JP2012042280A - Method for measuring absolute value of electric field enhancement degree, device for measuring absolute value of electric field enhancement degree, method for evaluating measurement member, device for evaluating measurement member, method for detecting analyte and device for detecting analyte - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and device capable of measuring an electric field enhancement degree on a plane (in a plane direction) of a metallic thin film of a real target of measurement, and also obtaining three dimensional distribution of electric field enhancement degrees corresponding to thickness of the metallic thin film and a height position of an analyte captured in a vicinity of the metallic thin film so as to measure (or estimate) an accurate electric field enhancement degree.SOLUTION: In the method and device, a first excitation light is irradiated from a light source to generate a surface plasmon light on a surface of a metallic thin film, and through a dielectric member, a second excitation light which is different from the first excitation light is irradiated toward the metallic thin film to generate a propagation light on the surface of the metallic thin film, so that an interference pattern is formed by the surface plasmon light and the propagation light, and while changing a light volume of the second excitation light, based on the light volume of the second excitation light when contrast of the interference pattern becomes strongest, an electric field enhancement degree at a predetermined height position of an electric field enhancement degree measurement member is converted for estimation.

Description

  The present invention uses a measurement member including a dielectric member and a metal thin film formed on the upper surface of the dielectric member, and from the outside of the dielectric member below the dielectric member via the dielectric member. When irradiating excitation light from the light source toward the metal thin film, generating surface plasmon light on the surface of the metal thin film, enhancing the electric field on the metal thin film, and obtaining the electric field enhancement effect of the surface plasmon light, The present invention relates to a method for measuring an absolute value of an electric field enhancement for measuring an absolute value of an electric field enhancement and a measuring device for an absolute value of an electric field enhancement.

  Further, the present invention evaluates the measurement member based on the absolute value data of the electric field enhancement obtained by using the method for measuring the absolute value of the electric field enhancement and the measurement device for the absolute value of the electric field enhancement. The present invention relates to a measurement member evaluation method and a measurement member evaluation device.

  Furthermore, the present invention captures near the metal thin film based on the data on the absolute value of the electric field enhancement obtained by using the method for measuring the absolute value of the electric field enhancement and the measurement device for the absolute value of the electric field enhancement. The present invention relates to an analyte detection method and an analyte detection apparatus for detecting an analyte.

2. Description of the Related Art Conventionally, when detecting a very small substance, various specimen detection apparatuses that can detect such a substance by applying a physical phenomenon of the substance have been used.
As one of such analyte detection devices, a phenomenon in which high light output is obtained by resonating electrons and light in a minute region such as a nanometer level (Surface Plasmon Resonance (SPR) phenomenon). For example, a surface plasmon resonance device (hereinafter referred to as “SPR device”) that detects minute analytes in a living body can be used.

  In addition, based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) using surface plasmon resonance (SPR) phenomenon, it is possible to detect analytes with higher accuracy than SPR equipment. The surface plasmon enhanced fluorescence spectrometer (hereinafter referred to as “SPFS device”) is one of such specimen detection devices.

  In this surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), surface plasmon light is applied to the surface of the metal thin film under the condition that the excitation light such as laser light irradiated from the light source is attenuated by total reflection (ATR) on the surface of the metal thin film. By generating (coherent wave), the amount of photons contained in the excitation light irradiated from the light source is increased to several tens to several hundred times, and the electric field enhancement effect of the surface plasmon light is obtained.

  This electric field enhancement effect efficiently excites the fluorescent substance bound (labeled) with the analyte trapped in the vicinity of the metal thin film in the vicinity of the metal thin film, and observes this fluorescence, thereby allowing an extremely low concentration of the analyte to be analyzed. This is a method for detecting light.

  By the way, to know the absolute value of the electric field enhancement intensity, which is an index of the electric field enhancement effect of such surface plasmon light, for example, in clinical tests such as blood tests using surface plasmon excitation enhanced fluorescence spectroscopy (SPFS). In such a field where high-precision detection is required, it is important to improve the measurement accuracy of analyte detection by correcting detection data using the absolute value of the electric field enhancement intensity.

However, since this enhanced electric field is non-propagating light, direct observation is impossible, and visualization of the electric field enhancement has been conventionally studied.
For example, in Non-Patent Document 1 ("Surface plasmons at single nanoholes in Au films", Yin et al., Appl. Phys. Lett., Vol. 85, No. 3, 19 July 2004, pp. 468-469), Attempts have been made to measure electric field enhancement by observing interference fringes caused by interference between plasmon light and excitation light generated by incident excitation light using a near-field probe.

  That is, in the method of Non-Patent Document 1, as shown in FIG. 17, a metal thin film (gold film) 102 is formed on the upper surface of the dielectric 100, and a pinhole 104 is formed in the central portion. Then, the excitation light 106 is irradiated onto the metal thin film (gold film) 102 formed on the upper surface of the dielectric 100 through the dielectric 100 from directly below the dielectric 100.

  Then, an interference fringe due to the interference between the plasmon light 101 and the excitation light 106 generated at the edge portion 104 a of the pinhole 104 by the incident excitation light 106 is observed by the near-field probe 110 arranged immediately above the pinhole 104. is doing.

As a result, as shown in FIG. 18, concentric interference fringes are observed, whereby the electric field enhancement is measured (estimated).
In Non-Patent Document 2 ("Local excitation of surface plasmon polaritons at discontinuities of a metal film: Theoretical analysis and optical near-field measurements", L Salomon et al., PHYSICAL REVIEW B. vol. B, 125409) Attempts have been made to measure the electric field enhancement by observing interference between the plasmon light generated by the excited excitation light and the incident excitation light using a near-field probe.

  That is, in the method of Non-Patent Document 2, as shown in FIG. 19, a metal thin film (gold film) 202 is formed on the upper surface of the dielectric 200 and the dielectric 200 is passed through the dielectric 200 from obliquely below. Then, the excitation light 206 is applied to the metal thin film (gold film) 202 formed on the upper surface of the dielectric 200.

  Thereby, the plasmon light 201 is generated at the edge portion 204 of the metal thin film (gold film) 202, and the interference between the plasmon light and the excitation light 206 is observed by the near-field probe 210.

  As a result, as shown in the graph of FIG. 20, an electric field enhancement distribution according to the distance from the edge portion 204 is obtained.

"Surface plasmons at single nanoholes in Au films", Yin et al., Appl. Phys. Lett., Vol. 85, No. 3, 19 July 2004, pp. 468-469 `` Local excitation of surface plasmon polaritons at discontinuities of a metal film: Theoretical analysis and optical near-field measurements '', L Salomon et al., PHYSICAL REVIEW B. vol. B, 125409

However, the conventional method for measuring the electric field enhancement has the following problems.
That is, in the method of measuring the electric field enhancement in Non-Patent Document 1, as shown in FIG. 17, the incident angle is changed from obliquely below the dielectric 100 and the top surface of the dielectric 100 is interposed via the dielectric 100. When the excitation light 108 is irradiated onto the metal thin film (gold film) 102 formed in the above, for example, as shown in FIG. 21, the interference fringes do not become concentric circles. It may be difficult to measure (estimate).

  Further, in the method of measuring the electric field enhancement in Non-Patent Document 1, since the interference fringes are caused by the interference between the plasmon light 101 generated at the edge portion 104a of the pinhole 104 and the excitation light 106, the metal thin film (true) to be measured ( The electric field enhancement on the plane (plane direction) of the (gold film) 102 cannot be measured.

Furthermore, since the electric field enhancement varies depending on the shape of the edge portion 104a of the pinhole 104, it is practically difficult to measure the absolute value of the electric field enhancement.
Moreover, the pinhole 104 must be provided in the metal thin film (gold film) 102. For example, when used as a measurement member such as a microchip, the manufacturing process becomes complicated and the cost is increased.

  On the other hand, in the method for measuring the electric field enhancement in Non-Patent Document 2, it is possible to obtain the electric field enhancement distribution according to the distance from the edge portion 204, but it is actually possible to measure the absolute value of the electric field enhancement. Is difficult.

  Further, the method for measuring the electric field enhancement in Non-Patent Document 2 is a measurement based on the interference between the plasmon light 201 generated at the edge portion 204 of the metal thin film (gold film) 202 and the excitation light 206, so it is desired to perform a true measurement. The electric field enhancement intensity on the plane (plane direction) of the metal thin film (gold film) 202 cannot be measured.

Further, since the electric field enhancement varies depending on the shape of the edge portion 204 of the metal thin film (gold film) 202, it is practically difficult to measure the absolute value of the electric field enhancement.
Further, the edge portion 204 of the metal thin film (gold film) 202 must be provided. For example, when used as a measuring member such as a microchip, the manufacturing process becomes complicated and the cost increases.

  Moreover, in any method for measuring the electric field enhancement, a three-dimensional distribution of the electric field enhancement corresponding to the influence of the thickness of the metal thin film and the height position of the analyte trapped in the vicinity of the metal thin film is obtained. Is impossible at all.

  In view of the current situation, the present invention does not require pinholes or edges in the metal thin film as in the prior art, and measures the electric field enhancement on the plane (in the plane direction) of the metal thin film to be truly measured. In addition, it is possible to obtain a three-dimensional distribution of the electric field enhancement corresponding to the thickness of the metal thin film and the height position of the analyte trapped in the vicinity of the metal thin film. It is an object of the present invention to provide a method for measuring the absolute value of the electric field enhancement and an apparatus for measuring the absolute value of the electric field enhancement that can be estimated.

  In addition, the present invention can accurately know the absolute value of the electric field enhancement intensity, which is an index of the electric field enhancement effect of surface plasmon light, and thereby, for example, based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS). Evaluation of measuring members that can be used for process inspection, management, and quality assurance that can check and sort the formation state of metal thin films when used as measuring members such as microchips used in SPFS devices It is an object of the present invention to provide a method and an evaluation device for a measurement member.

  Furthermore, the present invention can accurately know the absolute value of the electric field enhancement intensity, which is an index of the electric field enhancement effect of the surface plasmon light, and thereby, for example, based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS). When used as a measurement member such as a microchip used in a SPFS device, the detection signal measurement value in the detection process, which is a subsequent process, is corrected by grasping the electric field enhancement intensity for each substrate to improve the measurement accuracy. It is an object of the present invention to provide an analyte detection method and an analyte detection apparatus that can perform the above-described process.

The present invention was invented in order to achieve the above-described problems and objects in the prior art, and the method for measuring the absolute value of the electric field enhancement of the present invention is as follows.
When the electric field on the metal thin film is enhanced by irradiating excitation light from the light source toward the metal thin film formed on the dielectric member through the dielectric member and generating surface plasmon light on the surface of the metal thin film. And a measurement method for measuring the absolute value of the electric field enhancement intensity,
An electric field enhancement measuring member is disposed at a predetermined height position on the surface of the metal thin film,
Irradiating the first excitation light from the light source toward the metal thin film through the dielectric member, and generating surface plasmon light on the metal thin film surface,
Irradiating a second excitation light different from the first excitation light toward the metal thin film through the dielectric member to generate propagating light on the surface of the metal thin film,
Generating interference fringes by the surface plasmon light and the propagating light;
Based on the light quantity of the second excitation light when the contrast of the interference fringes is maximized while changing the light quantity of the second excitation light, the electric field enhancement intensity at a predetermined height position of the electric field enhancement measurement member. Is converted and estimated.

In addition, the measurement device for the absolute value of the electric field enhancement intensity of the present invention,
When the electric field on the metal thin film is enhanced by irradiating excitation light from the light source toward the metal thin film formed on the dielectric member through the dielectric member and generating surface plasmon light on the surface of the metal thin film. And a measuring device for measuring the absolute value of the electric field enhancement intensity,
A member for measuring the electric field strength arranged at a predetermined height position on the surface of the metal thin film;
A first excitation light generator that irradiates the metal thin film with the first excitation light from the light source through the dielectric member, and generates surface plasmon light on the metal thin film surface;
Second excitation light that emits propagating light on the surface of the metal thin film by irradiating the metal thin film with second excitation light different from the first excitation light through the dielectric member. A generator,
When generating interference fringes due to the surface plasmon light and the propagating light,
Based on the light quantity of the second excitation light when the contrast of the interference fringes is maximized while changing the light quantity of the second excitation light, the electric field enhancement intensity at a predetermined height position of the electric field enhancement measurement member. And an electric field enhancement conversion device for converting and estimating.

  With this configuration, the first excitation light is irradiated from the light source toward the metal thin film formed on the dielectric member through the dielectric member, and surface plasmon light is generated on the surface of the metal thin film. At the same time, a second excitation light different from the first excitation light can be irradiated through the dielectric member toward the metal thin film to generate propagation light on the surface of the metal thin film.

  Then, interference fringes of interference light are generated due to the difference in the wave number component in the direction parallel to the plane of the metal thin film, which is the direction in which the surface plasmon light travels, due to the surface plasmon light and the propagation light.

  At this time, the electric field enhancement at a predetermined height position of the electric field enhancement measuring member is performed based on the light quantity of the second excitation light when the contrast of the interference fringes is maximized while changing the light quantity of the second excitation light. It can be estimated by converting the degree.

  Accordingly, it is not necessary to provide pinholes or edges in the metal thin film as in the prior art, and the electric field enhancement on the plane (plane direction) of the metal thin film to be truly measured can be measured, and the thickness of the metal thin film can be measured. A three-dimensional distribution of the electric field enhancement corresponding to the height position of the analyte captured in the vicinity of the metal thin film can be obtained, and accurate electric field enhancement can be measured (estimated).

  In addition, since the second excitation light is irradiated from the same direction through the dielectric member below the dielectric member in the same manner as the first excitation light, the excitation light irradiation device is arranged below and aggregated. Therefore, the measuring device can be made compact.

  Further, since the second excitation light is irradiated from the same direction through the dielectric member below the dielectric member as in the case of the first excitation light, the coherence distance of the laser light is shortened, and the optical path Interference fringes can be generated by superimposing laser beams within the length and interfering with each other, improving the coherence and measuring (estimating) more accurate electric field enhancement.

The method for measuring the absolute value of the electric field enhancement intensity according to the present invention is characterized in that the second excitation light is excitation light dispersed from the same light source as the first excitation light.
In the apparatus for measuring the absolute value of the electric field enhancement intensity according to the present invention, the second excitation light generator includes a spectroscopic unit that separates the second excitation light from the same light source as the first excitation light. It is characterized by providing.

By configuring in this way, for example, it is only necessary to provide spectroscopic means such as a half mirror, so that the apparatus can be made compact.
In addition, since the second excitation light is split from the same light source as the first excitation light, the second excitation light having the same phase as the first excitation light is formed on the upper surface of the dielectric member. Irradiation can be performed toward the metal thin film, and accurate electric field enhancement can be measured (estimated).

In the method for measuring the absolute value of the electric field enhancement intensity according to the present invention, the second excitation light is excitation light derived from a light source different from the light source of the first excitation light.
In the apparatus for measuring the absolute value of the electric field enhancement intensity according to the present invention, the second excitation light generation device generates the second excitation light from a light source different from the light source of the first excitation light. It is comprised as follows.

  With this configuration, the second excitation light can be the second excitation light derived from a light source different from the light source of the first excitation light, and the light amount of the second excitation light can be easily obtained. Based on the amount of the second excitation light when the interference fringe contrast is maximized, the electric field enhancement at the predetermined height position of the electric field enhancement measuring member is accurately converted and estimated. can do.

Further, in the method for measuring the absolute value of the electric field enhancement of the present invention, the electric field enhancement measuring member is a fluorescent substance,
The fluorescent substance is excited, and the fluorescence enhanced thereby is detected by a light detection means, and the electric field enhancement measurement is performed based on the light quantity of the second excitation light when the contrast of the interference fringes becomes maximum. The electric field enhancement at a predetermined height position of the member for use is converted and estimated.

Further, in the apparatus for measuring the absolute value of the electric field enhancement intensity of the present invention, the electric field enhancement measuring member is a fluorescent material,
The fluorescent substance is excited, and the fluorescence enhanced thereby is detected by a light detection means, and the electric field enhancement measurement is performed based on the light quantity of the second excitation light when the contrast of the interference fringes becomes maximum. It is configured to convert and estimate the electric field enhancement at a predetermined height position of the member for use.

  With this configuration, a fluorescent material is used as a member for measuring the electric field enhancement, for example, the fluorescent material is used as a coating film, the height position thereof is changed, or the DNA and the fluorescent material are bound (labeled). By forming a monolayer and changing the height position by changing the size of the protein molecule, the fluorescent substance can be excited at a predetermined height position, thereby enhancing the fluorescence. Is detected by the light detection means, and the electric field enhancement at a predetermined height position of the electric field enhancement measuring member is accurately determined based on the amount of the second excitation light when the contrast of the interference fringes becomes maximum. It can be estimated by conversion.

  Therefore, for example, when using a measurement member as a microchip substrate used in an SPFS apparatus based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), the electric field enhancement in a state close to the actual is accurately converted. Can be estimated.

Further, in the method for measuring the absolute value of the electric field enhancement of the present invention, the electric field enhancement measuring member is a near-field probe,
The intensity of the interference light at a predetermined height position of the near-field probe is measured, and based on the light quantity of the second excitation light when the contrast of the interference fringes is maximized, the predetermined value of the electric field enhancement measuring member is determined. It is characterized in that it is estimated by converting the electric field enhancement at the height position.

Further, in the apparatus for measuring an absolute value of electric field enhancement of the present invention, the electric field enhancement measuring member is a near-field probe,
The intensity of the interference light at a predetermined height position of the near-field probe is measured, and based on the light quantity of the second excitation light when the contrast of the interference fringes is maximized, the predetermined value of the electric field enhancement measuring member is determined. The electric field enhancement at the height position is converted and estimated.

  By configuring in this way, using a near-field probe as a member for electric field enhancement measurement, for example, by attaching a fluorescent substance to the tip of the near-field probe and changing the height position of the near-field probe, The fluorescent material can be excited at a predetermined height position, whereby the enhanced fluorescence is detected by the near-field probe, and the amount of the second excitation light when the contrast of the interference fringes becomes maximum Based on the above, it is possible to accurately convert and estimate the electric field enhancement at a predetermined height position of the electric field enhancement measurement member.

  Therefore, for example, when using a measurement member as a microchip substrate used in an SPFS apparatus based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), the electric field enhancement in a state close to the actual is accurately converted. Can be estimated.

Further, in the method for measuring the absolute value of the electric field enhancement of the present invention, the electric field enhancement measuring member is a light scattering material,
The light scattering material is excited, and the scattered light enhanced thereby is detected by the light detection means, and the electric field enhancement is based on the light quantity of the second excitation light when the contrast of the interference fringes becomes maximum. The electric field enhancement at a predetermined height position of the measurement member is converted and estimated.

Further, in the apparatus for measuring the absolute value of the electric field enhancement intensity of the present invention, the electric field enhancement measuring member is a light scattering material,
The light scattering material is excited, and the scattered light enhanced thereby is detected by the light detection means, and the electric field enhancement is based on the light quantity of the second excitation light when the contrast of the interference fringes becomes maximum. It is configured to convert and estimate the electric field enhancement at a predetermined height position of the measurement member.

  By configuring in this way, a light scattering material is used as a member for measuring the electric field enhancement, for example, the light scattering material is formed into a coating film using fine particles such as colloidal gold, calcium acid powder, etc. Change the position, form a monolayer by binding (labeling) microparticles such as DNA and colloidal gold, calcium acid powder, etc., and change the height position by changing the size of the protein molecule. The light scattering material can be excited at the height position of the second excitation light, so that the enhanced scattered light is detected by the light detecting means, and the second excitation light when the contrast of the interference fringes becomes maximum is detected. Based on the amount of light, the electric field enhancement at a predetermined height position of the electric field enhancement measuring member can be accurately converted and estimated.

  Therefore, for example, when using a measurement member as a microchip substrate used in an SPFS apparatus based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), the electric field enhancement in a state close to the actual is accurately converted. Can be estimated.

  In the method for measuring the absolute value of the electric field enhancement intensity according to the present invention, two-dimensional measurement is performed by changing at least one of the phase of the second excitation light and the height position of the electric field enhancement measurement member. It is characterized by obtaining data on the absolute value of the target or three-dimensional electric field enhancement.

  The apparatus for measuring the absolute value of the electric field enhancement intensity according to the present invention can measure two-dimensionally by changing at least one of the phase of the second excitation light and the height position of the electric field enhancement measuring member. It is configured to obtain data on the absolute value of the electric field enhancement strength in three dimensions.

  With this configuration, a two-dimensional and three-dimensional overall distribution of the electric field enhancement intensity corresponding to the influence of the thickness of the metal thin film and the height position of the analyte trapped in the vicinity of the metal thin film is obtained. It is possible to measure (estimate) an accurate electric field enhancement.

Further, the evaluation method of the measurement member of the present invention is based on the absolute value data of the electric field enhancement obtained by using the method for measuring the absolute value of the electric field enhancement described in any of the above,
A measurement member including a dielectric member and a metal thin film formed on the upper surface of the dielectric member is evaluated.

In addition, the evaluation device for the measurement member of the present invention is based on the absolute value data of the electric field enhancement obtained using the absolute value measurement device for the electric field enhancement described in any of the above,
The measuring member including the dielectric member and the metal thin film formed on the upper surface of the dielectric member is evaluated.

  With this configuration, it is possible to accurately know the absolute value of the electric field enhancement intensity, which is an index of the electric field enhancement effect of the surface plasmon light, and thereby, for example, the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) Evaluation of fixed members for process inspection, management, and quality assurance that can check and select the state of metal thin film formation when used as a measuring member such as a microchip used in SPFS devices based on It can be used as an evaluation apparatus for a method and a measuring member.

In the analyte detection method of the present invention, an analyte captured in the vicinity of a metal thin film is detected using a measurement member including an electric member and a metal thin film formed on the upper surface of the dielectric member. When
The detection of the analyte is performed by correcting the detection data based on the absolute value data of the electric field enhancement obtained using the method for measuring the absolute value of the electric field enhancement described in any of the above. .

The analyte detection apparatus of the present invention detects an analyte trapped in the vicinity of a metal thin film using a measurement member including a dielectric member and a metal thin film formed on the upper surface of the dielectric member. When
Based on the absolute value data of the electric field enhancement obtained by using the absolute value measuring device of the electric field enhancement described in any of the above, the detection data is corrected and the analyte is detected. It is characterized by being.

  With this configuration, it is possible to accurately know the absolute value of the electric field enhancement intensity, which is an index of the electric field enhancement effect of the surface plasmon light, and thereby, for example, the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) When using it as a measurement member such as a microchip used in an SPFS device based on the above, it is possible to correct the detection signal measurement value in the detection process which is a subsequent process by grasping the electric field enhancement intensity for each substrate, and to improve the measurement accuracy. It is possible to improve.

  According to the present invention, it is not necessary to provide pinholes and edges in the metal thin film as in the prior art, and the electric field enhancement on the plane (plane direction) of the metal thin film to be truly measured can be measured. It is possible to obtain a three-dimensional distribution of the electric field enhancement corresponding to the thickness of the metal thin film and the height position of the analyte captured in the vicinity of the metal thin film, and to accurately measure (estimate) the electric field enhancement. it can.

  Therefore, for example, when using a measurement member as a microchip substrate used in an SPFS apparatus based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), the electric field enhancement in a state close to the actual is accurately converted. Can be estimated.

  Moreover, since the second excitation light is irradiated from the same direction via the dielectric member below the dielectric member as in the case of the first excitation light, the excitation light irradiation device is arranged below and aggregated. Therefore, the measuring device can be made compact.

  Further, since the second excitation light is irradiated from the same direction through the dielectric member below the dielectric member as in the case of the first excitation light, the coherence distance of the laser light is shortened, and the optical path Interference fringes can be generated by superimposing laser beams within the length and interfering with each other, improving the coherence and measuring (estimating) more accurate electric field enhancement.

  Furthermore, according to the present invention, a two-dimensional or three-dimensional overall distribution of the electric field enhancement intensity corresponding to the influence of the thickness of the metal thin film and the height position of the analyte trapped in the vicinity of the metal thin film is obtained. It is possible to measure (estimate) an accurate electric field enhancement.

  Further, according to the present invention, the absolute value of the electric field enhancement intensity, which is an index of the electric field enhancement effect of the surface plasmon light, can be accurately known, and thus, for example, the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) Evaluation of fixed members for process inspection, management, and quality assurance that can check and select the state of metal thin film formation when used as a measuring member such as a microchip used in SPFS devices based on It can be used as an evaluation apparatus for a method and a measuring member.

  Furthermore, according to the present invention, the absolute value of the electric field enhancement intensity, which is an index of the electric field enhancement effect of surface plasmon light, can be accurately known. For example, blood using surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) In areas where high-precision detection is required, such as clinical trials such as tests, the measurement accuracy of analyte detection should be improved by correcting the detection data using the absolute value of the electric field enhancement intensity. Can do.

FIG. 1 is a schematic diagram schematically showing an outline of an apparatus for measuring an absolute value of electric field enhancement for explaining a method for measuring an absolute value of electric field enhancement of the present invention. FIG. 2 is a partially enlarged view of FIG. FIG. 3 is a block diagram for explaining the outline of the method for measuring the absolute value of the electric field enhancement according to the present invention. FIG. 4 is a graph showing a pattern of emission interference, and is a graph showing the relationship between the distance in the irradiation beam area of the second lower excitation light 34 on the surface 14a of the metal thin film 14 and the emission intensity. FIG. 5 is a graph showing a light emission interference pattern when the contrast of the interference fringes is maximized, and the distance in the irradiation beam area of the second lower excitation light 34 on the surface 14a of the metal thin film 14 and the light emission intensity. It is a graph which shows the relationship. FIG. 6 is a graph showing the interference fringe contrast, in which the horizontal axis indicates the magnification of the first lower excitation light amount (electric field amplitude) with respect to the second lower excitation light amount, and the vertical axis indicates the interference fringe contrast. It is a graph. FIG. 7 is a graph showing an interference light pattern when the second lower excitation light amount / first lower excitation light amount = 0.5. FIG. 8 is a graph showing an interference light pattern when the second lower excitation light quantity / first lower excitation light quantity = 1. FIG. 9 is a graph showing the relationship between the height position of the electric field enhancement measuring member and the electric field enhancement. FIG. 10 is a schematic diagram schematically showing an outline of an apparatus for measuring an absolute value of electric field enhancement for explaining a method for measuring an absolute value of electric field enhancement of another embodiment of the present invention. FIG. 11 is a schematic view schematically showing an outline of a measurement apparatus for an absolute value of electric field enhancement for explaining a method for measuring an absolute value of electric field enhancement of another embodiment of the present invention. FIG. 12 is a partially enlarged view of FIG. FIG. 13 is a block diagram for explaining an outline of a method for measuring an absolute value of electric field enhancement in another embodiment of the present invention. FIG. 14 is a schematic diagram schematically showing an outline of a measurement apparatus for an absolute value of electric field enhancement, which explains a method for measuring an absolute value of electric field enhancement of another embodiment of the present invention. FIG. 15 is a partially enlarged view of FIG. FIG. 16 is a block diagram for explaining an outline of a method for measuring the absolute value of the electric field enhancement strength according to another embodiment of the present invention. FIG. 17 is a schematic diagram for explaining a conventional method for measuring electric field enhancement. FIG. 18 is a schematic diagram schematically showing interference fringes observed in the conventional method for measuring electric field enhancement in FIG. FIG. 19 is a schematic diagram illustrating a conventional method for measuring electric field enhancement. FIG. 20 is a graph showing the distribution of electric field enhancement by the conventional method for measuring electric field enhancement of FIG. FIG. 21 is a schematic diagram schematically showing interference fringes observed in the conventional method for measuring electric field enhancement in FIG.

  Hereinafter, embodiments (examples) of the present invention will be described in more detail with reference to the drawings.

1. As the electric field enhancement measuring member, examples using a fluorescent substance Figure 1 shows a schematic of a measurement apparatus of the absolute value of the degree of electric field enhancement for explaining a method for measuring the absolute value of the degree of electric field enhancement of the present invention schematically FIG. 2 is a partial enlarged view of FIG. 1, and FIG. 3 is a block diagram for explaining the outline of the method for measuring the absolute value of the electric field enhancement according to the present invention.

1-1. 1 is a block diagram of the apparatus for measuring the absolute value of electric field enhancement according to the present invention.
As shown in FIG. 1 and FIG. 2, the device 10 for measuring the absolute value of the electric field enhancement has a substantially triangular prism-shaped dielectric member 12, and a horizontal upper surface 12 a of the dielectric member 12 A metal thin film 14 is formed. A fluorescent material layer 16 is formed on the upper surface of the metal thin film 14 as a member for measuring electric field enhancement.

The dielectric member 12, the metal thin film 14, and the fluorescent material layer 16 constitute a measuring member 18.
Further, a light source 20 is disposed on the side surface 12b below the dielectric member 12,
A spectroscope 24 configured by, for example, a half mirror is provided that transmits a part of the light 22 from the light source 20 and reflects the remaining light.

  The first lower excitation light 26, which is the first excitation light transmitted through the spectroscope 24, is adjusted so that the amount of light becomes a certain amount via a neutral density (ND) filter 28, and then the dielectric A certain angle (resonance) is incident on the side surface 12b of the dielectric member 12 from the lower outside of the body member 12 and through the dielectric member 12 toward the metal thin film 14 formed on the upper surface 12a of the dielectric member 12. (Angle) is irradiated at α1.

  As shown in the enlarged view of FIG. 2, the surface 14 a of the metal thin film 14 has a surface parallel to the surface 14 a of the metal thin film 14 by the first lower excitation light 26 irradiated to the metal thin film 14. Plasmon light (dense wave) 30 is generated.

  On the other hand, of the light 22 from the light source 20, the remaining light 32 split by the spectroscope 24 is reflected by the mirror 37, and passes through a variable attenuation (ND) filter 35 and a phase shift plate 36, and is a dielectric member. The light is incident on the side surface 12b of the dielectric member 12 from the lower outer side of the dielectric member 12, and passes through the dielectric member 12 toward the surface 14a of the metal thin film 14 formed on the upper surface 12a of the dielectric member 12. It is designed to irradiate at an angle below the critical angle.

  That is, the light incident on the side surface 12b of the dielectric member 12 from the lower outside of the dielectric member 12 via the variable dimming (ND) filter 35 and the phase shift plate 36 is the second excitation light. As shown in the enlarged view of FIG. 2, the lower excitation light 34 is irradiated onto the surface 14a of the metal thin film 14 at a constant angle α2.

  Further, on the other side surface 12 c below the dielectric member 12, a light receiving means 40 is provided for receiving the metal thin film reflected light 38 reflected by the metal thin film 14 by the first lower excitation light 26. Yes.

  Further, as will be described later, in order to detect the interference fringe contrast caused by the fluorescence excited by the fluorescent material layer 16, the emission intensity of the fluorescent light from the fluorescent material layer 16 is detected above the measuring member 18. A light detection means 42 such as a CCD is provided.

  The light source 20, the spectroscope 24, and the neutral density (ND) filter 28 constitute a first lower excitation light generator 46 as a first excitation light generator, and a variable neutral density (ND) filter. 35, the phase shift plate 36, and the mirror 37 constitute a second lower excitation light generator 48 as a second excitation light generator.

  Further, the light detection means 42 is connected to an electric field enhancement conversion device 50 such as a CPU of a computer, and converts the electric field enhancement at a predetermined height position of the electric field enhancement measurement member as will be described later. Thus, a calculation process to be estimated is performed.

  In this case, the excitation light emitted from the light source 20 is not particularly limited, but laser light is preferable, an LD laser having a wavelength of 200 to 900 nm, 0.001 to 1,000 mW, or a wavelength of 230 to 800 nm. A semiconductor laser of 0.01 to 100 mW is suitable.

In addition, the dielectric member 12 is not particularly limited, but optically transparent, for example, various inorganic substances such as glass and ceramics, natural polymers, and synthetic polymers can be used, and the chemical stability. From the viewpoints of production stability and optical transparency, it is preferable to contain silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ).

In this embodiment, the substantially triangular prism-shaped dielectric member 12 is used. However, the shape of the dielectric member 12 can be changed as appropriate, such as a semicircular shape or an elliptical shape.
The material of the metal thin film 14 is not particularly limited, but is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, more preferably It is made of gold and may be made of an alloy of these metals.

  That is, such a metal is suitable for the metal thin film 14 because it is stable against oxidation and, as will be described later, the electric field enhancement due to surface plasmon light (coherent wave) increases.

  Further, the method for forming the metal thin film 14 is not particularly limited, and examples thereof include sputtering, vapor deposition (resistance heating vapor deposition, electron beam vapor deposition, etc.), electrolytic plating, electroless plating, and the like. . It is preferable to use a sputtering method or a vapor deposition method because it is easy to adjust the thin film formation conditions.

  Further, the thickness of the metal thin film 14 is not particularly limited, but preferably, gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 ˜500 nm and their alloys: preferably in the range of 5 to 500 nm.

  From the viewpoint of the electric field enhancement effect, more preferably, gold: 20 to 70 nm, silver: 20 to 70 nm, aluminum: 10 to 50 nm, copper: 20 to 70 nm, platinum: 20 to 70 nm, and alloys thereof: It is desirable to be within the range of 10 to 70 nm.

  If the thickness of the metal thin film 14 is within the above range, it is preferable that surface plasmon light (coherent wave) is easily generated. In addition, as long as the metal thin film 14 has such a thickness, the size (vertical x horizontal) size and shape are not particularly limited.

  On the other hand, the fluorescent material constituting the fluorescent material layer 16 is not particularly limited as long as it is a material that emits fluorescence by irradiating predetermined excitation light or using a field effect to emit fluorescence. In the present specification, “fluorescence” includes various types of light emission such as phosphorescence.

A fluorescent material layer 16 is formed to a predetermined thickness on the upper surface of the metal thin film 14 in a state where such a fluorescent material is contained alone or in a base material.
In this case, the method for forming the fluorescent material layer 16 with a predetermined thickness on the upper surface of the metal thin film 14 is not particularly limited, and examples thereof include printing, spin coating, dipping, brush coating, spray coating, static coating, and the like. Known methods such as electrocoating and electrocoating can be used.

1-2. The method for measuring the absolute value of the electric field enhancement using the apparatus 10 for measuring the absolute value of the electric field enhancement of the present invention configured as described above will be described below.

  First, the first lower excitation light 26, which is the first excitation light irradiated from the light source 20 and transmitted through the spectroscope 24, has a constant light quantity via a neutral density (ND) filter 28. After the adjustment is made, the amount of light is made incident on the side surface 12 b of the dielectric member 12 from the lower outside of the dielectric member 12. Then, the light is irradiated through the dielectric member 12 toward the metal thin film 14 formed on the upper surface 12a of the dielectric member 12 at a constant incident angle (resonance angle) α1.

As a result, as shown in the enlarged view of FIG. 2, surface plasmon light (coherent wave) 30 is generated on the surface 14 a of the metal thin film 14 in a direction parallel to the surface 14 a of the metal thin film 14.
In this case, the following method may be used to determine the incident angle (resonance angle) α1 having a constant angle.

  That is, when the surface plasmon light (coherent wave) 30 is generated on the metal thin film 14, the first lower excitation light 26 and the electronic vibration in the metal thin film 14 are coupled, and the signal of the metal thin film reflected light 38. Therefore, it is sufficient to find a point where the signal of the metal thin film reflected light 38 received by the light receiving means 40 changes (decreases the amount of light).

  On the other hand, of the light 22 from the light source 20, the remaining light 32 split by the spectroscope 24 is reflected by a mirror 37 disposed at a predetermined angle, and is a variable attenuation (ND) filter 35 and a phase shift plate 36. The surface 14a of the metal thin film 14 formed on the upper surface 12a of the dielectric member 12 through the dielectric member 12 is incident on the side surface 12b of the dielectric member 12 from the lower outside of the dielectric member 12. The light is irradiated at a constant angle α2 (an angle less than the critical angle).

  That is, light incident on the side surface 12 b of the dielectric member 12 from the lower outside of the dielectric member 12 through the variable dimming (ND) filter 35 and the phase shift plate 36 is used as the second lower excitation light 34. As shown in the enlarged view of FIG. 2, the surface 14a of the metal thin film 14 is irradiated as another second excitation light at a constant angle α2.

In this case, the second lower excitation light 34 irradiates the surface 14 a of the metal thin film 14 with the second lower excitation light 34 having the same phase as the first lower excitation light 26. Yes.
Thereby, as shown in the enlarged view of FIG. 2, the propagation light 44 is generated as the second light at a predetermined height position of the member for measuring the electric field strength on the surface 14a of the metal thin film 14. .

In this case, the phase shift plate 36 is adjusted so that the phase of the second lower pumping light 34 is the same as the phase of the first lower pumping light 26.
Or, further, by moving the phase shift plate 36 in a direction orthogonal to the optical axis, it is possible to see changes in the brightness of the interference fringes at one measurement point.

  The angle α2 is not particularly limited as long as it is equal to or smaller than a critical angle for generating the propagation light 44 at a predetermined height position of the electric field enhancement measuring member on the surface 14a of the metal thin film 14. It is not a thing.

  Then, as shown in the enlarged view of FIG. 2, the propagation light 44 by the second lower excitation light 34 and the surface plasmon light 30 are parallel to the plane 14a of the metal thin film 14 in the direction in which the surface plasmon light 30 travels. Interference fringes of interference light are generated due to different wave number components in the direction.

  By the way, since this interference fringe is near-field light, it cannot be seen directly as it is. For this reason, in the present invention, the fluorescent material layer 16 is formed on the upper surface of the metal thin film 14 as a member for measuring the electric field strength.

  That is, the fluorescent substance layer on the metal thin film 14 is generated by the propagation light 44 by the second lower excitation light 34 and the interference fringes (interference light) by the surface plasmon light (coherent wave) 30 by the first lower excitation light 26. Sixteen fluorescent substances are efficiently excited.

  Using this principle, the electric field enhancement measuring member is changed based on the light quantity of the second lower excitation light 34 when the contrast of the interference fringes is maximized while changing the light quantity of the second lower excitation light 34. Thus, the electric field enhancement at a predetermined height position of the fluorescent material layer 16 is converted and estimated.

  More specifically, the light intensity of the second lower excitation light 34 is changed by the variable dimming (ND) filter 35, so that the fluorescence emission intensity of the fluorescent material layer 16 on the metal thin film 14 is changed to, for example, a CCD or the like. The light detection means 42 detects the light.

The fluorescence emission interference patterns detected by the light detection means 42 are as shown in the graph of FIG. 4 and the graph of FIG.
4 and 5, the vertical axis represents the fluorescence emission intensity by the fluorescent material layer 16, and the horizontal axis represents the irradiation beam area of the second lower excitation light 34 on the surface 14 a of the metal thin film 14. The distance in is shown.

  As shown in the graph of FIG. 5, the light amount of the second lower excitation light 34 when the contrast of the interference fringes becomes maximum is the light amount of the surface plasmon light 30 (the second lower excitation light 34 As the light quantity = the light quantity of the surface plasmon light 30), the electric field enhancement intensity at a predetermined height position of the fluorescent material layer 16 is converted and estimated based on the light quantity of the second lower excitation light 34 at this time. It has become.

  In the case of the graph of FIG. 4, since the contrast of the interference fringes is smaller than that of the graph of FIG. 5, (the light amount of the second lower excitation light 34> the light amount of the surface plasmon light 30 or the second lower light This shows the case where the light quantity of the side excitation light 34 <the light quantity of the surface plasmon light 30).

  Then, from the graph of FIG. 5, the electric field enhancement intensity at a predetermined height position of the fluorescent material layer 16 is converted based on the light amount of the second lower excitation light 34 when the contrast of the interference fringes becomes maximum. The estimation method is as follows, for example.

  That is, when the interference point is at the position of the surface 14 a of the metal thin film 14 (interference point height Z = 0 μm), the propagation light 44 by the second lower excitation light 34 and the first lower excitation light 26. The interference fringes of two beams of interference fringes (interference light) due to surface plasmon light (coherent wave) 30 due to

However, since there is generally metal quenching on the surface of the metal thin film, the electric field enhancement considering this quenching amount is estimated.
In this case, in order to estimate the electric field enhancement intensity of the surface plasmon light 30 from the ratio of the light amount of the second lower excitation light 34 and the light amount of the first lower excitation light 26 when the contrast of the interference fringes is the highest. Is calculated using a well-known wave equation or the like, and the electric field enhancement is estimated.

  That is, the graphs of FIGS. 7 and 8 are obtained by standardizing the results obtained by the above calculation, and the graph of FIG. 7 is the second lower excitation light amount / first lower excitation light amount = 0.5. In this case, the interference fringe contrast is approximately 0.8, and the graph of FIG. 8 shows that the second lower excitation light amount / first lower excitation light amount = 1. In this case, the interference fringe contrast = 1.

Therefore, specifically, as shown in the graph of FIG. 6, the electric field (amplitude) ratio of the second lower excitation light 34 and the first lower excitation light 26 when the contrast of the interference fringes becomes maximum. Is a conversion factor K = 1. However, since the second lower excitation light 34 is transmitted through the metal thin film, the amount of light at the interference point includes the transmittance T (%) of the metal thin film, and the correction ((100 / T) ² ). Is required.

Therefore, surface plasmon electric field enhancement (energy intensity)
= K ² × (second lower excitation light amount at interference fringe contrast MAX / (100 / T) ² ) / first lower excitation light amount = (second lower excitation light amount at interference fringe contrast MAX / (100 / T) ² ) / first lower excitation light quantity.

The electric field enhancement at a predetermined height position of the fluorescent material layer 16 may be converted and estimated by this calculation formula.
6 is a graph showing the interference fringe contrast, the horizontal axis indicates the magnification of the first lower excitation light amount (electric field amplitude) with respect to the second lower excitation light amount, and the vertical axis indicates the interference. It is a graph which shows a fringe contrast.

  Also, in the graphs of FIGS. 6 to 8, θ1 is an angle formed between the second lower excitation light 34 and a vertical line in the height direction, and θ2 is a vertical line between the first lower excitation light 26 and the height direction. The angle formed, n1 represents the refractive index of the dielectric member 12 (in this example, glass), and n2 represents the refractive index of a medium such as air (in this example: water).

  The process of converting and estimating the electric field enhancement at the predetermined height position of the above-described member for measuring electric field enhancement is based on the detection data from the light detection means 42 and is connected to the light detection means 42. In the electric field enhancement conversion device 50 such as a CPU, arithmetic processing is performed based on a predetermined program.

  When a large number of phosphors are arranged on the above plane, the interference light between the excitation light and the surface plasmon light includes not only light from the target phosphor but also other nearby phosphors. Is considered to be fluorescence.

However, in this case, the light quantity ratio at which the interference fringe contrast is maximized does not change, so that no particular problem occurs.
In this embodiment, the second lower excitation light 34 irradiates the surface 14a of the metal thin film 14 with the second lower excitation light 34 having the same phase as the first lower excitation light 26. By irradiating the surface 14 a of the metal thin film 14 with the second lower excitation light 34 having a phase different from that of the first lower excitation light 26 by the phase shift plate 36, It is possible to obtain a two-dimensional distribution in the horizontal position (direction parallel to the surface 14a of the metal thin film 14) of the member for measuring the electric field enhancement strength.

  Furthermore, by changing the height position of the electric field enhancement measuring member, that is, the film thickness of the fluorescent material layer 16, it corresponds to the height position of the electric field enhancement measuring member as shown in the graph of FIG. A two-dimensional distribution of the electric field enhancement intensity can be obtained.

  Furthermore, the above combination corresponds to the distribution of the electric field enhancement member in the horizontal position (direction parallel to the surface 14a of the metal thin film 14) of the electric field enhancement member and the height position of the electric field enhancement member. A three-dimensional overall distribution of the electric field enhancement combined with the distribution of the electric field enhancement can be obtained.

  In this case, although not shown, when changing the height position of the electric field enhancement measuring member, that is, when changing the film thickness of the fluorescent material layer 16, a measurement member 18 having a plurality of different fluorescent material layer 16 thicknesses is prepared. In addition to the measurement, the film thickness of the fluorescent material layer 16 in the direction of the surface 14a of the metal thin film 14 of the same measurement member 18 can be changed in stages and measured simultaneously.

  In addition, DNA and fluorescent substances are bound (labeled) to form a monomolecular layer, and the fluorescent substance is excited at a predetermined height position by changing the height position by changing the size of protein molecules. It can also be made.

  Further, in this embodiment, the second lower pumping light 34 is the second lower pumping light 34 dispersed from the same light source 20 as the first lower pumping light 26, but is shown in FIG. As described above, the second lower excitation light 34 may be the second lower excitation light 34 derived from the light source 52 different from the light source 20 of the first lower excitation light 26. Although not shown, also in the following second and third embodiments, the second lower excitation light 34 is derived from a light source 52 different from the light source 20 of the first lower excitation light 26. The second lower excitation light 34 can also be used.

  With this configuration, the second lower excitation light 34 can be the second lower excitation light derived from the light source 52 different from the light source 20 of the first lower excitation light 26. The light quantity of the second lower excitation light 34 can be easily changed, and the electric field enhancement measuring member is determined based on the light quantity of the second lower excitation light when the contrast of the interference fringes is maximized. The electric field enhancement at the height position of can be accurately converted and estimated.

  With this configuration, according to the method for measuring the absolute value of the electric field enhancement and the apparatus for measuring the absolute value of the electric field enhancement of the present invention, for example, the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) is used. When the measurement member 18 is used as a microchip substrate used in the SPFS device based on the electric field enhancement in a state close to the actual, it can be accurately converted and estimated.

  Further, according to the present invention, the absolute value of the electric field enhancement intensity, which is an index of the electric field enhancement effect of the surface plasmon light, can be accurately known, and thus, for example, the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) Evaluation of fixed members for process inspection, management, and quality assurance that can check and select the state of metal thin film formation when used as a measuring member such as a microchip used in SPFS devices based on It can be used as an evaluation apparatus for a method and a measuring member.

  Furthermore, according to the present invention, the absolute value of the electric field enhancement intensity, which is an index of the electric field enhancement effect of surface plasmon light, can be accurately known. For example, blood using surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) In areas where high-precision detection is required, such as clinical trials such as tests, the measurement accuracy of analyte detection should be improved by correcting the detection data using the absolute value of the electric field enhancement intensity. Can do.

  Furthermore, according to the present invention, a two-dimensional and three-dimensional overall distribution of the electric field enhancement corresponding to the influence of the thickness of the metal thin film and the height position of the analyte trapped in the vicinity of the metal thin film is obtained. It is possible to measure (estimate) an accurate electric field enhancement.

  In this case, although not shown, a flow path is provided in the measurement member 18 so that a sample solution containing the analyte to be detected flows, and the analyte is bound in the vicinity of the metal thin film 14. What is necessary is just to comprise so that a trace amount and an extremely low concentration analyte may be detected by exciting the (labeled) fluorescent substance efficiently and observing this fluorescence.

  The sample solution used here is a solution prepared using a specimen, for example, a mixture of a specimen and a reagent and a treatment for binding a fluorescent substance to an analyte contained in the specimen. It is done.

Examples of such specimens include blood, serum, plasma, urine, nasal fluid, saliva, stool, body cavity fluid (eg, cerebrospinal fluid, ascites, pleural effusion).
The analyte contained in the sample is, for example, a nucleic acid (DNA that may be single-stranded or double-stranded, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., or nucleoside Nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modified molecules thereof, Specific examples thereof include a complex, and may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signal transduction substance, a hormone, and the like, and is not particularly limited.

2. Example Using Near Field Probe as Member for Measuring Electric Field Intensity FIG. 11 is a diagram of an apparatus for measuring an absolute value of electric field enhancement for explaining a method for measuring an absolute value of electric field enhancement in another embodiment of the present invention. FIG. 12 is a partially enlarged view of FIG. 11, and FIG. 13 is a block diagram for explaining an outline of a method for measuring an absolute value of electric field enhancement in another embodiment of the present invention. .

  The apparatus 10 for measuring the absolute value of the electric field enhancement in this embodiment has basically the same configuration as that of the apparatus 10 for measuring the absolute value of the electric field enhancement shown in FIGS. Therefore, the same components are denoted by the same reference numerals, and detailed description thereof is omitted.

  In Example 1 shown in FIGS. 1 to 3, a fluorescent material layer 16 is formed on the upper surface of the metal thin film 14 as a member for measuring electric field enhancement, and the emission intensity of fluorescence from the fluorescent material layer 16 is detected. For example, a light detecting means 42 such as a CCD is provided. In this embodiment, the fluorescent material layer 16 is not formed on the upper surface of the metal thin film 14, and the near field is used as a member for measuring the electric field enhancement. A probe 54 is used.

  For example, by attaching the fluorescent material 55 to the tip of the near-field probe 54 and changing the height position of the near-field probe 54, the fluorescent material can be excited at a predetermined height position. The detected fluorescence is detected by the near-field probe 54, and the electric field is determined based on the light amount of the second lower excitation light 34 when the contrast of the interference fringes becomes maximum using the same principle as in the first embodiment. It is configured to accurately convert and estimate the electric field enhancement at a predetermined height position of the member for measuring the enhancement, that is, the near-field probe 54.

2-2. The method for measuring the absolute value of the electric field enhancement using the apparatus 10 for measuring the absolute value of the electric field enhancement of the present invention configured as described above will be described below.

  In the same manner as in the first embodiment, as shown in FIG. 11, first, light 22 is irradiated from the light source 20, and a certain angle is directed toward the metal thin film 14 formed on the upper surface 12 a of the dielectric member 12. As shown in the enlarged view of FIG. 12, surface plasmon light (dense wave) is emitted in a direction parallel to the surface 14a of the metal thin film 14 on the surface 14a of the metal thin film 14 as shown in the enlarged view of FIG. ) 30 is generated.

  On the other hand, as shown in the enlarged views of FIGS. 11 and 12, the second lower excitation light 34 is irradiated onto the surface 14a of the metal thin film 14 at a certain angle α2 (an angle less than the critical angle), and the metal Propagating light 44 is generated at a predetermined height position of the member for measuring electric field enhancement on the surface 14 a of the thin film 14.

  Then, as shown in the enlarged view of FIG. 12, the propagation light 44 by the second lower excitation light 34 and the surface plasmon light 30 are parallel to the plane 14a of the metal thin film 14 in the direction in which the surface plasmon light 30 travels. Interference fringes of interference light are generated due to different wave number components in the direction.

  By the way, since this interference fringe is near-field light and cannot be seen directly as it is, for example, the fluorescent substance 55 is attached to the tip of the near-field probe 54 and the second light is placed at a predetermined height position of the near-field probe 54. At the tip of the near-field probe 54 on the metal thin film 14 by the propagation light 44 by the lower excitation light 34 and the interference fringes (interference light) by the surface plasmon light (coherent wave) 30 by the first lower excitation light 26. The fluorescent material 55 is excited.

  Using this principle, the electric field enhancement measuring member is changed based on the light quantity of the second lower excitation light 34 when the contrast of the interference fringes is maximized while changing the light quantity of the second lower excitation light 34. In other words, the electric field enhancement intensity at a predetermined height position of the fluorescent material 55 attached to the tip of the near-field probe 54 is converted and estimated.

  Specifically, the light intensity of the second lower excitation light 34 is changed by the variable dimming (ND) filter 35, so that the fluorescence emission intensity of the fluorescent material 55 is changed at the tip of the near-field probe 54. 54.

  Then, based on the light amount of the second lower excitation light 34 when the contrast of the interference fringe is maximized, using the same principle as the graphs of FIGS. The electric field enhancement at a predetermined height position of the electric field enhancement measurement member can be accurately converted and estimated.

3. Example using light scattering material as member for measuring electric field enhancement FIG. 14 is a diagram of an apparatus for measuring the absolute value of electric field enhancement for explaining the method for measuring the absolute value of electric field enhancement according to another embodiment of the present invention. FIG. 15 is a partially enlarged view of FIG. 14, and FIG. 16 is a block diagram for explaining an outline of a method for measuring the absolute value of electric field enhancement in another embodiment of the present invention. .

  The apparatus 10 for measuring the absolute value of the electric field enhancement in this embodiment has basically the same configuration as that of the apparatus 10 for measuring the absolute value of the electric field enhancement shown in FIGS. Therefore, the same components are denoted by the same reference numerals, and detailed description thereof is omitted.

  In Example 1 shown in FIGS. 1 to 3, the fluorescent material layer 16 is formed on the upper surface of the metal thin film 14 as a member for measuring the electric field strength. In this embodiment, on the upper surface of the metal thin film 14. Uses a light scattering material layer 56 as a member for measuring the electric field enhancement instead of the fluorescent material layer 16.

  Then, the light scattering material of the light scattering material layer 56 is excited, and the scattered light that is enhanced by this is detected by the light detection means 42, and the second lower excitation light when the contrast of the interference fringes becomes maximum. Based on the amount of light 34, the electric field enhancement measuring member, that is, the electric field enhancement at a predetermined height position of the light scattering material layer 56 is converted and estimated.

  In this case, for example, the light scattering material is made of a fine particle such as colloidal gold or calcium acid powder, like the fluorescent material layer 16 of Example 1, and the height position is changed, or DNA and gold are changed. Light scattering at a predetermined height position by binding (labeling) fine particles such as colloid and calcium acid powder to form a monolayer and changing the height position by changing the size of the protein molecule. The second lower side when the contrast of the interference fringes is maximized by exciting the substance and detecting the enhanced scattered light by the light detection means 42 using the same principle as in the first embodiment. Based on the light quantity of the excitation light 34, it is configured to accurately convert and estimate the electric field enhancement at a predetermined height position of the electric field enhancement measurement member.

3-2. The method for measuring the absolute value of the electric field enhancement using the apparatus 10 for measuring the absolute value of the electric field enhancement of the present invention configured as described above will be described below.

  As in the first embodiment, as shown in FIG. 14, first, the light source 20 is irradiated with the light 22, and is directed toward the metal thin film 14 formed on the upper surface 12 a of the dielectric member 12. As shown in the enlarged view of FIG. 15, surface plasmon light (dense wave) is emitted in a direction parallel to the surface 14a of the metal thin film 14 at the surface 14a of the metal thin film 14 as shown in the enlarged view of FIG. ) 30 is generated.

  On the other hand, as shown in the enlarged views of FIGS. 14 and 15, the second lower excitation light 34 is irradiated onto the surface 14 a of the metal thin film 14 at a constant angle α 2 (an angle less than the critical angle), and the metal Propagating light 44 is generated at a predetermined height position of the member for measuring electric field enhancement on the surface 14 a of the thin film 14.

  Then, as shown in the enlarged view of FIG. 11, the propagation light 44 by the second lower excitation light 34 and the surface plasmon light 30 are parallel to the plane 14a of the metal thin film 14 in the direction in which the surface plasmon light 30 travels. Interference fringes of interference light are generated due to different wave number components in the direction.

  By the way, since this interference fringe is near-field light and cannot be seen directly as it is, in the present invention, the light scattering material layer 56 is formed on the upper surface of the metal thin film 14 as a member for measuring the electric field intensity. The light scattering material layer 56 on the metal thin film 14 is caused to propagate by interference light (interference light) caused by propagation light 44 by the lower excitation light 34 and surface plasmon light (coherent wave) 30 by the first lower excitation light 26. Excited.

  Using this principle, the electric field enhancement measuring member is changed based on the light quantity of the second lower excitation light 34 when the contrast of the interference fringes is maximized while changing the light quantity of the second lower excitation light 34. That is, the electric field enhancement intensity at a predetermined height position of the light scattering material layer 56 is converted and estimated.

  More specifically, the light intensity of the scattered light from the light scattering material layer 56 is detected by, for example, a CCD or the like by changing the amount of the second lower excitation light 34 by the variable dimming (ND) filter 35. Detected by means 42.

  Then, based on the light amount of the second lower excitation light 34 when the contrast of the interference fringe is maximized, using the same principle as the graphs of FIGS. The electric field enhancement at a predetermined height position of the electric field enhancement measurement member can be accurately converted and estimated.

In this case, in the graphs of FIGS. 4 and 5, the vertical axis indicates the fluorescence emission intensity by the light scattering material layer 56.
The preferred embodiment of the present invention has been described above. However, the present invention is not limited to this. For example, in the above embodiment, surface plasmon excitation used in detection of an analyte trapped in the vicinity of a metal thin film. Although enhanced fluorescence spectroscopy (SPFS) is given as an example, in the field where high-precision detection is required, it is a field where surface plasmon light is used and the absolute value of electric field enhancement is used to correct detection data. For example, the present invention can be used in an industrial field such as an optical inspection, and is not limited to any field, can be applied, and various changes can be made without departing from the object of the present invention.

  The present invention uses the absolute value of the electric field enhancement intensity in a field where high-precision detection is required, such as a clinical test such as blood test using surface plasmon excitation enhanced fluorescence spectroscopy (SPFS). By correcting the detection data, the measurement accuracy of the analyte detection can be improved.

DESCRIPTION OF SYMBOLS 10 Measuring apparatus 12 Dielectric member 12a Upper surface 12b Side surface 12c Side surface 14 Metal thin film 14a Surface 16 Fluorescent substance layer 18 Measuring member 20 Light source 22 Light 24 Spectrometer 26 First lower excitation light 28 Filter 30 Surface plasmon light 32 Light 34 First 2 lower excitation light 35 filter 36 phase shift plate 37 mirror 38 metal thin film reflected light 40 light receiving means 42 light detection means 44 propagating light 46 first lower excitation light generator 48 second lower excitation light generator 50 Electric field enhancement conversion device 52 Light source 54 Near-field probe 55 Fluorescent material 56 Light scattering material layer 100 Dielectric 101 Plasmon light 104 Pinhole 104a Edge portion 106 Excitation light 108 Excitation light 110 Near-field probe 200 Dielectric 201 Plasmon light 204 Edge portion 206 Excitation light 210 Near-field probe

Claims (18)

When the electric field on the metal thin film is enhanced by irradiating excitation light from the light source toward the metal thin film formed on the dielectric member through the dielectric member and generating surface plasmon light on the surface of the metal thin film. And a measurement method for measuring the absolute value of the electric field enhancement intensity,
An electric field enhancement measuring member is disposed at a predetermined height position on the surface of the metal thin film,
Irradiating the first excitation light from the light source toward the metal thin film through the dielectric member, and generating surface plasmon light on the metal thin film surface,
Irradiating a second excitation light different from the first excitation light toward the metal thin film through the dielectric member to generate propagating light on the surface of the metal thin film,
Generating interference fringes by the surface plasmon light and the propagating light;
Based on the light quantity of the second excitation light when the contrast of the interference fringes is maximized while changing the light quantity of the second excitation light, the electric field enhancement intensity at a predetermined height position of the electric field enhancement measurement member. A method for measuring the absolute value of the electric field enhancement, characterized by converting and estimating.   The method of measuring an absolute value of an electric field enhancement intensity according to claim 1, wherein the second excitation light is excitation light that is dispersed from the same light source as the first excitation light.   The method for measuring an absolute value of an electric field enhancement intensity according to claim 1, wherein the second excitation light is excitation light derived from a light source different from the light source of the first excitation light. The electric field enhancement measuring member is a fluorescent material,
The fluorescent substance is excited, and the fluorescence enhanced thereby is detected by a light detection means, and the electric field enhancement measurement is performed based on the light quantity of the second excitation light when the contrast of the interference fringes becomes maximum. The method for measuring an absolute value of electric field enhancement according to any one of claims 1 to 3, wherein the electric field enhancement at a predetermined height position of the member is converted and estimated. The electric field enhancement measuring member is a near-field probe,
The intensity of the interference light at a predetermined height position of the near-field probe is measured, and based on the light quantity of the second excitation light when the contrast of the interference fringes is maximized, the predetermined value of the electric field enhancement measuring member is determined. The method for measuring the absolute value of the electric field enhancement according to any one of claims 1 to 3, wherein the electric field enhancement at the height position is estimated by conversion. The electric field enhancement measuring member is a light scattering material,
The light scattering material is excited, and the scattered light enhanced thereby is detected by the light detection means, and the electric field enhancement is based on the light quantity of the second excitation light when the contrast of the interference fringes becomes maximum. The method for measuring the absolute value of the electric field enhancement according to any one of claims 1 to 3, wherein the electric field enhancement at a predetermined height position of the measurement member is converted and estimated.   By measuring at least one of the phase of the second excitation light and the height position of the electric field enhancement measuring member, data on the absolute value of the two-dimensional or three-dimensional electric field enhancement is obtained. The method for measuring an absolute value of the electric field enhancement intensity according to any one of claims 1 to 6. When the electric field on the metal thin film is enhanced by irradiating excitation light from the light source toward the metal thin film formed on the dielectric member through the dielectric member and generating surface plasmon light on the surface of the metal thin film. And a measuring device for measuring the absolute value of the electric field enhancement intensity,
A member for measuring the electric field strength arranged at a predetermined height position on the surface of the metal thin film;
A first excitation light generator that irradiates the metal thin film with the first excitation light from the light source through the dielectric member, and generates surface plasmon light on the metal thin film surface;
Second excitation light that emits propagating light on the surface of the metal thin film by irradiating the metal thin film with second excitation light different from the first excitation light through the dielectric member. A generator,
When generating interference fringes due to the surface plasmon light and the propagating light,
Based on the light quantity of the second excitation light when the contrast of the interference fringes is maximized while changing the light quantity of the second excitation light, the electric field enhancement intensity at a predetermined height position of the electric field enhancement measurement member. An apparatus for measuring the absolute value of electric field enhancement, comprising: an electric field enhancement conversion device for converting and estimating the electric field.   9. The electric field enhancement intensity according to claim 8, wherein the second excitation light generation device includes a spectroscopic unit that splits the second excitation light from the same light source as the first excitation light. Absolute value measuring device.   9. The second excitation light generation device is configured to generate the second excitation light from a light source different from the light source of the first excitation light. For measuring the absolute value of the electric field enhancement intensity. The electric field enhancement measuring member is a fluorescent material,
The fluorescent substance is excited, and the fluorescence enhanced thereby is detected by a light detection means, and the electric field enhancement measurement is performed based on the light quantity of the second excitation light when the contrast of the interference fringes becomes maximum. The apparatus for measuring the absolute value of the electric field enhancement according to any one of claims 8 to 10, wherein the electric field enhancement at a predetermined height position of the member is converted and estimated. The electric field enhancement measuring member is a near-field probe,
The interference light intensity at a predetermined height position of the near-field probe is measured, and based on the amount of the second excitation light when the contrast of the interference fringe is maximized, a predetermined value of the electric field enhancement measurement member is determined. The apparatus for measuring an absolute value of an electric field enhancement according to any one of claims 8 to 10, wherein the electric field enhancement at a height position is converted and estimated. The electric field enhancement measuring member is a light scattering material,
The light scattering material is excited, and the scattered light enhanced thereby is detected by the light detection means, and the electric field enhancement is based on the light quantity of the second excitation light when the contrast of the interference fringes becomes maximum. The apparatus for measuring an absolute value of electric field enhancement according to any one of claims 8 to 10, wherein the electric field enhancement at a predetermined height position of the measurement member is converted and estimated. .   By measuring at least one of the phase of the second excitation light and the height position of the electric field enhancement measuring member, data on the absolute value of the two-dimensional or three-dimensional electric field enhancement is obtained. The apparatus for measuring an absolute value of the electric field enhancement intensity according to any one of claims 8 to 13, wherein the apparatus is configured as described above. Based on the absolute value data of the electric field enhancement obtained by using the method for measuring the absolute value of the electric field enhancement according to any one of claims 1 to 7,
A measurement member evaluation method comprising: evaluating a measurement member comprising a dielectric member and a metal thin film formed on an upper surface of the dielectric member. Based on the absolute value data of the electric field enhancement obtained by using the absolute value measuring device of the electric field enhancement according to any one of claims 1 to 7,
An apparatus for evaluating a measurement member, characterized in that the measurement member includes a dielectric member and a metal thin film formed on an upper surface of the dielectric member. When detecting an analyte trapped in the vicinity of a metal thin film using a measurement member comprising a dielectric member and a metal thin film formed on the upper surface of the dielectric member,
The detection of the analyte is performed by correcting the detection data based on the absolute value data of the electric field enhancement obtained using the method for measuring the absolute value of the electric field enhancement according to any one of claims 1 to 7. Analyte detection method characterized by the above. When detecting an analyte trapped in the vicinity of a metal thin film using a measurement member comprising a dielectric member and a metal thin film formed on the upper surface of the dielectric member,
The detection of the analyte is performed by correcting the detection data based on the absolute value data of the electric field enhancement obtained using the absolute value measurement apparatus for the electric field enhancement according to any one of claims 8 to 14. An analyte detecting device characterized by comprising:

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