JP2014032148A - Surface plasmon excitation enhanced fluorescence acquisition structure and surface plasmon excitation enhanced fluorescence measurement system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To eliminate the need for a collimator in surface plasmon excitation enhanced fluorescence measurement.SOLUTION: A dielectric substrate 1 has a metal thin film 2 formed on a surface of a base material transmitting light. A reaction layer 3 including a fluorescent substance is formed on a surface of the metal thin film 2. A reverse surface of the metal thin film 2 at the part where the reaction layer 3 is formed is irradiated with excitation light at an angle where a surface plasmon is excited from the base material side of the dielectric substrate 1, and the fluorescent substance emits fluorescent light. A light guide member 5 transmits the fluorescent light emitted by the reaction layer 3 from the reaction layer 3 to a light detection part 25. A wavelength selection member 6 is arranged in an optical path of the fluorescent light transmitted from the reaction layer 3 to the light detection part 25. The wavelength selection member 6 transmits light having the wavelength of the fluorescent light at a predetermined rate or higher without reference to the angle of incidence, and attenuates light having the wavelength of the excitation light to a predetermined rate or lower.

Description

  The present invention relates to a surface plasmon excitation enhanced fluorescence acquisition structure and a surface plasmon excitation enhanced fluorescence measurement system.

  For example, a minute amount of a substance generated by a biological reaction is detected using surface plasmon-excited fluorescence spectroscopy (SPFS). In SPFS, the light intensity of fluorescence is extremely low compared to the excitation light that excites the fluorescent substance, so that it is necessary to discriminate and detect the fluorescence with high accuracy from the reflected excitation light when detecting the fluorescence.

  For example, in Patent Document 1, a wavelength selection function member that removes unnecessary light other than fluorescence is arranged on the upper or lower surface of a total reflection function member (condensing member) that causes fluorescence to reach the light detection means under total reflection conditions. Is disclosed. In the surface plasmon enhanced fluorescence sensor of Patent Document 1, it is described that an interference filter is used as a wavelength selection function member.

  Patent Document 3 discloses a technique for detecting the characteristics of an object with high accuracy by combining a surface plasmon resonance angle detection (SPR) method and an SPFS method. The surface plasmon resonance angle and fluorescence simultaneous detection device of Patent Document 3 irradiates light from a light source with a predetermined angle width to the boundary of a metal thin film on a substrate, and detects the surface plasmon resonance angle detected from reflected light and the substrate. The analyte bound to the sample probe is detected based on the fluorescence from the fluorescent material excited by the evanescent wave generated when the light is totally reflected at the boundary of the metal thin film.

  When analyzing cells fluorescently labeled with a fluorescent substance such as a fluorescent dye or a fluorescent-labeled monoclonal antibody, a colored glass filter may be used to select the fluorescence wavelength (see, for example, Patent Document 2). However, since the fluorescence light intensity is extremely low in SPFS, an interference filter is used to effectively attenuate the excitation light without effectively attenuating the fluorescence to be measured and to select the wavelength effectively.

International Publication No. 2010/101052 JP 05-45355 A JP 2002-62255 A

  The interference filter has higher wavelength selectivity than the colored glass filter. Further, the attenuation rate of transmitted light is small, and the attenuation rate of light to be cut is extremely high. However, the interference filter has a wavelength selection characteristic that greatly depends on the incident angle, and has high wavelength selectivity for light incident on the surface of the interference filter at a specified angle. However, if the incident angle changes from the specified angle, it will be cut. The wavelength band shifts to the short wavelength side, and the transmittance also decreases.

  Therefore, in order to select a wavelength with the interference filter, it is necessary to make the light to be observed be a parallel light beam with a collimator and to enter the interference filter. It is necessary to provide and adjust the lens to configure the collimator. With a collimator, it is difficult to collimate fluorescence of a reaction field having a certain area, not a point light source. Furthermore, it is necessary to make the excitation light scattered and incident on the collimator parallel, but it is difficult to adjust such an optical system. When collimated light is formed, the light density decreases, and it becomes difficult to guide sufficient light to the limited light receiving area of the light receiving element.

  In addition, in order to introduce weak fluorescence to the light receiving element, it is necessary to assemble a multistage lens in an optical system using a lens. In this case, not only is the optical system bulky, but it is difficult to adjust the optical axis, and the optical axis may be shifted during use. Therefore, the surface plasmon excitation enhanced fluorescence measuring device is increased in size and the manufacturing cost is increased. Since the light to be guided is limited by the numerical aperture based on the lens diameter and distance, it is disadvantageous for high sensitivity. Furthermore, it is difficult that fluorescence is not a point light source and that the scattered light of excitation light from multiple directions is ideally parallel light. Considering the intensity ratio of fluorescence and excitation light, the light component that does not become parallel light occupies a considerably high ratio.

  The present invention has been made in view of the above circumstances, and an object thereof is to eliminate the need for a collimator in surface plasmon excitation enhanced fluorescence measurement.

In order to achieve the above object, a surface plasmon excitation enhanced fluorescence acquisition structure according to the first aspect of the present invention comprises:
A substrate having a metal thin film formed on the surface of a base material that transmits light;
The fluorescent material is irradiated from the base material side by excitation light at an angle that excites surface plasmons on the back surface of the metal thin film where the reaction layer containing the fluorescent material is formed on the surface of the metal thin film. A light guide that transmits the generated fluorescence from the reaction layer to a light detection unit that converts the fluorescence into an electrical signal;
Arranged in the light path of the fluorescence of the light guide part, regardless of the incident angle, the light of the wavelength of the fluorescence is transmitted more than a predetermined ratio, the light of the wavelength of the excitation light is attenuated to a predetermined ratio or less, An incident angle independent wavelength selection member;
It is characterized by providing.

  Preferably, the wavelength selection member is characterized in that a ratio between the transmittance of light having the fluorescence wavelength and the transmittance of light having a wavelength other than the fluorescence is 2 or more.

  Preferably, the light having a wavelength other than the fluorescence includes at least the excitation light.

  Preferably, light having a wavelength other than the fluorescence is the excitation light.

  Preferably, a part of the light guide part includes a light guide member, and the wavelength selection member is integrally formed with the light guide member.

  Alternatively, a light guide member may be included in a part of the light guide unit, and at least a part of the light guide member may be formed of a material having the property of the wavelength selection member.

Preferably, it has a light guide member holding portion for holding the light guide member,
The light guide member holding portion functions as a light shielding member of the light guide member.
It is characterized by that.

  Furthermore, the light guide member holding portion may be composed of a plurality of members and assembled by fitting the plurality of members.

  Preferably, a condensing lens is disposed between the light guide member and the substrate to collect fluorescence generated by the fluorescent material on the light guide member.

Preferably, a cover is provided that covers a surface of the substrate on which the metal thin film is to be formed and has a flow path that passes through a portion on which the reaction layer is to be formed.
The condensing lens is fixed to or integrally formed with the cover.

  Alternatively, the condensing lens may be fixed or integrally formed on the light guide member side.

  Preferably, the light guide member includes a light shielding member that shields a side surface other than the surface on which the fluorescence is incident and the surface on which the fluorescence is emitted.

Preferably, a cover is provided that covers a surface of the substrate on which the metal thin film is to be formed and has a flow path that passes through a portion on which the reaction layer is to be formed.
The cover is formed with a hole for inserting a nozzle or a tube through which the liquid passes.

The surface plasmon excitation enhanced fluorescence measurement system according to the second aspect of the present invention is:
A surface plasmon excitation enhanced fluorescence acquisition structure according to a first aspect;
A light source that emits excitation light at an angle that excites surface plasmons on the back surface of the reaction layer in the metal thin film from the base material side,
A light detection unit that converts fluorescence generated by the fluorescent material into an electrical signal;
It is characterized by providing.

  According to the present invention, a collimator can be dispensed with in surface plasmon excitation enhanced fluorescence measurement.

It is a figure which shows the cross section of the surface plasmon excitation enhanced fluorescence measuring system which concerns on Embodiment 1 of this invention. It is a figure which shows the conceptual characteristic of a filter. It is a figure which shows the cross section of the surface plasmon excitation enhanced fluorescence measuring system which concerns on Embodiment 2 of this invention. It is a figure which shows the cross section of the surface plasmon excitation enhanced fluorescence measuring system which concerns on Embodiment 3 of this invention. It is a figure which shows the cross section of the surface plasmon excitation enhanced fluorescence measuring system which concerns on Embodiment 4 of this invention. It is a figure which shows the cross section of the surface plasmon excitation enhanced fluorescence measuring system which concerns on Embodiment 5 of this invention.

  Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. In the drawings, the same or equivalent parts are denoted by the same reference numerals.

(Embodiment 1)
FIG. 1 shows a cross section of a surface plasmon excitation enhanced fluorescence measurement system according to Embodiment 1 of the present invention. The surface plasmon excitation enhanced fluorescence measurement system (hereinafter referred to as SPFS system) 20 includes a surface plasmon excitation enhanced fluorescence acquisition structure 21, a light source 22, an excitation light guide 23, a light receiver 24, and a light detector 25. A surface plasmon excitation enhanced fluorescence acquisition structure (hereinafter referred to as SPF acquisition structure) 21 includes a dielectric substrate 1, a reaction layer 3, a condensing lens 4, a rod lens (light guide member) 5, and a wavelength selection member (light filter). 6 is provided. In the dielectric substrate 1, a metal thin film 2 is formed on the surface of a base material (dielectric material) that transmits light (excitation light).

  The condenser lens 4 is a hemispherical lens, for example. The condensing lens 4 is opposed to the reaction layer 3 and is held by the cover 11 in contact with the reaction layer 3 or close to a certain distance. The condensing lens 4 and the cover 11 may be integrally formed. The rod lens 5 is held in contact with or close to the convex surface of the condenser lens 4 by a lens holder (light guide member holding portion) 12. The rod lens 5 is made of, for example, optical fiber glass or transparent resin. The optical axes of the condensing lens 4 and the rod lens 5 coincide with each other, and the optical axes pass through the center of the reaction layer 3. The condensing lens 4 and the rod lens 5 are light guide members.

  On the opposite side of the rod lens 5 from the condensing lens 4, a light detection unit 25 is disposed to face the wavelength selection member 6. In the light detection unit 25, the light receiving surface faces the end surface of the rod lens 5. The light detection unit 25 is, for example, a photomultiplier tube (PMT).

  The wavelength selection member 6 has a wavelength selection characteristic that does not depend on the incident angle of light. For the wavelength selection member 6, for example, a colored glass filter is used. As the wavelength selection member 6, in addition to a colored glass filter, colored water, oil, colored plastic, or the like can be used. For example, as the wavelength selection member 6, a dye solution having desired wavelength selection characteristics may be enclosed in a transparent container. Further, a colored flexible film or the like can be used. The wavelength selection member 6 may be integrated by being brought into close contact with the end surface of the rod lens 5.

  The dielectric substrate 1 is placed in close contact with the excitation light guide 23. The excitation light guide 23 is, for example, a prism or a cylindrical lens. In FIG. 1, a cylindrical lens is shown as the excitation light guide 23. In order to prevent the excitation light E from being reflected at the boundary between the excitation light guide 23 and the dielectric substrate 1, a transparent liquid having the same refractive index as that of the dielectric substrate 1 is applied and adhered between them. The dielectric substrate 1 and the excitation light guide 23 may be formed integrally.

  The light source 22 irradiates the metal thin film 2 with the excitation light E at the surface plasmon resonance angle from the excitation light guide part 23 side of the dielectric substrate 1. The light receiving unit 24 receives the excitation light E and detects whether surface plasmon resonance has occurred from the intensity of the excitation light E. Further, the excitation light E is absorbed so as not to be scattered outside.

  Fluorescence is generated from the fluorescent material contained in the reaction layer 3 by the evanescent wave generated when the light is totally reflected at the boundary between the dielectric substrate 1 and the metal thin film 2. For example, fluorescence is generated by exciting a labeled fluorescent molecule captured by an antigen-antibody reaction with localized polarization. The amount of fluorescence depends on the amount (concentration) of the reactive substance that generates fluorescence. By measuring the amount of fluorescent light, the amount (concentration) of the reactant can be known.

  The material of the metal thin film 2 formed on the surface of the dielectric substrate 1 is preferably at least one metal selected from the group consisting of gold, silver, aluminum, copper and platinum. More preferably, the metal thin film 2 is made of gold, and may be an alloy or a laminate of these metals.

  Fluorescence from the excited fluorescent material is condensed to the diameter of the rod lens 5 by the condenser lens 4 and is incident on the end surface of the rod lens 5. The condensing lens 4 condenses the fluorescence so that the fluorescence incident on the rod lens 5 is totally reflected on the side surface of the rod lens 5 or is emitted from the other end of the rod lens 5 without hitting the side surface. The light incident from the end surface of the rod lens 5 on the condenser lens 4 side is only attenuated by the material of the rod lens 5 and is almost emitted from the other end.

  Furthermore, a reflective layer, for example, a metal such as aluminum, gold, or silver may be formed on the side surface of the rod lens 5 by sputtering or the like, and a light beam that is less than the critical angle may be reflected and guided by the side surface. Further, the same surface treatment may be applied to the inner wall of the lens holder 12, or a material that reflects light may be filled in the gap between the rod lens 5 and the lens holder 12. In this way, it is desirable that the light incident from the end surface of the rod lens 5 on the condenser lens 4 side is emitted from the other end.

  The light emitted from the rod lens 5 is incident on the light receiving surface of the light detection unit 25 after the light having a wavelength other than fluorescence is greatly attenuated by the wavelength selection member 6. The light path from the reaction layer 3 (metal thin film 2) to the light detection unit 25 is referred to as a light guide unit. The SPF acquisition structure 21 of the present embodiment transmits the light having the fluorescence wavelength at a predetermined ratio or more in the optical path of the fluorescence of the light guide unit regardless of the incident angle, and transmits the light having the wavelength of the excitation light to the predetermined wavelength. An incident angle-independent wavelength selection member 6 that attenuates to a ratio or less is disposed.

  The reaction field that generates fluorescence has a certain area and is not a point light source, so the light emitted from the rod lens 5 does not become a parallel light beam. In addition to the fluorescence that is not parallel, light from which the excitation light has leaked and light from which the excitation light has been reflected or scattered are incident on the wavelength selection member 6 at various incident angles. However, since the wavelength selection member 6 has wavelength selection characteristics that do not depend on the incident angle, light other than fluorescence is uniformly attenuated regardless of the incident angle. As a result, the amount of fluorescent light can be detected by the light detection unit 25 without using a collimator.

  FIG. 2 is a diagram illustrating the conceptual characteristics of the filter. The transmittance A of the colored glass filter in FIG. 2 is the ratio (referred to as the selection ratio) of the transmittance of the wavelength having a small attenuation (transmitting) and the transmittance of the wavelength having a large attenuation (blocking) (referred to as a selection ratio). Is smaller than the selection ratio. Further, the transmittance B of the interference filter has a steep slope from the wavelength to be blocked to the wavelength to be transmitted, whereas the slope from the wavelength to be cut off by the colored glass filter is gentle. However, the transmittance B of the interference filter is in the case of a specified incident angle, and if there is a light component deviated from the specified incident angle, the transmittance B is as shown by the transmittance B ′ indicated by a one-dot chain line. Thus, the selection ratio such as the transmittance B cannot be obtained.

  The transmittance B ′ and the transmittance B ″ indicated by a two-dot chain line in FIG. 2 conceptually show an example in the case where there is light incident on the interference filter at an angle larger than a prescribed incident angle. θ (when the transmittance is B), θ ′ is the incident angle when the transmittance is B ′, and θ ″ is the incident angle when the transmittance is B ″, θ <θ ′ <θ ″. Usually θ = 0 ° (corresponding to the normal of the incident surface). If the incident angle of all rays incident on the filter is θ, the interference filter has the characteristic of transmittance B, but actually the incident light enters the interference filter at an arbitrary incident angle larger than θ, such as θ ′ and θ ″. The first reason for the generation of light rays incident on the interference filter at an angle greater than the specified incident angle is that the reaction field that generates fluorescence has a certain area and is not a point light source. However, the incident angle does not become constant, and the second is the scattered light component of the excitation light that is reflected from many directions. In the case of a multi-stage lens, a shift occurs due to a shift in the relative position of each element arranged on the optical axis, such as a shift in the optical axis between lenses, an inclination of an interference filter, or the like.

  Light rays having an incident angle larger than θ are not uniformly distributed, and the ratio of the light amounts of incident angles θ, θ ′, θ ″ is a deviation from the optical axis of the light emitting position, unevenness of light emission intensity on the light emitting surface, It varies depending on the intensity and direction of the scattered light of the excitation light, etc. In some cases (light quantity at incident angle θ)> (light quantity at incident angle θ ′)> (light quantity at incident angle θ ″). (Light quantity at angle θ) <(light quantity at incident angle θ ′) <(light quantity at incident angle θ ″), and sometimes (light quantity at incident angle θ ′)> (light quantity at incident angle θ)> (incident angle θ ″) The ratio of the light amount and the light amount varies randomly. That is, the ratio of the transmittances B, B ′, and B ″ is different for each measurement, and as a result, the measurement results vary.

  For convenience, the description has been made by modeling with three points of transmittances B, B ′, and B ″, but there are originally an infinite number of points, and it is clear that the ratio thereof varies for each measurement. In 'and transmittance B', the transmittance in the right wavelength region to be selected is lower than the transmittance B, and the transmittance in the left wavelength to be blocked is increased. In addition, the boundary between the wavelength to be blocked and the wavelength to be transmitted moves to the blocking side, and the inclination becomes gentle. As a result, the transmitted component of the wavelength of the excitation light indicated by the emission intensity IE increases.

  The wavelength selection characteristic of the wavelength selection member 6 can be represented by the ratio of the transmittance (S2) of the wavelength of the target fluorescence and the transmittance (S1) of the wavelength of the excitation light (transmittance relative ratio = S2 / S1). The value obtained by dividing the area of the hatched portion in FIG. 2 by the area of the fluorescence emission intensity IF is defined as the fluorescence wavelength transmittance S2. Further, the value obtained by dividing the area of the hatched portion by the right upward by the emission intensity IE of the excitation light is defined as the transmittance S1 of the wavelength of the excitation light. The wavelength selection characteristic of the wavelength selection member 6 is represented by transmittance relative ratio = S2 / S1.

  The wavelength selection characteristic of the wavelength selection member 6 is selected according to the wavelength of the fluorescence to be measured and the wavelength of the excitation light. The ratio of the transmittance of light having a wavelength of fluorescence and the transmittance of light having a wavelength other than fluorescence of the wavelength selection member 6 is preferably 2 or more. Preferably, the relative transmittance ratio (S2 / S1) of the wavelength selection member 6 is 2 or more. The transmittance relative ratio (S2 / S1) of the wavelength selection member 6 is more preferably 5 or more, still more preferably 10 or more, still more preferably 30 or more, and still more preferably 50 or more.

The concentration of C-reactive protein (CRP) was measured using the SPFS system 20 of the first embodiment. For example, when the relative transmittance ratio (S2 / S1) of the wavelength selection member 6 is 50 or more, the coefficient of variation (CV: Coefficient) is obtained when the CRP concentration ranges from 10 ⁻⁷ mg / dL to 10 ⁻³ mg / dL. of Variation, standard deviation / average value) were 10 or less, and very good results were obtained.

  In the conventional SPFS system, an interference filter is employed in order not to attenuate minute fluorescence, but collimation is difficult. And unless collimation is performed, excitation light cannot be removed effectively. On the other hand, the present embodiment is a concept for emphasizing the ratio (S / N ratio) between fluorescence and excitation light that cannot be removed, and performing wavelength selection independent of the incident angle. As a result, measurement accuracy equal to or better than that of an SPFS system using an interference filter and a collimator was obtained.

  Further, when non-uniform light emission in the reaction field having a certain area is passed through the rod lens 5, the light density distribution of the emitted light becomes Gaussian distribution due to the homogenization effect, for example, in the case of the rod lens 5 having a circular cross section. Therefore, even if the light receiving surface of the light detection unit 25 is small, it can be efficiently incident on the light detection unit 25. As a result, the sensitivity for detecting fluorescence is improved.

  The SPF acquisition structure 21 according to the first embodiment does not limit the unit for handling or replacement. That is, in the present specification, in the “SPFS acquisition structure”, all members constituting the structure may be integrally formed, or the members may be formed in a separable form. Alternatively, a part of the plurality of members constituting the SPFS acquisition structure may be formed in a separable form. For example, in this specification, a unit exchanged for each measurement including a portion where the reaction layer 3 is formed can be referred to as an SPFS device. For example, a configuration in which the SPFS device is removed from the SPFS system can be referred to as an SPFS device. In the present specification, “system” means an assembly or combination of members necessary for performing measurement / detection of a target substance using the SPFS acquisition structure of the present invention. The SPFS device can be replaced for each measurement. For example, only the derivative substrate 1 including the metal thin film 2 and the reaction layer 3 may be used. The dielectric substrate 1 and the excitation light guide 23 may be integrally formed, and the dielectric substrate 1 including the excitation light guide 23 and having the metal thin film 2 formed thereon and the reaction layer 3 may be used as an SPFS device. . FIG. 1 shows an example in which the SPFS device 26 extends from the excitation light guide 23 to the dielectric substrate 1 and the reaction layer 3. When the dielectric substrate 1 and the excitation light guide 23 are integrally formed, the step of aligning and closely contacting the dielectric substrate 1 and the excitation light guide 23 each time the SPFS device 26 is replaced can be omitted.

  Further, the SPFS device 26 may be formed by combining the condenser lens 4 and the cover 11 from the derivative substrate 1. Also in this case, the dielectric substrate 1 and the excitation light guide 23 can be formed integrally. Furthermore, the unit from the derivative substrate 1 to the rod lens 5 and the lens holder 12 can be combined to form an exchange unit.

  In the first embodiment, the rod lens 5, the lens holder 12, the wavelength selection member 6, and the light detection unit 25 are supported so as not to be relatively displaced. The wavelength selection member 6 can be exchanged according to the wavelength (fluorescence) to be selected. In the first embodiment, the same configuration from the condenser lens 4 to the rod lens 5 and the lens holder 12 can be obtained by exchanging the wavelength selection member 6 according to the measurement target.

(Embodiment 2)
FIG. 3 shows a cross section of a surface plasmon excitation enhanced fluorescence measurement system according to Embodiment 2 of the present invention. In the second embodiment, the rod lens also serves as a wavelength selection member that does not depend on the incident angle. The SPF acquisition structure 21 according to the second embodiment includes a rod lens (light guide member) 7 having wavelength selectivity instead of the rod lens 5 and the wavelength selection member 6 according to the first embodiment. Other configurations are the same as those in the first embodiment. The rod lens 7 is made of colored glass, for example. The rod lens 7 may be formed by mixing, applying, or coating a transparent resin with a component that provides a filter effect.

  Even in the SPFS system 20 of the second embodiment, the excitation light can be uniformly attenuated regardless of the incident angle of the light incident on the rod lens 7 from the condenser lens 4. As a result, similarly to the first embodiment, the light detection unit 25 can detect the amount of fluorescence without using a collimator. In addition, the rod lens 7 can be efficiently incident on the light detection unit 25 by the homogenization effect similarly to the rod lens 5 of the first embodiment.

  Since the SPFS system 20 of the second embodiment does not have the wavelength selection member 6 as compared with the first embodiment, the number of parts is small and the structure can be simplified. As in the first embodiment, the unit of handling or replacement may be, for example, a portion of the derivative substrate 1 including the metal thin film 2 and the reaction layer 3. FIG. 3 shows an example in which the SPFS device 26 can be exchanged from the excitation light guide 23 to the condenser lens 4 and the cover 11.

(Embodiment 3)
FIG. 4 shows a cross section of a surface plasmon excitation enhanced fluorescence measurement system according to Embodiment 3 of the present invention. In Embodiment 3, the sheet 9 on which the flow path 10 is formed is attached on the metal thin film 2, and the flow path 10 is used as a reaction part. The sheet 9 is formed of a slightly adhesive film or the like. Further, the cover 11, the lens holder 12, and the like are formed of a light shielding member, and a hole for inserting the liquid passing nozzle (or tube) 8 is formed in the cover 11.

  From one nozzle 8, a solution containing a fluorescent substance and a reactive substance is injected into the flow path 10, and the gas in the flow path 10 is extracted from the other nozzle 8 to introduce the solution into the flow path 10. By irradiating the metal thin film 2 with the excitation light E from the light source 22, surface plasmons are excited in the portion of the channel 10 on the opposite side. Due to the evanescent wave generated when the light is totally reflected, fluorescence is generated from the fluorescent material contained in the nearby solution (reaction layer: not shown). Similarly to the second embodiment, the fluorescence is collected by the condenser lens 4, the excitation light is removed by the rod lens 7, and enters the light detection unit 25.

  The cover 11 and the lens holder 12 are not only shielded from light, but the surface is painted black or colored to absorb excitation light (appears black when irradiated with excitation light only) so that light is not reflected, film pasting, plating or sputtering. For example, it is desirable to provide a thin film that can be shielded by light and does not reflect light. Or you may use the material of the color which absorbs excitation light except the window which permeate | transmits fluorescence. Although not shown in FIG. 4, the cover 11 is formed so that the rod lens 7 can be inserted, and the contact surface with the lens holder 12 does not have a gap. Thereby, the optical axis alignment of the rod lens 7 and the distance (contact or constant interval) with the condenser lens 4 are set, and the light from the outside is blocked.

  In the third embodiment, an AR coat (Anti Reflection Coat) 23a is further applied to the surface of the excitation light guide 23. The surface of the rod lens 7 is also provided with an AR coat 7a. The AR coats 23a and 7a can reduce stray light components due to surface reflection.

  With the wavelength selectivity that does not depend on the incident angle of the rod lens 7, as in the second embodiment, the light amount of the fluorescence can be detected by the light detection unit 25 without using a collimator. In the SPFS system 20 of the third embodiment, unnecessary light is prevented from entering the light detection unit 25 by shielding and preventing reflection of the cover 11 and the lens holder 12, and further preventing reflection of the AR coats 23a and 7a. Can do. As a result, the measurement accuracy of the SPFS system can be improved.

(Embodiment 4)
FIG. 5 shows a cross section of a surface plasmon excitation enhanced fluorescence measurement system according to Embodiment 4 of the present invention. In the fourth embodiment, the structure for forming the flow path 10 is changed from the third embodiment. As shown in FIG. 5, a flow path 10 is configured by a cover 11 on a dielectric substrate 1 (metal thin film 2). A flow path 10 that passes through a reaction layer (not shown) is formed in the cover 11. The condenser lens 4 is fixed to or integrally formed with the cover 11. By integrating the dielectric substrate 1, the cover 11, and the condenser lens 4, the number of components can be reduced. Other configurations are the same as those of the third embodiment.

  In the fourth embodiment, the cover 11 is further provided with a recess in which the lens holder 12 can be accommodated, and the lens holder 12 is fitted therein. In other words, the member that holds the condensing lens 4 and the rod lens 7 that are light guide members is composed of two members, the cover 11 and the lens holder 12, and can be assembled by fitting together. With this structure, light shielding around the condensing lens 4 and the rod lens 7, and axial alignment and distance adjustment between the rod lens 7 and the condensing lens 4 can be performed.

  The cover 11 is fixed to the dielectric substrate 1 by adhesion or the like. Accordingly, the condenser lens 4 is fixed to the dielectric substrate 1 via the cover 11. Also in the fourth embodiment, the SPFS device 26 can be formed from the excitation light guide 23 to the condenser lens 4 and the cover 11 (not shown).

  In the SPFS system of the fourth embodiment, the number of parts is reduced and the light shielding performance is further improved as compared with the third embodiment. As a result, the handling of the SPF acquisition structure 21 is facilitated.

(Embodiment 5)
FIG. 6 shows a cross section of a surface plasmon excitation enhanced fluorescence measurement system according to Embodiment 5 of the present invention. In the fifth embodiment, a configuration in which the lens holder 12 holds the condenser lens 4 is employed. In the fifth embodiment, the condensing lens 4 is moved to the lens holder 12 from the configuration of the fourth embodiment, and the fluorescent transmission member 13 in contact with the flow path 10 is provided. The fluorescent transmission member 13 is made of glass, quartz, plastic (PMMA (Poly (Methyl Methacrylate)), PS (polystyrene), PC (Polycarbonate), or the like). Other configurations of the SPF acquisition structure 21 are the same as those in the fourth embodiment.

  The condenser lens 4 is installed in the lens holder 12. The condensing lens 4 is fixed by the lens holder 12 so as to be in contact with the rod lens 7 or to be kept at a constant distance. The condenser lens 4 may be formed integrally with the rod lens 7. An AR coat 4a is applied to the surface of the condenser lens 4 on the flow path 10 side. In the fifth embodiment, for example, the condenser lens 4 is not included, and the portion from the excitation light guide 23 to the cover 11 can be used as the SPFS device 26 (not shown).

  Since the condensing lens 4 is held by the lens holder 12 together with the rod lens 7, the optical axes of the condensing lens 4 and the rod lens 7 do not shift. Then, the AR coat 4a can suppress reflection, and can effectively collect the fluorescence on the rod lens 7. By these, the structure (the cover 11 and the fluorescence transmission member 13) which forms the dielectric substrate 1 and the flow path 10 can be simplified. That is, the dielectric substrate 1 including the metal thin film 2, the cover 11, and the fluorescent transmission member 13 can be easily integrated into an exchange unit.

DESCRIPTION OF SYMBOLS 1 Dielectric substrate 2 Metal thin film 3 Reaction layer 4 Condensing lens 5 Rod lens (light guide member)
6 Wavelength selection member 7 Rod lens (light guide member)
8 Tube 9 Sheet 10 Flow path 11 Cover 12 Lens holder (light guide member holding portion)
13 Fluorescent transmission member 20 Surface plasmon excitation enhanced fluorescence measurement system (SPFS system)
21 Surface plasmon excitation enhanced fluorescence acquisition structure (SPF acquisition structure)
Reference Signs List 22 Light source 23 Excitation light guide unit 24 Light receiver 25 Photodetector 26 SPFS device

Claims (14)

A substrate having a metal thin film formed on the surface of a base material that transmits light;
The fluorescent material is irradiated from the base material side by excitation light at an angle that excites surface plasmons on the back surface of the metal thin film where the reaction layer containing the fluorescent material is formed on the surface of the metal thin film. A light guide that transmits the generated fluorescence from the reaction layer to a light detection unit that converts the fluorescence into an electrical signal;
Arranged in the light path of the fluorescence of the light guide part, regardless of the incident angle, the light of the wavelength of the fluorescence is transmitted more than a predetermined ratio, the light of the wavelength of the excitation light is attenuated to a predetermined ratio or less, An incident angle independent wavelength selection member;
A surface plasmon excitation enhanced fluorescence acquisition structure comprising:   2. The surface plasmon excitation enhancement according to claim 1, wherein the wavelength selection member has a ratio of a transmittance of light having a wavelength of the fluorescence to a transmittance of light having a wavelength other than the fluorescence of 2 or more. Fluorescence acquisition structure.   The surface plasmon excitation-enhanced fluorescence acquisition structure according to claim 2, wherein the light having a wavelength other than the fluorescence includes at least the excitation light.   3. The surface plasmon excitation enhanced fluorescence acquisition structure according to claim 2, wherein the light having a wavelength other than the fluorescence is the excitation light.   5. The surface according to claim 1, wherein a part of the light guide part includes a light guide member, and the wavelength selection member is formed integrally with the light guide member. 6. Plasmon excitation enhanced fluorescence acquisition structure.   The light guide member is included in a part of the light guide part, and at least a part of the light guide member is formed of a material having properties of the wavelength selection member. 2. The surface plasmon excitation enhanced fluorescence acquisition structure according to claim 1. A light guide member holding portion for holding the light guide member;
The light guide member holding portion functions as a light shielding member of the light guide member.
The surface plasmon excitation-enhanced fluorescence acquisition structure according to claim 5 or 6.   8. The surface plasmon excitation enhanced fluorescence acquisition structure according to claim 7, wherein the light guide member holding portion is composed of a plurality of members and can be assembled by fitting the plurality of members.   The condensing lens which collects the fluorescence which the said fluorescent substance generate | occur | produces in the said light guide member is arrange | positioned between the said light guide member and the said board | substrate, The any one of Claim 5 thru | or 8 characterized by the above-mentioned. The surface plasmon excitation-enhanced fluorescence acquisition structure described in 1. Covering a surface of the substrate on which the metal thin film is formed, and having a cover in which a flow path passing through a portion where the reaction layer is formed is formed;
The surface plasmon excitation enhanced fluorescence acquisition structure according to claim 9, wherein the condenser lens is fixed to or integrally formed with the cover.   The surface plasmon excitation-enhanced fluorescence acquisition structure according to claim 9, wherein the condenser lens is fixed or integrally formed on the light guide member side.   The surface according to any one of claims 5 to 11, further comprising a light shielding member that shields a side surface of the light guide member other than a surface on which the fluorescence is incident and a surface on which the fluorescence is emitted. Plasmon excitation enhanced fluorescence acquisition structure. Covering a surface of the substrate on which the metal thin film is formed, and having a cover in which a flow path passing through a portion where the reaction layer is formed is formed;
The surface plasmon excitation-enhanced fluorescence acquisition structure according to any one of claims 1 to 12, wherein the cover is formed with a hole for inserting a nozzle or a tube through which a liquid passes through the flow path. . The surface plasmon excitation enhanced fluorescence acquisition structure according to any one of claims 1 to 13,
A light source that emits excitation light at an angle that excites surface plasmons on the back surface of the reaction layer in the metal thin film from the base material side,
A light detection unit that converts fluorescence generated by the fluorescent material into an electrical signal;
A surface plasmon excitation enhanced fluorescence measurement system comprising:

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