of about 40 nm, the fluorescence Lb has a peak wavelength of 672 nm and a full width at half maximum of about 40 nm.

  The fluorescent filter 10 is formed of a multilayer interference filter. As shown in FIG. 3D, the fluorescent filter 10 transmits light in the wavelength band W2 of the fluorescent light Lb emitted from the fluorescent dye 35, and transmits light of other wavelengths with an attenuation factor of, for example, 50 to 70 dB. It is configured to block (reflect).

The neutral density filter 12 is configured to allow light of all wavelengths to pass through while being reduced at a predetermined rate. The neutral density filter 12 is a kind of absorption filter, and is also called an ND (Neutral Density) filter. The absorption filter includes a color filter. Even if a plurality of interference filters are stacked, the noise removal effect is small. However, the combination of the fluorescent filter 10 that is an interference filter and the neutral density filter 12 that is an absorption filter increases the noise removal effect.
Further, the neutral density filter 12 may be composed of a single neutral density filter or may be a laminate of a plurality of neutral density filters having the same or different configurations. It is preferable to be configured. If a plurality of layers are stacked, problems such as interference occur depending on the arrangement, and the desired effect cannot be obtained.

  In general, the neutral density filter has no wavelength dependency and is considered to be uniformly dimmed at each wavelength. However, in practice, the transmittance tends to be higher at the longer wavelength side. The attenuation factor of the neutral density filter 12 is, for example, 20 to 30 dB. FIG. 4 is a graph showing an example of the dimming characteristic of the neutral density filter 12. FIG. 4 also shows an example of the attenuation rate of the color filter and an example of the peak wavelength of the excitation light La. As can be seen from FIG. 4, the color filter has a higher attenuation rate on the shorter wavelength side than the peak wavelength of the excitation light La (635 nm in FIG. 4), but the attenuation rate decreases as the wavelength increases. On the other hand, the neutral density filter 12 has a certain degree of attenuation at all wavelengths, although the attenuation becomes smaller as the wavelength becomes longer.

  FIG. 5 shows the measurement results of the light intensity when the excitation light is attenuated using the neutral density filter and the color filter shown in FIG. The horizontal axis of the graph in FIG. 5 indicates the incident angle of the excitation light to the filter. As can be seen from FIG. 5, the excitation light cannot be removed sufficiently when the color filter is used, but the excitation light can be substantially removed when the neutral density filter is used.

  As the photodetector 14, a sensor capable of detecting light in units of photons, such as a PMT (Photomultiplier Tube) is used. The sensitivity of the photodetector 14 varies depending on the wavelength of light to be detected.

  The fluorescence Lb emitted from the fluorescent dye 35 by the surface plasmon resonance passes through the collimator lens 7 together with a part of the excitation light La that has passed through the sensor chip 20, and then is reflected by the right-angle mirror 8, and then the diaphragm 9 After the light beam width is narrowed through, light other than the wavelength band W2 of the fluorescence Lb is removed by the fluorescence filter 10 (see FIG. 3D). Thereby, most of the excitation light La is removed.

  Then, the light (fluorescence Lb and excitation light La) that has passed through the fluorescent filter 10 is collected by the condenser lens 11 and then light of all wavelengths is emitted by the neutral density filter 12 as shown in FIG. The light is attenuated, and then enters the photodetector 14 through the diaphragm 13.

  When the wavelength band of the excitation light La and the wavelength band of the fluorescence Lb are close, the excitation light La leaks from the light shielding area of the excitation filter 4 and passes through the transmission area of the fluorescence filter 10 (wavelength band W2 of the fluorescence Lb). Although the intensity of light increases, the excitation light La can be attenuated to such an extent that it is not detected by the photodetector 14 by the neutral density filter 12. Further, since the neutral density of the neutral density filter 12 is larger on the shorter wavelength side, the excitation light La leaking from the light shielding area of the fluorescent filter 10 can be attenuated to the extent that it is not detected by the photodetector 14. Therefore, the fluorescence can be reliably detected by the photodetector 14 without being affected by noise due to the excitation light La.

In the surface plasmon enhanced fluorescence measuring apparatus 1 of the present embodiment, the light including the fluorescence Lb emitted from the fluorescent dye 35 and the excitation light La that has passed through the substrate 21 is transmitted by the fluorescence filter 10 other than the wavelength band W2 of the fluorescence Lb. After most of the light has been removed, the light of all wavelengths is attenuated by a predetermined ratio by the neutral density filter 12 and then enters the photodetector 14.
When the wavelength band of the excitation light La and the wavelength band of the fluorescence Lb are close, the excitation light La cannot be sufficiently removed only by the filter that allows only the light in the fluorescence wavelength band to pass. However, the fluorescence filter 10 and the neutral density filter In combination with 12, excitation light La can be sufficiently removed. Further, the neutral density filter 12 tends to have higher transmittance as the wavelength is longer, and the fluorescence Lb is longer than the excitation light La, so that the SN ratio of fluorescence measurement is higher. In addition, the color filter can attenuate only light on the short wavelength side from the peak wavelength of the excitation light La, but the neutral density filter 12 can attenuate light on the short wavelength side and long wavelength side of the peak wavelength of the excitation light La. Noise can be removed more than when color filters are used.
Further, although the neutral density filter 12 also attenuates the fluorescence Lb, since the photodetector 14 can detect light in photon units, even the attenuated fluorescence Lb can be detected.
Therefore, even when the peak wavelengths of the fluorescence Lb and the excitation light La are close, it is possible to reliably detect the fluorescence Lb by removing the excitation light and noise on the short wavelength side and the long wavelength side thereof.

  In the present embodiment, the excitation filter 4 that passes only the light in the wavelength band W1 that generates the evanescent wave that generates surface plasmon resonance is disposed in the irradiation optical path from the light source 2 to the sensor chip 20. For this reason, light having a wavelength that causes noise in the irradiation optical path can be removed by the excitation filter 4, so that noise can be further reduced.

  In this embodiment, in order to generate an evanescent wave by diffracting light by irradiating the diffraction grating 21a formed on the substrate 21 with the excitation light La, a prism is provided instead of the substrate 21 and Compared with the case where an evanescent wave is generated by reflection, the incident angle of the excitation light La to the metal film 23 can be reduced to about 10 to 35 degrees, so that the apparatus can be miniaturized with respect to the surface direction of the metal film 23.

  In the present embodiment, since the irradiation optical path and the detection optical path are arranged on both sides of the substrate 21, the configuration of the apparatus can be simplified and compared with the configuration in which the illumination optical path and the detection optical path are arranged on one side of the substrate 21. Can be miniaturized.

  In this embodiment, since the polarization maintaining fiber 6 is used in the illumination optical path, the polarization state in the optical fiber can be maintained even if stress is applied to the polarization maintaining fiber 6, so that the excitation power due to the fluctuation of the polarization state Variation can be suppressed, and measurement can be performed stably.

  Further, in the present embodiment, since a light source with an optical fiber output is used, the light emitting portion and the laser oscillating portion can be separated. Therefore, since the laser oscillation unit and the control unit can be arranged in a spaced part in the apparatus 1, the layout inside the apparatus 1 can be designed relatively freely, and the apparatus 1 can be downsized. it can.

  Further, the irradiation optical path should not be shifted from the collimator lens 3 onward. In this embodiment, since the polarization maintaining fiber 6 is connected to the light source 2 and the collimator lens 3, the incident optical system does not shift when the light source 2 is replaced. Therefore, realignment is not necessary when the light source 2 is replaced. Become.

  The preferred embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments, and various modifications can be made as long as they are described in the claims.

  In the above embodiment, the substrate 21 on which the diffraction grating 21a is formed is used as the substrate of the sensor chip 20, but the substrate may be a prism. When a prism is used, a first adhesive film (optional), a metal film, a second adhesive film (optional), and an antibody binding film may be laminated on the surface of the prism. It may be fixed to the prism. When a prism is used, surface plasmon resonance is generated by irradiating light under the condition of total reflection on the surface of the metal film on the prism side. The detection optical path for detecting fluorescence is arranged on the prism side of the sensor chip.

  Moreover, in the said embodiment, although the collimator lens 3, the excitation filter 4, and the polarizing plate 5 are arrange | positioned in the irradiation optical path, if the excitation filter 4 is arrange | positioned, the irradiation optical path is a structure other than the said embodiment. It may be. Further, the detection optical path may have a configuration other than the above embodiment as long as the fluorescent filter 10 and the neutral density filter 12 are arranged.

  Moreover, in the said embodiment, although the transmission region of the fluorescence filter 10 is substantially in agreement with the wavelength band W2 of the fluorescence Lb generated by surface plasmon resonance (see FIG. 3B), the transmission region of the fluorescence filter 10 is As long as the peak wavelength of the fluorescence Lb is included, they do not have to coincide completely.

DESCRIPTION OF SYMBOLS 1 Surface plasmon enhanced fluorescence measuring apparatus 2 Light source 4 Excitation filter 6 Polarization maintaining fiber 10 Fluorescence filter 12 Neutral filter 14 Photo detector 21 Substrate 21a Diffraction grating 23 Metal film 35 Fluorescent dye (fluorescent substance)
La excitation light Lb fluorescence

Claims (5)

Surface plasmon enhanced fluorescence in which fluorescence is generated from a fluorescent substance in the vicinity of the metal film by generating surface plasmon resonance on the metal film formed on the substrate based on light irradiation to the substrate and measuring the fluorescence. A measuring device,
A photodetector for detecting light containing fluorescence emitted from the fluorescent substance in photon units;
A fluorescent filter disposed in a detection optical path from the substrate to the photodetector, and passing only light in the fluorescence wavelength band; and
A surface plasmon enhanced fluorescence measuring apparatus, comprising: a neutral density filter disposed in a detection optical path from the fluorescence filter to the photodetector and allowing light of all wavelengths to pass through while being reduced at a predetermined rate. A light source that emits the light;
2. An excitation filter that is disposed in an irradiation light path from the light source to the substrate and passes only light in a wavelength band that generates an evanescent wave that generates the surface plasmon resonance. Surface plasmon enhanced fluorescence measurement device. The metal film is formed on a diffraction grating formed on the surface of the substrate;
The surface plasmon enhanced fluorescence measurement apparatus according to claim 1 or 2, wherein the light source is set to irradiate light to the diffraction grating of the substrate. The detection optical path is provided on the diffraction grating side of the substrate;
4. The surface plasmon enhanced fluorescence measuring apparatus according to claim 3, wherein the irradiation optical path is provided on a side of the substrate where the diffraction grating is not formed.   5. The surface plasmon enhanced fluorescence measurement apparatus according to claim 1, wherein the irradiation light optical system is formed of a polarization maintaining fiber.

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