WO2013080505A1 - Sensing device and sensing method using same - Google Patents

Sensing device and sensing method using same Download PDF

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Publication number
WO2013080505A1
WO2013080505A1 PCT/JP2012/007531 JP2012007531W WO2013080505A1 WO 2013080505 A1 WO2013080505 A1 WO 2013080505A1 JP 2012007531 W JP2012007531 W JP 2012007531W WO 2013080505 A1 WO2013080505 A1 WO 2013080505A1
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Prior art keywords
light
sample
excitation light
measurement
contact surface
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PCT/JP2012/007531
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French (fr)
Japanese (ja)
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昇吾 山添
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富士フイルム株式会社
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Publication of WO2013080505A1 publication Critical patent/WO2013080505A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons

Definitions

  • the present invention relates to a sensing apparatus and method for detecting the characteristics of a substance to be measured on the surface of an active substrate in a state where a sample containing the substance to be measured is brought into contact with a plasmon active substrate that is irradiated with excitation light to generate a plasmon enhancement field. Is.
  • a sensor device for detecting signal light such as Raman scattered light and fluorescence with high sensitivity using a plasmon active substrate having a metal concavo-convex structure on the surface that can generate a plasmon enhancement field by light irradiation, and a sensing device using them R & D is actively underway.
  • the plasmon enhancement field is strongest on the surface (sensing surface) where the plasmon enhancement field is generated, and its intensity decreases exponentially with respect to the distance from the surface. Sensation with higher sensitivity can be performed the closer to the sensing surface. Therefore, in sensing using a plasmon enhancement field, it is one of important issues to make the concentration of a substance to be measured in a sample solution as high as possible in the vicinity of the sensing surface.
  • a method of increasing the concentration of the substance to be measured in the vicinity of the sensing surface of the plasmon active substrate for example, a method of fixing a ligand that specifically binds to the substance to be measured on the sensing surface and binding the substance to be measured to the ligand. can give. Thereby, the substance to be measured in the sample is fixed near the sensing surface, and highly sensitive sensing can be performed.
  • Patent Document 1 discloses a device for collecting surface analytes on a metal film by electrophoresis and increasing the concentration of the substance to be measured on the metal film to measure surface plasmon resonance.
  • a dielectrophoresis detection device having a configuration in which the concentration of a substance to be measured at a measurement location is increased by controlling the voltage applied to the plurality of electrodes by installing the electrodes is disclosed.
  • Patent Document 2 and Patent Document 3 disclose a sensing device including a light capturing light irradiation optical system that irradiates a sensing surface that generates a plasmon enhancement field with capture light that captures a target substance in a sample solution by a light capturing effect. Is disclosed.
  • the present invention has been made in view of the above circumstances, and in a sensing device that detects signal light such as Raman scattered light or fluorescence with high sensitivity using a plasmon active substrate that can generate a plasmon enhancement field by light irradiation. It is an object of the present invention to provide a sensing device capable of sensing a substance to be measured with high sensitivity at a plurality of measurement locations in the same sample and a sensing method using the sensing device.
  • the sensing device of the present invention has a sample contact surface with which a sample containing a substance to be measured is brought into contact, and a plasmon active substrate that generates a plasmon enhancement field on the sample contact surface by irradiating the sample contact surface with excitation light
  • An excitation light irradiation optical system for irradiating the excitation light A measurement light irradiation optical system for irradiating the sample with measurement light including the excitation light
  • a physical property detection system for detecting the physical property of the signal light emitted from the sample on the sample contact surface by the irradiation of the measurement light and enhanced by the plasmon enhancement field generated on the sample contact surface by the irradiation; Prepared Irradiating a plurality of measurement points of the sample with the measurement light to detect the physical characteristics at the plurality of measurement points;
  • the excitation light irradiating optical system irradiates the excitation light capable of irradiating simultaneously a region including at least two of the measurement points
  • the “measurement point” is a point on an arbitrary measurement light irradiation surface where one sensing is performed, and means a spot irradiated with the measurement light by one sensing.
  • the excitation light irradiation optical system irradiates the excitation light that can simultaneously irradiate a region including all the measurement points on the sample contact surface.
  • the excitation light and the measurement light preferably have polarized light in substantially the same direction, and the polarized light is preferably linearly polarized light or circularly polarized light.
  • the excitation light preferably has a substantially uniform intensity at the sample contact surface, and preferably has a wavelength of 700 nm to 1600 nm.
  • the intensity is substantially uniform means that the root mean square roughness (RMS) of the intensity is more than 90% in the excitation light irradiation region on the sample contact surface.
  • the sensing device of the present invention is preferably used when the signal light emitted from the sample is any one of fluorescence, Raman scattered light, coherent anti-Stokes scattered light, second harmonic light, and third harmonic light. Can do.
  • the sensing method of the present invention includes a sample contact step in which a sample containing a substance to be measured is brought into contact with a sample contact surface that generates a plasmon enhancement field when irradiated with excitation light; Irradiating the sample with the excitation light to generate a plasmon enhancement field on the sample contact surface of the excitation light irradiation portion, and at least a part of the substance to be measured is adsorbed to the sample contact surface by the plasmon enhancement field An adsorption process, After the adsorption step, the sample on the sample contact surface is irradiated with measurement light including the excitation light to generate a plasmon enhancement field on the sample contact surface by the irradiation, and signal light emitted from the sample
  • a sensing method having a measurement process for detecting physical properties, The measurement step is performed for each of the plurality of measurement points of the sample, In the adsorption step, a region including at least two measurement points is simultaneously irradiated with the excitation light
  • the adsorption step it is preferable to irradiate the excitation light simultaneously to a region including all the measurement points. More preferably, at least a part of the substance to be measured is oriented and adsorbed on the sample contact surface by the plasmon enhancement field.
  • the sensing device of the present invention is to detect and detect signal light enhanced by a plasmon enhancement field at a plurality of measurement points of a sample on a plasmon active substrate, and an excitation light irradiation optical system that excites plasmons;
  • a measurement light irradiation optical system for irradiating the sample with measurement light including excitation light is provided, and the excitation light irradiation optical system can simultaneously irradiate a region including at least two measurement points on the sample contact surface.
  • the substance to be measured can be sensed in a short time and with high sensitivity at a plurality of measurement locations in the same sample. Further, by increasing the area that can be irradiated at the same time, sensing in a large-area sample can be performed efficiently and with high sensitivity.
  • FIG. 1 is a schematic configuration diagram of a sensing device according to an embodiment of the present invention.
  • Cross section in thickness direction showing preferred embodiment of plasmon active substrate
  • Cross-sectional view in the thickness direction showing an embodiment of a plasmon active substrate in the form of a microwell plate
  • the sensing method of the present invention is a sensing method for detecting signal light enhanced by a plasmon enhancement field.
  • the plasmon enhancement field is generated in a short time by generating the plasmon enhancement field with high efficiency. It realizes sensitive sensing.
  • the sensing method of the present invention includes a sample contact step in which a sample containing a substance to be measured is brought into contact with a sample contact surface that generates a plasmon enhancement field when irradiated with excitation light; An adsorption step of irradiating the sample with excitation light, generating a plasmon enhancement field on the sample contact surface of the excitation light irradiation portion, and adsorbing at least a part of the substance to be measured on the sample contact surface by the plasmon enhancement field; After the adsorption step, the sample on the sample contact surface is irradiated with measurement light including excitation light, thereby generating a plasmon enhancement field on the sample contact surface and detecting physical characteristics of the signal light emitted from the sample.
  • a sensing method having a measuring step The measurement process is performed for each of the plurality of measurement points of the sample, In the adsorption step, an area including at least two measurement points is irradiated with excitation light simultaneously.
  • the intensity of the plasmon enhancement field decreases exponentially with the distance from the surface where the plasmon is excited. Therefore, in order to effectively enhance the signal light, it is necessary to bring the substance to be measured closer to the sample contact surface to be plasmon excited.
  • an adsorption step is performed in which the substance to be measured is attracted to the sample contact surface by the electric field gradient of the plasmon enhancement field, and at least a part is adsorbed on the sample contact surface. Performed simultaneously at multiple measurement points.
  • the adsorption of the substance to be measured on the sample contact surface may be physical adsorption, chemical adsorption, or electrostatic adsorption.
  • the substance to be measured is a substance that is difficult to adsorb on the sample contact surface, a modification or the like for imparting a functional group to the substance to be measured and / or the sample contact surface may be performed.
  • the adsorbed substance to be measured may not be in an adsorbed state.
  • the measurement step can be carried out in a state where the electric field enhancement effect due to the plasmon enhancement field generated by the measurement light irradiation in is sufficiently present.
  • the substance to be measured has chemical reactivity with the substance constituting the sample contact surface (for example, when the metal on the sample contact surface is Au and the substance to be measured has S 2 ⁇ in the sample), chemical adsorption If this is not the case, surface modification capable of chemically bonding to the sample contact surface and / or the substance to be measured may be performed. However, even with physical adsorption or electrostatic adsorption, sufficiently sensitive sensing can be performed in the sensing method of the present invention.
  • the substance to be measured can be oriented and adsorbed on the sample contact surface by the plasmon enhancement field by using linearly polarized light as the excitation light.
  • linearly polarized light having the same polarization direction as the excitation light as the measurement light, sensing with higher sensitivity is possible.
  • the adsorption process is performed simultaneously at a plurality of measurement points.
  • an area including all measurement points in the adsorption process it is preferable to irradiate simultaneously with excitation light.
  • the measurement area is large and it is difficult to irradiate the excitation light at the same time, after performing the adsorption process in the area where the excitation light can be irradiated at the same time, move the excitation light irradiation position to the area where the adsorption process has not been performed and perform the same adsorption process. It is preferable to carry out the measurement step after the adsorption step is completed in the entire measurement region.
  • the excitation light preferably has substantially uniform intensity at the sample contact surface, and is preferably light having a wavelength of 700 nm to 1600 nm.
  • the adsorption step may be performed by irradiating the sample with excitation light until at least a part of the substance to be detected in the sample is adsorbed on the sample contact surface of the plasmon active substrate, but from the viewpoint of improving measurement accuracy, it is as wide as possible. It is preferable to carry out the process until many substances to be detected are adsorbed in the region.
  • the time required for adsorption varies depending on the Raman stimulated emission cross-sectional area and concentration of the substance to be detected, so these values, the output of the light source used, the beam diameter, etc. It is necessary to adjust the adsorption time in consideration.
  • a single mode semiconductor laser with a maximum output of 300 mW and a peak wavelength of 808 nm is used to expand the beam diameter to a diameter of about 10 mm, and then the adsorption process is performed with excitation light whose beam in-plane intensity is made uniform with a diffusion plate Irradiation may take about 2 to 5 minutes.
  • the sample on the sample contact surface is irradiated with measurement light including excitation light, and this irradiation generates a plasmon enhancement field on the sample contact surface, and physical characteristics of the signal light emitted from the sample Is detected.
  • sensing is performed at a plurality of measurement points, and the measurement process is performed for each of the plurality of measurement points of the sample.
  • the signal light to be detected and its physical characteristics are not particularly limited.
  • Examples of signal light include Raman scattered light, fluorescence, coherent anti-Stokes light, Rayleigh scattered light, Mie scattered light, second harmonic light, third harmonic light, and the like.
  • the sensing method of the present invention uses signal light. It can be applied to sensing that detects physical characteristics according to the.
  • the substance to be measured can be specified by detecting the wavelength shift specific to the substance from the Raman spectrum (physical characteristics) obtained from the Raman scattered light.
  • the sensing device of the present invention suitable for the sensing method of the present invention will be described by taking a Raman spectroscopic device whose signal light is Raman scattered light as an example.
  • FIG. 1 is a schematic configuration diagram of a sensing device (a Raman spectroscopic device) of the present embodiment.
  • the scale of each component is appropriately changed and shown for easy visual recognition.
  • the Raman spectroscopic device (sensing device) 1 has a sample contact surface 10s with which a sample S containing a substance R to be measured is brought into contact, and a plasmon that generates a plasmon enhancement field on the sample contact surface 10s when irradiated with excitation light L1.
  • the measurement light L2 is irradiated onto a plurality of measurement points of the sample S, and the Raman spectrum (physical characteristics) of the Raman scattered light L3 at the plurality of measurement points is detected.
  • the excitation light irradiation optical system 20 irradiates the excitation light L1 that can simultaneously irradiate a region including at least two measurement points on the sample contact surface 10s.
  • a Raman spectroscopic device 1 shown in FIG. 1 uses an excitation light irradiation optical system 20 to adsorb a sample S placed on a sample contact surface 10s of a plasmon active substrate 10 in the sensing method of the present invention. Then, the irradiation of the excitation light L1 is canceled, the measurement light irradiation optical system 30 emits the measurement light L2 including the excitation light, and the irradiation generates a plasmon enhancement field on the sample contact surface 10s. A measurement step of detecting the Raman spectrum (physical characteristics) of the Raman scattered light L3 emitted from S by the physical characteristic detection system 30 is performed.
  • the excitation light irradiation optical system 20 irradiates excitation light L1 that can simultaneously irradiate a region including all measurement points on the sample contact surface 10s, and the adsorption in the sensing method of the present invention described above.
  • the mode for carrying out the process is shown, it is not possible to irradiate a region including all measurement points at the same time by providing a scanning means capable of relatively moving the plasmon active substrate 10 in the illustrated x-axis direction. Even so, by performing the adsorption process by changing the irradiation position of the excitation light L1, the adsorption process of the region including all measurement points can be performed before the measurement process.
  • the scanning means may be, for example, one that scans excitation light L1 such as a galvanometer mirror, or may make the plasmon active substrate 10 movable in the x direction.
  • one measurement point is a point where the measurement light L2 is condensed on the sample contact surface 10s.
  • the Raman spectroscopic device 1 is configured to detect Raman scattered light from the back surface of the plasmon active substrate 10.
  • the Raman spectroscopic device 1 is configured to detect Raman scattered light from the back surface of the plasmon active substrate 10.
  • the sample contact surface 10s is a substrate on which plasmon can be excited by the excitation light L1, and irradiation of the measurement light L2 and Raman scattering from the back side of the substrate 10
  • the light L3 can be detected.
  • Plasmons are excited when metal free electrons resonate with the electric field of light and vibrate.
  • the free electrons of the convex part resonate with the electric field of light. It is said that a strong electric field is generated around the convex portion by the vibration, and the localized plasmon resonance occurs effectively.
  • FIG. 2 is a cross-sectional view in the thickness direction showing an example of a preferred embodiment of the plasmon active substrate 10.
  • the plasmon active substrate 10 includes a transparent substrate 11, a fine concavo-convex structure 12 provided on the surface of the transparent substrate 11, and a metal film 13 formed on the surface of the fine concavo-convex structure 12.
  • a metal film 13 is formed along the fine concavo-convex structure 12 to form a metal fine concavo-convex structure, and has a surface with a metal fine concavo-convex structure, which can obtain a photoelectric enhancement effect by local plasmon resonance. It functions as a field enhancement device.
  • the plasmon active surface has translucency for each light.
  • the translucency here means that the excitation light L1 is capable of plasmon excitation, the measurement light L2 is capable of exciting Raman scattered light that can be detected with high sensitivity, and the Raman scattered light L3.
  • the light detection unit 41 has such a translucency that the light detection unit 41 has an intensity capable of high-sensitivity detection after being emitted from the substrate 10.
  • the transparent substrate 11 for the plasmon active substrate 10 by using the transparent substrate 11 for the plasmon active substrate 10, light can be irradiated from either the front surface side of the metal film 13 or the back surface side of the transparent substrate 11, and by this light irradiation, Light generated from the sample can also be detected from either the front surface side of the metal film 13 or the back surface side of the transparent substrate 11. Therefore, the excitation light L1, the measurement light L2, and the Raman scattered light L3 are detected from either the front surface side of the metal film 13 or the back surface side of the transparent substrate 11 depending on the type, size, etc. of the substance to be detected. Therefore, the degree of freedom in measurement is high, and detection can be performed with a higher S / N.
  • the transparent substrate 11 is not particularly limited, and can be selected from glass, transparent resin, and the like.
  • the fine concavo-convex structure 12 may be any structure that can generate plasmons on the surface of the metal fine structure formed by forming the metal film 13 on the surface of the fine concavo-convex structure 12. And it is preferable that it is a fine concavo-convex structure with an average pitch that is shorter than the wavelength of the excitation light.
  • the fine concavo-convex structure 12 desirably has an average depth of 200 nm or less from the top of the convex portion to the bottom of the adjacent concave portion, and an average pitch between the vertices of the most adjacent convex portions separating the concave portions.
  • the average pitch of the unevenness is obtained by binarizing a surface image taken by a scanning electron microscope (SEM) by image processing and performing statistical processing.
  • the material of the fine concavo-convex structure 12 is not particularly limited as long as it has the above-described translucency, but a boehmite layer or an anodized alumina layer is preferable. Further, the fine concavo-convex structure 12 is not only made of a material different from that of the transparent substrate 11, but may be made of the same material as the substrate body by processing the surface of the transparent substrate body. For example, the surface of the glass substrate may be used by forming a fine uneven structure on the surface by performing lithography and dry etching treatment.
  • the fine uneven structure 12 is more preferably a boehmite layer.
  • Boehmite is a hydrate of aluminum.
  • the boehmite layer 12 is formed on the transparent substrate 11 by, for example, preparing a transparent substrate 11 washed with pure water, depositing aluminum on the surface thereof by about several tens of nanometers by sputtering, and then boiling pure water.
  • the transparent substrate 11 with aluminum can be immersed in water and taken out after a few minutes (about 5 minutes) (boiling treatment (boehmite treatment)).
  • the metal film 13 may be made of a metal that can generate localized plasmons when irradiated with excitation light.
  • a metal that can generate localized plasmons when irradiated with excitation light.
  • it is made of Au, Ag, Cu, Al, Pt, or an alloy containing these as a main component. It consists of at least one metal selected from the group.
  • Au or Ag is preferable.
  • the main component means a component having a content of 90% by mass or more.
  • the thickness of the metal film 13 is such that when formed on the surface of the fine concavo-convex structure 12, the concavo-convex shape capable of generating localized plasmons upon irradiation with excitation light as the metal fine concavo-convex structure can be maintained. If there is no particular limitation, it is preferably 10 to 100 nm.
  • the metal film 13 may be formed on the fine concavo-convex structure 12 by vapor deposition or the like.
  • the plasmon active substrate 10 may be in the form of a microwell plate as shown in FIG. By setting it as this aspect, an adsorption process can be simultaneously implemented with respect to the sample S inject
  • a typical microwell plate has, for example, a top view area of about 100 mm ⁇ 50 mm, and has 96 wells.
  • the application of the present invention dramatically improves the measurement efficiency. Can be made.
  • the microwell plate-shaped plasmon active substrate 10 has, for example, a configuration including a transparent substrate 11, a fine uneven structure 12 and a metal film 13 disposed thereon.
  • the metal fine concavo-convex structure may be formed only on the surface of each well serving as the sample contact surface.
  • the excitation light irradiation optical system 20 simultaneously excites the entire sample S by expanding the beam diameter of the excitation light source 21, the mirror 22 for guiding the excitation light L1 emitted from the excitation light source 21 toward the plasmon active substrate 10, and the excitation light L1.
  • the beam expander 23 enables the light L1 to be irradiated, and the diffusion plate 24 that uniformizes the intensity of the excitation light L1 whose beam diameter is expanded by the beam expander 23 at the sample contact surface 10s.
  • the beam expander 23 is not particularly limited as long as the beam expander 23 can be expanded so as to have a desired beam diameter.
  • the beam expander 23 can be configured by two types of lenses having different curvatures.
  • the beam shape is made elliptic by enlarging one axis of the beam shape using two types of cylindrical lenses having different curvatures. It is preferable.
  • the excitation light source 21 is not particularly limited as long as it is a light source that emits light including a wavelength capable of exciting plasmons to the sample contact surface 10 s of the plasmon active substrate 10. Since a suitable wavelength for exciting plasmons varies depending on the type of metal that excites plasmons in the plasmon active substrate 10, it is preferable to select a light source with a suitable wavelength according to the type of metal.
  • the substance R to be measured is a biological substance, it is preferable that it is not damaged as much as possible. Therefore, it is preferably visible to near-infrared light having a relatively low energy of 700 nm to 2 ⁇ m.
  • the measurement light irradiation optical system (Raman scattering excitation light irradiation optical system) 30 emits measurement light (Raman scattering excitation light) L2 including excitation light, and the light L2 to the sample contact surface 10s of the plasmon active substrate 10.
  • measurement light Raman scattering excitation light
  • the mirror 33 (43) reflects the measurement light L2, and includes light from the plasmon active substrate 10 side including the enhanced Raman scattered light L3 generated from the detection target substance R of the sample S by irradiation of the measurement light L2.
  • the light source 31 of the measurement light L2 is a single wavelength light source such as a laser that emits light of a specific wavelength, and a distributed feedback (DFB) semiconductor laser or the like can be preferably used.
  • a distributed feedback (DFB) semiconductor laser or the like can be preferably used.
  • plasmon is excited on the sample contact surface 10s of the plasmon active substrate 10 to generate a plasmon enhancement field, and light (scattering) containing Raman scattered light L3 in the sample S containing the substance R to be measured Light).
  • the light including the Raman scattered light L3 is enhanced by the generated plasmon enhancement field.
  • the Raman spectroscopic device 1 performs measurement on a plurality of measurement points.
  • the movement of the measurement point may be manually performed.
  • the scanning unit can be relatively moved in the x direction shown in the figure. It is preferable to provide.
  • the scanning means for example, the measuring light L2 such as a galvanometer mirror may be scanned, or the plasmon active substrate 10 may be movable in the x direction.
  • the physical property detection system 40 transmits the light including the Raman scattered light L3 emitted from the sample S including the Raman scattered light derived from the measurement target R to the dichroic mirror 43 and the dichroic mirror 43.
  • the pinhole plate having a notch filter 46 that absorbs light of the wavelength of the measurement light L2 and transmits other light, and a pinhole for removing noise light 48, a lens 47 for condensing the light including the Raman scattered light L3 transmitted through the notch filter 46 into the pinhole of the pinhole plate 48, and the Raman scattered light L3 that has passed through the pinhole are converted into parallel light.
  • a lens 49 that leads to a detection unit (spectrometer) 41 and a light detection unit (spectrometer) 41 that obtains a Raman spectrum by splitting the Raman scattered light L3 are provided.
  • the light including the Raman scattered light L3 is displayed as L3, and the path including the light including the Raman scattered light L3 and the measurement light L2 is displayed in the same way, but actually includes the Raman scattered light L3. Since the light includes various lights such as the reflected light of the measuring light L2, the Raman scattered light, the Rayleigh scattered light, and the Mie scattered light on the sample S, the light including the Raman scattered light L3 is illustrated in the lens 45. It is incident at a wider angle than the angle that is being used. Therefore, the dichroic mirror 43 cannot reflect light having the same wavelength as all the measurement light L2.
  • the notch filter 46 cuts light (reflected light, Rayleigh scattered light, Mie scattered light, etc.) having the same wavelength as the measurement light L2 that has passed through the dichroic mirror 43.
  • Light having a wavelength different from that of the excitation light (Raman scattered light) passes through the notch filter 46, is collected by the lens 47, passes through the pinhole 48, is collimated again by the lens 49, and enters the spectroscope 41.
  • a Raman spectrum (physical characteristic) is detected.
  • the Raman spectroscopic device (sensing device) 1 is configured.
  • the Raman spectroscopic device (sensing device) 1 performs sensing by detecting the signal light L3 enhanced in the plasmon enhancement field at a plurality of measurement points of the sample S on the plasmon active substrate 10, and excites the plasmon.
  • the excitation light irradiation optical system 20 includes at least two measurements on the sample contact surface 10s. It is assumed that an area including a point can be irradiated simultaneously.
  • the plasmon enhancement field can be generated simultaneously at a plurality of measurement points to adsorb the substance R to be measured on the sample contact surface 10s, and after the substance R to be measured is adsorbed to the sample contact surface 10s. Detecting a Raman spectrum (physical characteristic) of Raman scattered light (signal light) L3 emitted from the substance R to be measured by the irradiation and enhanced by the plasmon enhancement field generated on the sample contact surface 10s by the irradiation. Can do. Therefore, according to the present invention, the substance to be measured can be sensed in a short time and with high sensitivity at a plurality of measurement locations in the same sample. Further, by increasing the area that can be irradiated at the same time, sensing in a large-area sample can be performed efficiently and with high sensitivity.
  • the Raman spectroscopic device in which the signal light is Raman scattered light has been described.
  • the signal light is not limited to Raman scattered light, but fluorescence, coherent anti-Stokes light, Rayleigh scattered light, Mie scattered light, second
  • the present invention can be applied to a device that detects second-order harmonics, third-order harmonic light, and the like, and detects physical characteristics corresponding to them.
  • sensing can be performed with a simple optical system by using a filter matched to the fluorescence and using a cooled CCD, a photomultiplier tube (PMT), or the like as a detector.
  • PMT photomultiplier tube
  • at least two measurement points are simultaneously applied.
  • the effect of the present invention can be obtained simply by irradiating.
  • the measurement process may be performed after the adsorption process is performed at all measurement points, or the measurement process is performed after one adsorption process, and the excitation light irradiation region is shifted after the measurement process is completed.
  • a region adsorption step and a measurement step may be performed.
  • the said embodiment demonstrated the aspect which injects excitation light and measurement light from the opposite side (back surface of a plasmon active base

Abstract

[Problem] To make possible sensing in a plurality of measurement locations within the same sample in a short time with high sensitivity in sensing using a plasmon enhanced field. [Solution] A sensing device (1) is provided with: a plasmon active base substance (10) that gives rise to a plasmon enhanced field on a sample contact surface (10s) because of irradiation by excitation light (L1, L2); an excitation light irradiating optical system (20) that irradiates excitation light (L1); a measurement light irradiating optical system (30) that irradiates measurement light (L2) that includes the excitation light onto a sample (S); and a physical property detecting system (40) that detects the physical properties of signal light (L3) that is generated by the sample (S) on the sample contact surface (10s) by the irradiation of the measurement light (L2) and amplified by the plasmon enhanced field arising on the sample contact surface (10s) because of the irradiation. The measurement light (L2) is irradiated onto a plurality of measurement points on the sample (S) and the physical properties are detected at the plurality of measurement points. The excitation light irradiating optical system (20) irradiates excitation light (L1) that can be emitted simultaneously onto a region that includes at least two measurement points on the sample contact surface (10s).

Description

センシング装置およびこれを用いたセンシング方法Sensing device and sensing method using the same
　本発明は、被測定物質を含む試料を、励起光が照射されプラズモン増強場を生じさせるプラズモン活性基体と接触させた状態で、活性基体表面の被測定物質の特性を検出するセンシング装置および方法に関するものである。 The present invention relates to a sensing apparatus and method for detecting the characteristics of a substance to be measured on the surface of an active substrate in a state where a sample containing the substance to be measured is brought into contact with a plasmon active substrate that is irradiated with excitation light to generate a plasmon enhancement field. Is.
　光照射によりプラズモン増強場を生じうる金属微細凹凸構造等を表面に備えたプラズモン活性基体を利用して、ラマン散乱光や蛍光等の信号光を感度良く検出するセンサデバイスやそれらを用いたセンシング装置の研究開発が活発に行われている。 A sensor device for detecting signal light such as Raman scattered light and fluorescence with high sensitivity using a plasmon active substrate having a metal concavo-convex structure on the surface that can generate a plasmon enhancement field by light irradiation, and a sensing device using them R & D is actively underway.
　プラズモン増強場は、プラズモン増強場を生じる表面（センシング面）上が最も強く、該面からの距離に対して指数関数的にその強度が減少することが知られており、センシング時に、被測定物質がセンシング面の近くにあればあるほど高感度なセンシングを行うことができる。従って、プラズモン増強場を利用するセンシングにおいては、試料溶液中の被測定物質のセンシング面近傍における濃度をできるだけ高くすることが重要な課題の１つとなっている。 It is known that the plasmon enhancement field is strongest on the surface (sensing surface) where the plasmon enhancement field is generated, and its intensity decreases exponentially with respect to the distance from the surface. Sensation with higher sensitivity can be performed the closer to the sensing surface. Therefore, in sensing using a plasmon enhancement field, it is one of important issues to make the concentration of a substance to be measured in a sample solution as high as possible in the vicinity of the sensing surface.
　プラズモン活性基体のセンシング面近傍において、被測定物質濃度を高くする方法としては、例えば、センシング面上に被測定物質と特異結合するリガンドを固定しておき、リガンドに被測定物質を結合させる方法があげられる。これによりセンシング面近傍に試料中の被測定物質が固定され、高感度なセンシングを行うことできる。 As a method of increasing the concentration of the substance to be measured in the vicinity of the sensing surface of the plasmon active substrate, for example, a method of fixing a ligand that specifically binds to the substance to be measured on the sensing surface and binding the substance to be measured to the ligand. can give. Thereby, the substance to be measured in the sample is fixed near the sensing surface, and highly sensitive sensing can be performed.
　また、特許文献１には、電気泳動法により金属膜上に被測定物質を集めることにより金属膜上における被測定物質濃度を高めて、表面プラズモン共鳴の測定を行う装置として、試料溶液中に複数の電極を設置して、この複数の電極への印加電圧を制御することにより測定箇所での被測定物質の濃度を上昇させる構成の誘電泳動検出装置が開示されている。 Further, Patent Document 1 discloses a device for collecting surface analytes on a metal film by electrophoresis and increasing the concentration of the substance to be measured on the metal film to measure surface plasmon resonance. A dielectrophoresis detection device having a configuration in which the concentration of a substance to be measured at a measurement location is increased by controlling the voltage applied to the plurality of electrodes by installing the electrodes is disclosed.
　特許文献２及び特許文献３には、プラズモン増強場を生じるセンシング面に、試料溶液中の被測定物質を光捕捉効果により捕捉させる捕捉光を照射する、光捕捉光照射光学系を備えたセンシング装置が開示されている。 Patent Document 2 and Patent Document 3 disclose a sensing device including a light capturing light irradiation optical system that irradiates a sensing surface that generates a plasmon enhancement field with capture light that captures a target substance in a sample solution by a light capturing effect. Is disclosed.
特開2005-195397号公報JP 2005-195397 特開2009-42112号公報JP 2009-42112 JP 特開2009-222484号公報JP 2009-222484 A
　しかしながら、リガンドを利用して被測定物質をセンシング面に固定させる方法では、リガンドと被測定物質との反応を充分に待たなければ、充分な量の結合量を確保できず、測定を迅速に実施することが難しい。しかも、リガンドと結合する物質は限られており、被測定物質の種類が限定されてしまう。 However, in the method of immobilizing a substance to be measured on the sensing surface using a ligand, a sufficient amount of binding cannot be ensured unless the reaction between the ligand and the substance to be measured is sufficiently waited, and the measurement is carried out quickly. Difficult to do. In addition, substances that bind to the ligand are limited, and the types of substances to be measured are limited.
　また、特許文献１に記載の装置では、溶液中に配置した複数の電極に対して印加電圧を制御する必要があり、また試料毎に印加電圧を調整しなければ基体表面での被測定物質濃度を高めることができないという問題がある。また、印加電圧をかける場合、被測定物質に対して電荷をチャージする必要があり、電荷をチャージすることで被測定物質の物性が変化する恐れもある。 Moreover, in the apparatus described in Patent Document 1, it is necessary to control the applied voltage for a plurality of electrodes arranged in the solution, and the concentration of the substance to be measured on the substrate surface unless the applied voltage is adjusted for each sample. There is a problem that can not be raised. In addition, when applying an applied voltage, it is necessary to charge the substance to be measured, and the physical properties of the substance to be measured may be changed by charging the charge.
　特許文献２や特許文献３のように、光捕捉効果を用いて捕捉光により被測定物質をセンシング面に捕捉させてからセンシングを行う場合、被測定物質の種類の限定や印加電圧の付与をすることなくセンシング面における被測定物質の濃度を高めてセンシングを実施することができる。しかしながら、かかる方法では、複数の測定点におけるセンシングをする場合には、各測定点毎に被測定物質の光捕捉を行う必要があり、測定点の数に比例してその測定時間が増加する。従って、大面積の試料に対して面内で一様にセンシングを行う場合や、データ信頼性向上を目的として同一試料に対して光照射箇所を変えて測定を実施して複数のデータを取る場合には莫大な時間を要することになり実用的でない。 When sensing is performed after the substance to be measured is captured on the sensing surface by the captured light using the light capturing effect as in Patent Document 2 and Patent Document 3, the type of the substance to be measured is limited and an applied voltage is applied. Without increasing the concentration of the substance to be measured on the sensing surface, sensing can be performed. However, in this method, when sensing at a plurality of measurement points, it is necessary to capture light of the substance to be measured at each measurement point, and the measurement time increases in proportion to the number of measurement points. Therefore, when performing uniform sensing within a surface for a large-area sample, or when performing multiple measurements with different light irradiation locations on the same sample for the purpose of improving data reliability. Takes a lot of time and is not practical.
　本発明は、上記事情を鑑みてなされたものであり、光照射によりプラズモン増強場を生じうるプラズモン活性基体を利用して、ラマン散乱光や蛍光等の信号光を感度良く検出するセンシング装置において、同一試料内の複数の測定箇所に対して短時間且つ高感度に被測定物質のセンシングが可能なセンシング装置及びそれを用いたセンシング方法を提供することを目的とするものである。 The present invention has been made in view of the above circumstances, and in a sensing device that detects signal light such as Raman scattered light or fluorescence with high sensitivity using a plasmon active substrate that can generate a plasmon enhancement field by light irradiation. It is an object of the present invention to provide a sensing device capable of sensing a substance to be measured with high sensitivity at a plurality of measurement locations in the same sample and a sensing method using the sensing device.
　本発明のセンシング装置は、被測定物質を含む試料が接触される試料接触面を有し、該試料接触面に励起光が照射されることにより該試料接触面にプラズモン増強場を生じるプラズモン活性基体と、
　前記励起光を照射する励起光照射光学系と、
　前記試料に前記励起光を含む測定光を照射する測定光照射光学系と、
　前記測定光の照射により前記試料接触面上の試料から発せられ、且つ、該照射により前記試料接触面に生じたプラズモン増強場により増強された信号光の物理特性を検出する物理特性検出系とを備えてなり、
　前記測定光を前記試料の複数の測定点に照射して、該複数の測定点における前記物理特性を検出するものであり、
　前記励起光照射光学系は、前記試料接触面の少なくとも２つの前記測定点を含む領域を同時に照射可能な前記励起光を照射するものであることを特徴とするものである。 The sensing device of the present invention has a sample contact surface with which a sample containing a substance to be measured is brought into contact, and a plasmon active substrate that generates a plasmon enhancement field on the sample contact surface by irradiating the sample contact surface with excitation light When,
An excitation light irradiation optical system for irradiating the excitation light;
A measurement light irradiation optical system for irradiating the sample with measurement light including the excitation light;
A physical property detection system for detecting the physical property of the signal light emitted from the sample on the sample contact surface by the irradiation of the measurement light and enhanced by the plasmon enhancement field generated on the sample contact surface by the irradiation; Prepared
Irradiating a plurality of measurement points of the sample with the measurement light to detect the physical characteristics at the plurality of measurement points;
The excitation light irradiating optical system irradiates the excitation light capable of irradiating simultaneously a region including at least two of the measurement points on the sample contact surface.
　本明細書において、「測定点」とは、１回のセンシングを実施する任意の測定光照射面における点であり、１回のセンシングで測定光が照射されるスポットを意味する。 In the present specification, the “measurement point” is a point on an arbitrary measurement light irradiation surface where one sensing is performed, and means a spot irradiated with the measurement light by one sensing.
　前記励起光照射光学系は、前記試料接触面の全ての前記測定点を含む領域を同時に照射可能な前記励起光を照射するものであることが好ましい。 It is preferable that the excitation light irradiation optical system irradiates the excitation light that can simultaneously irradiate a region including all the measurement points on the sample contact surface.
　本発明のセンシング装置において、前記励起光及び前記測定光は略同一方向の偏光を有していることが好ましく、偏光は直線偏光又は円偏光であることが好ましい。 In the sensing device of the present invention, the excitation light and the measurement light preferably have polarized light in substantially the same direction, and the polarized light is preferably linearly polarized light or circularly polarized light.
　また、前記励起光は、前記試料接触面における強度が略均一であることが好ましく、波長が７００ｎｍ～１６００ｎｍであることが好ましい。ここで、「強度が略均一である」とは、試料接触面における励起光の照射領域において、強度の自乗平均平方根粗さ（ＲＭＳ）が９０％超であることを意味する。 The excitation light preferably has a substantially uniform intensity at the sample contact surface, and preferably has a wavelength of 700 nm to 1600 nm. Here, “the intensity is substantially uniform” means that the root mean square roughness (RMS) of the intensity is more than 90% in the excitation light irradiation region on the sample contact surface.
　本発明のセンシング装置は、前記試料から発せられる信号光が、蛍光、ラマン散乱光、コヒーレントアンチストークス散乱光、第二次高調波光、第三次高調波光のいずれかである場合に好適に用いることができる。 The sensing device of the present invention is preferably used when the signal light emitted from the sample is any one of fluorescence, Raman scattered light, coherent anti-Stokes scattered light, second harmonic light, and third harmonic light. Can do.
　本発明のセンシング方法は、励起光が照射されることによりプラズモン増強場を生じる試料接触面に被測定物質を含む試料を接触させる試料接触工程と、
　該試料に前記励起光を照射して、該励起光照射部分の前記試料接触面にプラズモン増強場を生じさせて、該プラズモン増強場により前記被測定物質の少なくとも一部を前記試料接触面に吸着させる吸着工程と、
　該吸着工程後に、前記試料接触面上の該試料に前記励起光を含む測定光を照射して、該照射により前記試料接触面にプラズモン増強場を生じさせるとともに、前記試料から発せられる信号光の物理特性を検出する測定工程とを有するセンシング方法であって、
　前記測定工程は、前記試料の複数の測定点に対して該測定点毎に実施されるものであり、
　前記吸着工程において、少なくとも２つの前記測定点を含む領域に前記励起光を同時に照射することを特徴とするものである。 The sensing method of the present invention includes a sample contact step in which a sample containing a substance to be measured is brought into contact with a sample contact surface that generates a plasmon enhancement field when irradiated with excitation light;
Irradiating the sample with the excitation light to generate a plasmon enhancement field on the sample contact surface of the excitation light irradiation portion, and at least a part of the substance to be measured is adsorbed to the sample contact surface by the plasmon enhancement field An adsorption process,
After the adsorption step, the sample on the sample contact surface is irradiated with measurement light including the excitation light to generate a plasmon enhancement field on the sample contact surface by the irradiation, and signal light emitted from the sample A sensing method having a measurement process for detecting physical properties,
The measurement step is performed for each of the plurality of measurement points of the sample,
In the adsorption step, a region including at least two measurement points is simultaneously irradiated with the excitation light.
　前記吸着工程において、全ての前記測定点を含む領域に前記励起光を同時に照射することが好ましい。また、前記プラズモン増強場により前記被測定物質の少なくとも一部を前記試料接触面上に配向させて吸着させることがより好ましい。 In the adsorption step, it is preferable to irradiate the excitation light simultaneously to a region including all the measurement points. More preferably, at least a part of the substance to be measured is oriented and adsorbed on the sample contact surface by the plasmon enhancement field.
　本発明のセンシング装置は、プラズモン活性基体上の試料の複数の測定点においてプラズモン増強場により増強された信号光を検出してセンシングを行うものであり、プラズモンを励起する励起光照射光学系と、試料に励起光を含む測定光を照射する測定光照射光学系とをそれぞれ備え、励起光照射光学系を、試料接触面の少なくとも２つの測定点を含む領域を同時に照射可能なものとしている。かかる構成によれば、複数の測定点において同時にプラズモン増強場を生じさせて被測定物質を試料接触面に吸着させることができる上、試料接触面に被測定物質を吸着させた後に、測定光を照射して、該照射により被測定物質から発せられ、且つ、該照射により試料接触面に生じたプラズモン増強場により増強された信号光の物理特性の検出を行うことができる。従って、本発明によれば、同一試料内の複数の測定箇所に対して、短時間且つ高感度に被測定物質のセンシングを行うことができる。また、同時に照射可能な面積を大きくすることにより、大面積の試料におけるセンシングを効率的かつ高感度に実施することができる。 The sensing device of the present invention is to detect and detect signal light enhanced by a plasmon enhancement field at a plurality of measurement points of a sample on a plasmon active substrate, and an excitation light irradiation optical system that excites plasmons; A measurement light irradiation optical system for irradiating the sample with measurement light including excitation light is provided, and the excitation light irradiation optical system can simultaneously irradiate a region including at least two measurement points on the sample contact surface. According to such a configuration, it is possible to simultaneously generate a plasmon enhancement field at a plurality of measurement points to adsorb the substance to be measured on the sample contact surface, and to absorb the measurement substance after adsorbing the substance to be measured on the sample contact surface. It is possible to detect the physical characteristics of the signal light emitted from the substance to be measured by the irradiation and enhanced by the plasmon enhancement field generated on the sample contact surface by the irradiation. Therefore, according to the present invention, the substance to be measured can be sensed in a short time and with high sensitivity at a plurality of measurement locations in the same sample. Further, by increasing the area that can be irradiated at the same time, sensing in a large-area sample can be performed efficiently and with high sensitivity.
本発明に係る一実施形態のセンシング装置の概略構成図1 is a schematic configuration diagram of a sensing device according to an embodiment of the present invention. プラズモン活性基体の好適な態様を示す厚み方向断面図Cross section in thickness direction showing preferred embodiment of plasmon active substrate マイクロウェルプレート状のプラズモン活性基体の態様を示す厚み方向断面図Cross-sectional view in the thickness direction showing an embodiment of a plasmon active substrate in the form of a microwell plate
「センシング方法」
　本発明のセンシング方法について詳述する。 "Sensing method"
The sensing method of the present invention will be described in detail.
　本発明のセンシング方法は、プラズモン増強場により増強された信号光を検出するセンシング方法において、試料の複数箇所におけるセンシングを行う際に、プラズモン増強場を高効率に発生させることにより、短時間に高感度なセンシングを実現するものである。 The sensing method of the present invention is a sensing method for detecting signal light enhanced by a plasmon enhancement field. When sensing at a plurality of locations of a sample, the plasmon enhancement field is generated in a short time by generating the plasmon enhancement field with high efficiency. It realizes sensitive sensing.
　本発明のセンシング方法は、励起光が照射されることによりプラズモン増強場を生じる試料接触面に被測定物質を含む試料を接触させる試料接触工程と、
　該試料に励起光を照射して、励起光照射部分の試料接触面にプラズモン増強場を生じさせて、該プラズモン増強場により被測定物質の少なくとも一部を試料接触面に吸着させる吸着工程と、
　該吸着工程後に、試料接触面上の試料に励起光を含む測定光を照射して、該照射により試料接触面にプラズモン増強場を生じさせるとともに、試料から発せられる信号光の物理特性を検出する測定工程とを有するセンシング方法であって、
　測定工程は、試料の複数の測定点に対して該測定点毎に実施されるものであり、
　吸着工程において、少なくとも２つの測定点を含む領域に励起光を同時に照射することを特徴としている。 The sensing method of the present invention includes a sample contact step in which a sample containing a substance to be measured is brought into contact with a sample contact surface that generates a plasmon enhancement field when irradiated with excitation light;
An adsorption step of irradiating the sample with excitation light, generating a plasmon enhancement field on the sample contact surface of the excitation light irradiation portion, and adsorbing at least a part of the substance to be measured on the sample contact surface by the plasmon enhancement field;
After the adsorption step, the sample on the sample contact surface is irradiated with measurement light including excitation light, thereby generating a plasmon enhancement field on the sample contact surface and detecting physical characteristics of the signal light emitted from the sample. A sensing method having a measuring step,
The measurement process is performed for each of the plurality of measurement points of the sample,
In the adsorption step, an area including at least two measurement points is irradiated with excitation light simultaneously.
　プラズモン増強場は、プラズモンが励起されている面からの距離に対して指数関数的にその強度が減少する。従って、効果的に信号光を増強するためには、より被測定物質をプラズモン励起される試料接触面に近づけておく必要がある。 The intensity of the plasmon enhancement field decreases exponentially with the distance from the surface where the plasmon is excited. Therefore, in order to effectively enhance the signal light, it is necessary to bring the substance to be measured closer to the sample contact surface to be plasmon excited.
　本発明では、測定工程の前に、被測定物質をプラズモン増強場の電場勾配により試料接触面に引き寄せ、少なくとも一部を試料接触面に吸着させる吸着工程を実施し、この吸着工程を少なくとも２つの複数の測定点において同時に実施する。 In the present invention, before the measurement step, an adsorption step is performed in which the substance to be measured is attracted to the sample contact surface by the electric field gradient of the plasmon enhancement field, and at least a part is adsorbed on the sample contact surface. Performed simultaneously at multiple measurement points.
　被測定物質の試料接触面における吸着は、物理吸着、化学吸着、静電吸着のいずれであってもよい。被測定物質が、試料接触面に吸着しにくい物質である場合は、被測定物質及び／又は試料接触面に官能基を付与する修飾等を施せばよい。 The adsorption of the substance to be measured on the sample contact surface may be physical adsorption, chemical adsorption, or electrostatic adsorption. When the substance to be measured is a substance that is difficult to adsorb on the sample contact surface, a modification or the like for imparting a functional group to the substance to be measured and / or the sample contact surface may be performed.
　吸着態様が物理吸着である場合は、吸着工程後、測定工程を実施する際に、吸着させた被測定物質が吸着状態でなくなることもあるが、一度吸着した被測定物質であれば、測定工程における測定光照射により生じるプラズモン増強場による電場増強効果を充分に受けられる領域に存在した状態で測定工程を実施することができる。 When the adsorption mode is physical adsorption, when the measurement process is performed after the adsorption process, the adsorbed substance to be measured may not be in an adsorbed state. The measurement step can be carried out in a state where the electric field enhancement effect due to the plasmon enhancement field generated by the measurement light irradiation in is sufficiently present.
　より強固な吸着状態とするには、化学吸着させることが好ましい。被測定物質が試料接触面を構成する物質と化学反応性を有する場合（例えば、試料接触面の金属がＡｕであり、被測定物質が試料中においてＳ２－を有する場合等）は、化学吸着となりうるが、そうでない場合は、試料接触面及び／又は被測定物質に化学結合可能な表面修飾を施しておけばよい。しかしながら、物理吸着や静電吸着であっても、本発明のセンシング方法においては充分に高感度なセンシングを実施することができる。 In order to obtain a stronger adsorption state, it is preferable to perform chemical adsorption. When the substance to be measured has chemical reactivity with the substance constituting the sample contact surface (for example, when the metal on the sample contact surface is Au and the substance to be measured has S 2− in the sample), chemical adsorption If this is not the case, surface modification capable of chemically bonding to the sample contact surface and / or the substance to be measured may be performed. However, even with physical adsorption or electrostatic adsorption, sufficiently sensitive sensing can be performed in the sensing method of the present invention.
　被測定物質が非対称性を有する場合は、励起光として直線偏光を用いることによりプラズモン増強場により被測定物質を試料接触面上に配向させて吸着させることができる。かかる態様とし、測定光として励起光と同じ偏光方向の直線偏光を用いることで、より高感度なセンシングが可能となる。 When the substance to be measured has asymmetry, the substance to be measured can be oriented and adsorbed on the sample contact surface by the plasmon enhancement field by using linearly polarized light as the excitation light. By adopting such an aspect and using linearly polarized light having the same polarization direction as the excitation light as the measurement light, sensing with higher sensitivity is possible.
　ただし、ラマン分光法によるセンシング等、配向によりスペクトル内のピークが特異的に変化してしまう場合は、被測定物質が配向して吸着されないように、例えば、励起光及び測定光として円偏光を用いる等工夫することが好ましい。 However, when the peak in the spectrum changes specifically due to orientation, such as sensing by Raman spectroscopy, circularly polarized light is used as excitation light and measurement light, for example, so that the substance to be measured is not oriented and adsorbed It is preferable to devise.
　上記のとおり、吸着工程を複数の測定点において同時に実施するが、より大面積な測定領域に対して高感度なセンシングを短時間に実施するためには、吸着工程において全ての測定点を含む領域に励起光を同時に照射することが好ましい。 As described above, the adsorption process is performed simultaneously at a plurality of measurement points. However, in order to perform highly sensitive sensing in a short time for a larger measurement area, an area including all measurement points in the adsorption process. It is preferable to irradiate simultaneously with excitation light.
　測定領域が広く、同時に励起光を照射することが難しい場合は、同時に励起光照射可能な領域において吸着工程を実施した後、励起光照射位置を吸着工程未実施領域に移動させて同様に吸着工程を実施し、測定領域全体において吸着工程が完了した後に測定工程を実施することが好ましい。 If the measurement area is large and it is difficult to irradiate the excitation light at the same time, after performing the adsorption process in the area where the excitation light can be irradiated at the same time, move the excitation light irradiation position to the area where the adsorption process has not been performed and perform the same adsorption process. It is preferable to carry out the measurement step after the adsorption step is completed in the entire measurement region.
　励起光は、試料接触面における強度が略均一であることが好ましく、また、波長７００ｎｍ～１６００ｎｍの光であることが好ましい。 The excitation light preferably has substantially uniform intensity at the sample contact surface, and is preferably light having a wavelength of 700 nm to 1600 nm.
　吸着工程は、試料中の被検出物質の少なくとも一部が、プラズモン活性基体の試料接触面に吸着されるまで、励起光を試料に照射すればよいが、測定精度の向上の観点から、できるだけ広い領域において多くの被検出物質が吸着されるまで実施することが好ましい。 The adsorption step may be performed by irradiating the sample with excitation light until at least a part of the substance to be detected in the sample is adsorbed on the sample contact surface of the plasmon active substrate, but from the viewpoint of improving measurement accuracy, it is as wide as possible. It is preferable to carry out the process until many substances to be detected are adsorbed in the region.
　吸着に要する時間は、例えば、信号光がラマン散乱光である場合は、被検出物質のラマン誘導放出断面積や濃度によって変わるため、これらの数値、及び、使用する光源の出力、ビーム径等を考慮して吸着時間は調整する必要がある。最大出力３００ｍＷ、ピーク波長８０８ｎｍのシングルモード半導体レーザを用いて、直径約１０ｍｍにビーム径を拡大した後、拡散板にてビーム面内強度を一様化した励起光により吸着工程を実施する場合は、２～５分程度照射すればよい。 For example, when the signal light is Raman scattered light, the time required for adsorption varies depending on the Raman stimulated emission cross-sectional area and concentration of the substance to be detected, so these values, the output of the light source used, the beam diameter, etc. It is necessary to adjust the adsorption time in consideration. When a single mode semiconductor laser with a maximum output of 300 mW and a peak wavelength of 808 nm is used to expand the beam diameter to a diameter of about 10 mm, and then the adsorption process is performed with excitation light whose beam in-plane intensity is made uniform with a diffusion plate Irradiation may take about 2 to 5 minutes.
　測定工程は、吸着工程後に、試料接触面上の試料に励起光を含む測定光を照射して、該照射により試料接触面にプラズモン増強場を生じさせるとともに、試料から発せられる信号光の物理特性を検出するものである。 In the measurement process, after the adsorption process, the sample on the sample contact surface is irradiated with measurement light including excitation light, and this irradiation generates a plasmon enhancement field on the sample contact surface, and physical characteristics of the signal light emitted from the sample Is detected.
　本発明のセンシング方法では、複数の測定点においてセンシングを行うものであり、測定工程は、試料の複数の測定点に対して測定点毎に実施する。 In the sensing method of the present invention, sensing is performed at a plurality of measurement points, and the measurement process is performed for each of the plurality of measurement points of the sample.
　測定工程において、検出する信号光及びその物理特性は特に制限されない。信号光としては、例えば、ラマン散乱光、蛍光、コヒーレントアンチストークス光、レイリー散乱光、ミー散乱光、第２高調波光、第３次高調波光等が挙げられ、本発明のセンシング方法は、信号光に応じた物理特性を検出するセンシングに適用することができる。例えば、信号光がラマン散乱光である場合は、ラマン散乱光から得られるラマンスペクトル（物理特性）から物質固有の波長シフトを検出することにより被測定物質を特定することができる。 In the measurement process, the signal light to be detected and its physical characteristics are not particularly limited. Examples of signal light include Raman scattered light, fluorescence, coherent anti-Stokes light, Rayleigh scattered light, Mie scattered light, second harmonic light, third harmonic light, and the like. The sensing method of the present invention uses signal light. It can be applied to sensing that detects physical characteristics according to the. For example, when the signal light is Raman scattered light, the substance to be measured can be specified by detecting the wavelength shift specific to the substance from the Raman spectrum (physical characteristics) obtained from the Raman scattered light.
　以下に、本発明のセンシング方法に好適な、本発明のセンシング装置について、信号光がラマン散乱光であるラマン分光装置を例に説明する。 Hereinafter, the sensing device of the present invention suitable for the sensing method of the present invention will be described by taking a Raman spectroscopic device whose signal light is Raman scattered light as an example.
「センシング装置」
　図面を参照して本発明に係る一実施形態のセンシング装置について説明する。図１は、本実施形態のセンシング装置（ラマン分光装置）の概略構成図である。本明細書の図面において、各構成要素の縮尺は、視認しやすくするため適宜変更して示してある。 "Sensing device"
A sensing device according to an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a schematic configuration diagram of a sensing device (a Raman spectroscopic device) of the present embodiment. In the drawings of this specification, the scale of each component is appropriately changed and shown for easy visual recognition.
　ラマン分光装置（センシング装置）１は、被測定物質Ｒを含む試料Ｓが接触される試料接触面１０ｓを有し、励起光Ｌ１が照射されることにより試料接触面１０ｓにプラズモン増強場を生じるプラズモン活性基体１０と、励起光Ｌ１を照射する励起光照射光学系２０と、試料Ｓに励起光を含む測定光Ｌ２を照射する測定光照射光学系３０と、測定光Ｌ２の照射により試料接触面１０ｓ上の試料Ｓから発せられ、且つ、該照射により前記試料接触面に生じたプラズモン増強場により増強された信号光（ラマン散乱光）Ｌ３の物理特性を検出する物理特性検出系４０とを備えてなり、測定光Ｌ２を試料Ｓの複数の測定点に照射して、複数の測定点におけるラマン散乱光Ｌ３のラマンスペクトル（物理特性）を検出するものである。ラマン分光装置１において、励起光照射光学系２０は、試料接触面１０ｓの少なくとも２つの測定点を含む領域を同時に照射可能な励起光Ｌ１を照射するものである。 The Raman spectroscopic device (sensing device) 1 has a sample contact surface 10s with which a sample S containing a substance R to be measured is brought into contact, and a plasmon that generates a plasmon enhancement field on the sample contact surface 10s when irradiated with excitation light L1. The active substrate 10, the excitation light irradiation optical system 20 for irradiating the excitation light L1, the measurement light irradiation optical system 30 for irradiating the sample S with the measurement light L2 including the excitation light, and the sample contact surface 10s by irradiation with the measurement light L2. A physical property detection system 40 for detecting physical properties of signal light (Raman scattered light) L3 emitted from the sample S on the top and enhanced by a plasmon enhancement field generated on the sample contact surface by the irradiation. Thus, the measurement light L2 is irradiated onto a plurality of measurement points of the sample S, and the Raman spectrum (physical characteristics) of the Raman scattered light L3 at the plurality of measurement points is detected. In the Raman spectroscopic device 1, the excitation light irradiation optical system 20 irradiates the excitation light L1 that can simultaneously irradiate a region including at least two measurement points on the sample contact surface 10s.
　図１に示されるラマン分光装置１は、プラズモン活性基体１０の試料接触面１０ｓ上に裁置された試料Ｓに対して、励起光照射光学系２０を用いて上記本発明のセンシング方法における吸着工程を実施した後に、励起光Ｌ１の照射を解除し、測定光照射光学系３０により励起光を含む測定光Ｌ２を照射して、該照射により試料接触面１０ｓにおいてプラズモン増強場を生じさせるとともに、試料Ｓから発せられるラマン散乱光Ｌ３のラマンスペクトル（物理特性）を物理特性検出系３０により検出する測定工程を実施するものである。 A Raman spectroscopic device 1 shown in FIG. 1 uses an excitation light irradiation optical system 20 to adsorb a sample S placed on a sample contact surface 10s of a plasmon active substrate 10 in the sensing method of the present invention. Then, the irradiation of the excitation light L1 is canceled, the measurement light irradiation optical system 30 emits the measurement light L2 including the excitation light, and the irradiation generates a plasmon enhancement field on the sample contact surface 10s. A measurement step of detecting the Raman spectrum (physical characteristics) of the Raman scattered light L3 emitted from S by the physical characteristic detection system 30 is performed.
　図１に示されるラマン分光装置１では、励起光照射光学系２０が試料接触面１０ｓの全ての測定点を含む領域を同時に照射可能な励起光Ｌ１を照射し、上記本発明のセンシング方法における吸着工程を実施する態様を示してあるが、プラズモン活性基体１０を図示ｘ軸方向に相対的に移動させることができる走査手段を備えておくことにより、同時に全ての測定点を含む領域を照射できない場合であっても、励起光Ｌ１の照射位置を変えて吸着工程を実施することにより、測定工程前に、全ての測定点を含む領域の吸着工程を実施することができる。走査手段としては、例えば、ガルバノミラー等の励起光Ｌ１を走査させるものであってもよいし、プラズモン活性基体１０をｘ方向に可動とするものであってもよい。 In the Raman spectroscopic device 1 shown in FIG. 1, the excitation light irradiation optical system 20 irradiates excitation light L1 that can simultaneously irradiate a region including all measurement points on the sample contact surface 10s, and the adsorption in the sensing method of the present invention described above. Although the mode for carrying out the process is shown, it is not possible to irradiate a region including all measurement points at the same time by providing a scanning means capable of relatively moving the plasmon active substrate 10 in the illustrated x-axis direction. Even so, by performing the adsorption process by changing the irradiation position of the excitation light L1, the adsorption process of the region including all measurement points can be performed before the measurement process. The scanning means may be, for example, one that scans excitation light L1 such as a galvanometer mirror, or may make the plasmon active substrate 10 movable in the x direction.
　なお、図１において、個々の測定点については図示していないが、測定光Ｌ２が試料接触面１０ｓにて集光している点が１つの測定点である。 In FIG. 1, although individual measurement points are not shown, one measurement point is a point where the measurement light L2 is condensed on the sample contact surface 10s.
　また、ラマン分光装置１は、プラズモン活性基体１０の裏面からラマン散乱光を検出する構成としている。かかる構成とすることにより、試料Ｓ中に細胞のような大きな検体を配し、検体からしみだす被検出物質Ｒを検出するような場合に、試料接触面１０ｓと被検出物質Ｒとの界面で最も強く生じる増強ラマン散乱光が検体自身により遮蔽されることなく基体１０の裏面側から検出することができる。 Further, the Raman spectroscopic device 1 is configured to detect Raman scattered light from the back surface of the plasmon active substrate 10. With such a configuration, when a large specimen such as a cell is arranged in the sample S and the detected substance R exuding from the specimen is detected, at the interface between the sample contact surface 10s and the detected substance R. The most strongly generated enhanced Raman scattered light can be detected from the back side of the substrate 10 without being shielded by the specimen itself.
　以下、ラマン分光装置１の各構成要素について説明する。 Hereinafter, each component of the Raman spectroscopic device 1 will be described.
　本実施形態において、試料Ｓを裁置するプラズモン活性基体１０は、試料接触面１０ｓが、励起光Ｌ１によりプラズモンが励起されうる基体であり、基体１０の裏面側から測定光Ｌ２の照射及びラマン散乱光Ｌ３の検出を実施可能なものであれば特に制限されない。 In the present embodiment, in the plasmon active substrate 10 on which the sample S is placed, the sample contact surface 10s is a substrate on which plasmon can be excited by the excitation light L1, and irradiation of the measurement light L2 and Raman scattering from the back side of the substrate 10 There is no particular limitation as long as the light L3 can be detected.
　プラズモンは、金属の自由電子が光の電場に共鳴して振動することで励起されるものであり、微細な凹凸構造を有する金属層等では、凸部の自由電子が光の電場に共鳴して振動することで凸部周辺に強い電場を生じ、局在プラズモン共鳴が効果的に起こるとされている。 Plasmons are excited when metal free electrons resonate with the electric field of light and vibrate. In a metal layer or the like having a fine concavo-convex structure, the free electrons of the convex part resonate with the electric field of light. It is said that a strong electric field is generated around the convex portion by the vibration, and the localized plasmon resonance occurs effectively.
　図２は、プラズモン活性基体１０の好適な態様の一例を示す厚み方向断面図である。図２に示されるように、プラズモン活性基体１０は、透明基板１１と、透明基板１１の表面に備えられた微細凹凸構造１２と、その微細凹凸構造１２表面に形成された金属膜１３とからなる。金属膜１３が微細凹凸構造１２に沿って形成されて金属の微細凹凸構造を構成するものとなり、表面に金属微細凹凸構造を備えた、局在プラズモン共鳴による光電増強効果を得ることが可能な光電場増強デバイスとして機能するものである。 FIG. 2 is a cross-sectional view in the thickness direction showing an example of a preferred embodiment of the plasmon active substrate 10. As shown in FIG. 2, the plasmon active substrate 10 includes a transparent substrate 11, a fine concavo-convex structure 12 provided on the surface of the transparent substrate 11, and a metal film 13 formed on the surface of the fine concavo-convex structure 12. . A metal film 13 is formed along the fine concavo-convex structure 12 to form a metal fine concavo-convex structure, and has a surface with a metal fine concavo-convex structure, which can obtain a photoelectric enhancement effect by local plasmon resonance. It functions as a field enhancement device.
　本実施形態のように、試料接触面１０ｓの裏面側から励起光Ｌ１又は測定光Ｌ２の照射、あるいはラマン散乱光Ｌ３の検出を実施する場合、プラズモン活性面各光に対して透光性を有する必要がある。ここでいう透光性は、励起光Ｌ１であればプラズモン励起が可能な強度、測定光Ｌ２であれば高感度検出が可能なラマン散乱光の励起が可能な強度、そしてラマン散乱光Ｌ３であれば、光検出部４１において高感度な検出が可能な強度を、基体１０から出射後にそれぞれ有するような透光性とする。 When the excitation light L1 or the measurement light L2 is irradiated from the back surface side of the sample contact surface 10s or the Raman scattered light L3 is detected as in the present embodiment, the plasmon active surface has translucency for each light. There is a need. The translucency here means that the excitation light L1 is capable of plasmon excitation, the measurement light L2 is capable of exciting Raman scattered light that can be detected with high sensitivity, and the Raman scattered light L3. For example, the light detection unit 41 has such a translucency that the light detection unit 41 has an intensity capable of high-sensitivity detection after being emitted from the substrate 10.
　このように、プラズモン活性基体１０に透明基板１１を用いることにより、金属膜１３の表面側、あるいは透明基板１１の裏面側のいずれからでも光を照射することができ、また、この光の照射により試料から生じた光についても、金属膜１３の表面側あるいは透明基板１１の裏面側のいずれからでも検出することができる。そのため、被検出物質の種類、サイズ等に応じて、励起光Ｌ１、測定光Ｌ２の照射、ラマン散乱光Ｌ３の検出を金属膜１３の表面側あるいは透明基板１１の裏面側のいずれからでも行うことができるので、測定における自由度が高く、より高いＳ／Ｎで検出することが可能となる。 Thus, by using the transparent substrate 11 for the plasmon active substrate 10, light can be irradiated from either the front surface side of the metal film 13 or the back surface side of the transparent substrate 11, and by this light irradiation, Light generated from the sample can also be detected from either the front surface side of the metal film 13 or the back surface side of the transparent substrate 11. Therefore, the excitation light L1, the measurement light L2, and the Raman scattered light L3 are detected from either the front surface side of the metal film 13 or the back surface side of the transparent substrate 11 depending on the type, size, etc. of the substance to be detected. Therefore, the degree of freedom in measurement is high, and detection can be performed with a higher S / N.
　透明基板１１としては特に制限されず、ガラスや透明樹脂等から選択することができる。 The transparent substrate 11 is not particularly limited, and can be selected from glass, transparent resin, and the like.
　微細凹凸構造１２は、この微細凹凸構造１２表面に金属膜１３が形成されて構成される金属微細構造の表面においてプラズモンを生じうる構造であればよいが、凹凸の凸部の平均的な大きさおよび平均ピッチが励起光の波長より短いものとなる程度の微細な凹凸構造であることが好ましい。特には、微細凹凸構造１２は、凸部頂点から隣接する凹部の底部までの平均深さが２００ｎｍ以下、凹部を隔てた最隣接凸部の頂点同士の平均ピッチが２００ｎｍ以下であることが望ましい。凹凸の平均ピッチは、走査型電子顕微鏡（ＳＥＭ）により撮影された表面画像を、画像処理により２値化して統計的処理によって求めるものとする。 The fine concavo-convex structure 12 may be any structure that can generate plasmons on the surface of the metal fine structure formed by forming the metal film 13 on the surface of the fine concavo-convex structure 12. And it is preferable that it is a fine concavo-convex structure with an average pitch that is shorter than the wavelength of the excitation light. In particular, the fine concavo-convex structure 12 desirably has an average depth of 200 nm or less from the top of the convex portion to the bottom of the adjacent concave portion, and an average pitch between the vertices of the most adjacent convex portions separating the concave portions. The average pitch of the unevenness is obtained by binarizing a surface image taken by a scanning electron microscope (SEM) by image processing and performing statistical processing.
　かかる微細凹凸構造１２としては、上記透光性を有していればその材質及びその製造方法は特に制限されないが、ベーマイト層や陽極酸化アルミナ層が好ましい。また、微細凹凸構造１２は透明基板１１と異なる材料により構成されたもののみならず、透明基板本体の表面を加工することにより基板本体と同一の材料により構成されていてもよい。例えば、ガラス基板の表面をリソグラフィーとドライエッチング処理することにより、表面に微細凹凸構造を形成して用いてもよい。 The material of the fine concavo-convex structure 12 is not particularly limited as long as it has the above-described translucency, but a boehmite layer or an anodized alumina layer is preferable. Further, the fine concavo-convex structure 12 is not only made of a material different from that of the transparent substrate 11, but may be made of the same material as the substrate body by processing the surface of the transparent substrate body. For example, the surface of the glass substrate may be used by forming a fine uneven structure on the surface by performing lithography and dry etching treatment.
　特に、製造が容易で低コストであることから、微細凹凸構造１２はベーマイト層がより好ましい。ベーマイトとは、アルミニウムの水和物である。透明基板１１上へのベーマイト層１２の形成は、例えば、純水洗浄した透明基板１１を用意し、その表面にスパッタ法によりアルミニウムを数十ｎｍ程度成膜した後、純水を沸騰させた中に、アルミニウム付き透明基板１１を浸水させ、数分（５分程度）後に取り出す（煮沸処理（ベーマイト処理））ことにより実施することができる。 Especially, since the manufacturing is easy and the cost is low, the fine uneven structure 12 is more preferably a boehmite layer. Boehmite is a hydrate of aluminum. The boehmite layer 12 is formed on the transparent substrate 11 by, for example, preparing a transparent substrate 11 washed with pure water, depositing aluminum on the surface thereof by about several tens of nanometers by sputtering, and then boiling pure water. The transparent substrate 11 with aluminum can be immersed in water and taken out after a few minutes (about 5 minutes) (boiling treatment (boehmite treatment)).
　金属膜１３は、励起光の照射を受けて局在プラズモンを生じうる金属からなるものであればよいが、例えば、Ａｕ、Ａｇ、Ｃｕ、Ａｌ、Ｐｔ、およびこれらを主成分とする合金からなる群より選択される少なくとも１種の金属からなるものである。特には、ＡｕあるいはＡｇが好ましい。ここで主成分とは、含量９０質量％以上の成分を意味する。 The metal film 13 may be made of a metal that can generate localized plasmons when irradiated with excitation light. For example, it is made of Au, Ag, Cu, Al, Pt, or an alloy containing these as a main component. It consists of at least one metal selected from the group. In particular, Au or Ag is preferable. Here, the main component means a component having a content of 90% by mass or more.
　金属膜１３の膜厚は、微細凹凸構造１２の表面に形成されたときに、金属微細凹凸構造として励起光の照射を受けて局在プラズモンを生じうる凹凸形状を維持することができる程度の厚みであれば特に制限はないが、１０～１００ｎｍであることが好ましい。 The thickness of the metal film 13 is such that when formed on the surface of the fine concavo-convex structure 12, the concavo-convex shape capable of generating localized plasmons upon irradiation with excitation light as the metal fine concavo-convex structure can be maintained. If there is no particular limitation, it is preferably 10 to 100 nm.
　金属膜１３は、微細凹凸構造１２上に蒸着等により成膜すればよい。 The metal film 13 may be formed on the fine concavo-convex structure 12 by vapor deposition or the like.
　プラズモン活性基体１０は、図３に示されるようなマイクロウェルプレートの態様としてもよい。かかる態様とすることにより、複数のウェルに注入された試料Ｓに対して同時に吸着工程を実施し、短時間に高感度なセンシングを実施することができる。 The plasmon active substrate 10 may be in the form of a microwell plate as shown in FIG. By setting it as this aspect, an adsorption process can be simultaneously implemented with respect to the sample S inject | poured into the several well, and highly sensitive sensing can be implemented in a short time.
　一般的なマイクロウェルプレートは、例えば上面視面積１００ｍｍ×５０ｍｍ程度の大きさを有し、ウェル数９６個のもの等が挙げられることから、本発明を適用することにより測定効率を劇的に向上させることができる。 A typical microwell plate has, for example, a top view area of about 100 mm × 50 mm, and has 96 wells. The application of the present invention dramatically improves the measurement efficiency. Can be made.
　マイクロウェルプレート状プラズモン活性基体１０は、例えば、透明基板１１とその上に配された微細凹凸構造１２及び金属膜１３を備えた構成となっている。マイクロウェルプレート状プラズモン活性基体１０では、金属微細凹凸構造は試料接触面となる各ウェルの表面にのみ形成されていればよい。 The microwell plate-shaped plasmon active substrate 10 has, for example, a configuration including a transparent substrate 11, a fine uneven structure 12 and a metal film 13 disposed thereon. In the microwell plate-shaped plasmon active substrate 10, the metal fine concavo-convex structure may be formed only on the surface of each well serving as the sample contact surface.
　励起光照射光学系２０は、励起光源２１と、励起光源２１から出射された励起光Ｌ１をプラズモン活性基体１０方向に導くミラー２２と、励起光Ｌ１のビーム径を拡げて試料Ｓ全体に同時に励起光Ｌ１を照射可能にするビームエクスパンダー２３と、ビームエクスパンダー２３によりビーム径が拡大された励起光Ｌ１の、試料接触面１０ｓにおける強度を均一化する拡散板２４とから構成されている。 The excitation light irradiation optical system 20 simultaneously excites the entire sample S by expanding the beam diameter of the excitation light source 21, the mirror 22 for guiding the excitation light L1 emitted from the excitation light source 21 toward the plasmon active substrate 10, and the excitation light L1. The beam expander 23 enables the light L1 to be irradiated, and the diffusion plate 24 that uniformizes the intensity of the excitation light L1 whose beam diameter is expanded by the beam expander 23 at the sample contact surface 10s.
　ビームエクスパンダー２３は、所望のビーム径となるように拡大可能な構成であれば特に制限されないが、例えば曲率の異なる２種のレンズにより構成することができる。特に、上記マイクロウェルプレート状のプラズモン活性基体に適用する場合等の拡大率が大きい場合は、曲率の異なる２種類のシリンドリカルレンズを用いて、ビーム形状の一軸を拡大してビーム形状を楕円化することが好ましい。 The beam expander 23 is not particularly limited as long as the beam expander 23 can be expanded so as to have a desired beam diameter. For example, the beam expander 23 can be configured by two types of lenses having different curvatures. In particular, when the enlargement ratio is large, such as when applied to the plasmon active substrate in the form of a microwell plate, the beam shape is made elliptic by enlarging one axis of the beam shape using two types of cylindrical lenses having different curvatures. It is preferable.
　励起光源２１としては、プラズモン活性基体１０の試料接触面１０ｓにプラズモンを励起可能な波長を含む光を射出する光源であれば特に制限されない。プラズモンを励起するのに好適な波長は、プラズモン活性基体１０において、プラズモンを励起する金属の種類に応じて異なることから、金属の種類に応じて、好適な波長の光源を選択することが好ましい。被測定物質Ｒが生体物質である場合は、できるだけダメージを与えない方が好ましいことから、エネルギーの比較的低い７００ｎｍ～２μｍの可視～近赤外光であることが好ましい。 The excitation light source 21 is not particularly limited as long as it is a light source that emits light including a wavelength capable of exciting plasmons to the sample contact surface 10 s of the plasmon active substrate 10. Since a suitable wavelength for exciting plasmons varies depending on the type of metal that excites plasmons in the plasmon active substrate 10, it is preferable to select a light source with a suitable wavelength according to the type of metal. When the substance R to be measured is a biological substance, it is preferable that it is not damaged as much as possible. Therefore, it is preferably visible to near-infrared light having a relatively low energy of 700 nm to 2 μm.
　測定光照射光学系（ラマン散乱励起光照射光学系）３０は、励起光を含む測定光（ラマン散乱励起光）Ｌ２を射出する光源３１と、光Ｌ２をプラズモン活性基体１０の試料接触面１０ｓへ導くミラー３２～３４と、ミラー３４により反射された光Ｌ２をプラズモン活性基体１０の試料Ｓが載置された領域に集光するレンズ３５とを備えている。 The measurement light irradiation optical system (Raman scattering excitation light irradiation optical system) 30 emits measurement light (Raman scattering excitation light) L2 including excitation light, and the light L2 to the sample contact surface 10s of the plasmon active substrate 10. There are provided mirrors 32 to 34 for guiding, and a lens 35 for condensing the light L2 reflected by the mirror 34 in a region where the sample S of the plasmon active substrate 10 is placed.
　なお、ミラー３３（４３）は、測定光Ｌ２を反射し、且つ、測定光Ｌ２の照射により試料Ｓの被検出物質Ｒから生じ増強されたラマン散乱光Ｌ３を含むプラズモン活性基体１０側からの光を光検出部（分光器）４１側へ透過するダイクロイックミラーとなっている。 The mirror 33 (43) reflects the measurement light L2, and includes light from the plasmon active substrate 10 side including the enhanced Raman scattered light L3 generated from the detection target substance R of the sample S by irradiation of the measurement light L2. Is a dichroic mirror that transmits the light to the light detection unit (spectrometer) 41 side.
　ラマン分光法において、測定光Ｌ２の光源３１は、特定波長の光を射出するレーザ等の単波長光源であり、分布帰還型（ＤＦＢ）半導体レーザ等を好ましく用いることができる。 In Raman spectroscopy, the light source 31 of the measurement light L2 is a single wavelength light source such as a laser that emits light of a specific wavelength, and a distributed feedback (DFB) semiconductor laser or the like can be preferably used.
　測定光Ｌ２の照射により、プラズモン活性基体１０の試料接触面１０ｓにはプラズモンが励起され、プラズモン増強場を生じると共に、被測定物質Ｒが含まれる試料Ｓにおいてラマン散乱光Ｌ３が含まれる光（散乱光等）を生じる。ラマン散乱光Ｌ３を含む光は、生じたプラズモン増強場により増強されたものとなる。 By irradiation with the measurement light L2, plasmon is excited on the sample contact surface 10s of the plasmon active substrate 10 to generate a plasmon enhancement field, and light (scattering) containing Raman scattered light L3 in the sample S containing the substance R to be measured Light). The light including the Raman scattered light L3 is enhanced by the generated plasmon enhancement field.
　ラマン分光装置１は、複数の測定点に対して測定を実施するものである。ラマン分光装置１において、測定点（測定光Ｌ２の試料接触面１０ｓにおける集光位置）の移動を、手動により実施する態様としてもよいが、図示ｘ方向に相対的に移動させることができる走査手段を備えていることが好ましい。走査手段としては、例えば、ガルバノミラー等の測定光Ｌ２を走査させるものであってもよいし、プラズモン活性基体１０をｘ方向に可動とするものであってもよい。 The Raman spectroscopic device 1 performs measurement on a plurality of measurement points. In the Raman spectroscopic device 1, the movement of the measurement point (the condensing position of the measurement light L <b> 2 on the sample contact surface 10 s) may be manually performed. However, the scanning unit can be relatively moved in the x direction shown in the figure. It is preferable to provide. As the scanning means, for example, the measuring light L2 such as a galvanometer mirror may be scanned, or the plasmon active substrate 10 may be movable in the x direction.
　物理特性検出系４０は、被測定物質Ｒ由来のラマン散乱光を含む、試料Ｓから発せられたラマン散乱光Ｌ３を含む光をダイクロイックミラー４３へ導くレンズ４５及びミラー４４と、ダイクロイックミラー４３から透過してきたラマン散乱光Ｌ３を含む光のうち、測定光Ｌ２の波長の光を吸収し、それ以外の光を透過するノッチフィルタ４６と、ノイズ光を除去するためのピンホールを備えたピンホール板４８と、ノッチフィルタ４６を透過したラマン散乱光Ｌ３を含む光をピンホール板４８のピンホールに集光するためのレンズ４７と、ピンホールを通ってきたラマン散乱光Ｌ３を平行光化して光検出部（分光器）４１へ導くレンズ４９と、ラマン散乱光Ｌ３を分光してラマンスペクトルを得る光検出部（分光器）４１とを備えている。 The physical property detection system 40 transmits the light including the Raman scattered light L3 emitted from the sample S including the Raman scattered light derived from the measurement target R to the dichroic mirror 43 and the dichroic mirror 43. Of the light including the Raman scattered light L3, the pinhole plate having a notch filter 46 that absorbs light of the wavelength of the measurement light L2 and transmits other light, and a pinhole for removing noise light 48, a lens 47 for condensing the light including the Raman scattered light L3 transmitted through the notch filter 46 into the pinhole of the pinhole plate 48, and the Raman scattered light L3 that has passed through the pinhole are converted into parallel light. A lens 49 that leads to a detection unit (spectrometer) 41 and a light detection unit (spectrometer) 41 that obtains a Raman spectrum by splitting the Raman scattered light L3 are provided.
　図１において、わかりやすくするためにラマン散乱光Ｌ３を含む光をＬ３と表示し、ラマン散乱光Ｌ３を含む光と測定光Ｌ２の経路を同じで表示したが、実際、ラマン散乱光Ｌ３を含む光には、測定光Ｌ２の反射光、試料Ｓにおけるラマン散乱光、レイリー散乱光、ミー散乱光等の様々な光が含まれているため、ラマン散乱光Ｌ３を含む光は、レンズ４５に図示されている角度よりも幅広い角度で入射される。従って、ダイクロイックミラー４３にて、全ての測定光Ｌ２と同じ波長の光を反射することはできない。 In FIG. 1, for the sake of clarity, the light including the Raman scattered light L3 is displayed as L3, and the path including the light including the Raman scattered light L3 and the measurement light L2 is displayed in the same way, but actually includes the Raman scattered light L3. Since the light includes various lights such as the reflected light of the measuring light L2, the Raman scattered light, the Rayleigh scattered light, and the Mie scattered light on the sample S, the light including the Raman scattered light L3 is illustrated in the lens 45. It is incident at a wider angle than the angle that is being used. Therefore, the dichroic mirror 43 cannot reflect light having the same wavelength as all the measurement light L2.
　ノッチフィルタ４６は、ダイクロイックミラー４３を透過してきた測定光Ｌ２と同じ波長の光（反射光、レイリー散乱光、ミー散乱光等）をカットするものである。励起光とは波長が異なる光（ラマン散乱光）はノッチフィルタ４６を透過し、レンズ４７で集光され、ピンホール４８を通り、再度レンズ４９により平行光化され、分光器４１へ入射され、ラマンスペクトル（物理特性）の検出がなされる。 The notch filter 46 cuts light (reflected light, Rayleigh scattered light, Mie scattered light, etc.) having the same wavelength as the measurement light L2 that has passed through the dichroic mirror 43. Light having a wavelength different from that of the excitation light (Raman scattered light) passes through the notch filter 46, is collected by the lens 47, passes through the pinhole 48, is collimated again by the lens 49, and enters the spectroscope 41. A Raman spectrum (physical characteristic) is detected.
　以上のように、ラマン分光装置（センシング装置）１は構成されている。 As described above, the Raman spectroscopic device (sensing device) 1 is configured.
　ラマン分光装置（センシング装置）１は、プラズモン活性基体１０上の試料Ｓの複数の測定点においてプラズモン増強場におより増強された信号光Ｌ３を検出してセンシングを行うものであり、プラズモンを励起する励起光照射光学系２０と、試料Ｓに励起光を含む測定光Ｌ２を照射する測定光照射光学系３０とをそれぞれ備え、励起光照射光学系２０を、試料接触面１０ｓの少なくとも２つの測定点を含む領域を同時に照射可能なものとしている。かかる構成によれば、複数の測定点において同時にプラズモン増強場を生じさせて被測定物質Ｒを試料接触面１０ｓに吸着させることができる上、試料接触面１０ｓに被測定物質Ｒを吸着させた後に、該照射により被測定物質Ｒから発せられ、且つ、該照射により試料接触面１０ｓに生じたプラズモン増強場により増強されたラマン散乱光（信号光）Ｌ３のラマンスペクトル（物理特性）を検出することができる。従って、本発明によれば、同一試料内の複数の測定箇所に対して、短時間且つ高感度に被測定物質のセンシングを行うことができる。また、同時に照射可能な面積を大きくすることにより、大面積の試料におけるセンシングを効率的かつ高感度に実施することができる。 The Raman spectroscopic device (sensing device) 1 performs sensing by detecting the signal light L3 enhanced in the plasmon enhancement field at a plurality of measurement points of the sample S on the plasmon active substrate 10, and excites the plasmon. Each of the excitation light irradiation optical system 20 and the measurement light irradiation optical system 30 that irradiates the sample S with the measurement light L2 including excitation light. The excitation light irradiation optical system 20 includes at least two measurements on the sample contact surface 10s. It is assumed that an area including a point can be irradiated simultaneously. According to such a configuration, the plasmon enhancement field can be generated simultaneously at a plurality of measurement points to adsorb the substance R to be measured on the sample contact surface 10s, and after the substance R to be measured is adsorbed to the sample contact surface 10s. Detecting a Raman spectrum (physical characteristic) of Raman scattered light (signal light) L3 emitted from the substance R to be measured by the irradiation and enhanced by the plasmon enhancement field generated on the sample contact surface 10s by the irradiation. Can do. Therefore, according to the present invention, the substance to be measured can be sensed in a short time and with high sensitivity at a plurality of measurement locations in the same sample. Further, by increasing the area that can be irradiated at the same time, sensing in a large-area sample can be performed efficiently and with high sensitivity.
「設計変更」
　上記実施形態では、信号光がラマン散乱光であるラマン分光装置について説明したが、信号光は、ラマン散乱光に限定されず、蛍光、コヒーレントアンチストークス光、レイリー散乱光、ミー散乱光、第２次高調波、第３次高調波光等とし、それに応じた物理特性を検出する装置に適用することができる。例えば、蛍光の場合はその蛍光に合わせたフィルターを用い、検出器として冷却ＣＣＤや光電子増倍管（ＰＭＴ）等を用いることで、シンプルな光学系でセンシングを行うことができる。 "Design changes"
In the above embodiment, the Raman spectroscopic device in which the signal light is Raman scattered light has been described. However, the signal light is not limited to Raman scattered light, but fluorescence, coherent anti-Stokes light, Rayleigh scattered light, Mie scattered light, second The present invention can be applied to a device that detects second-order harmonics, third-order harmonic light, and the like, and detects physical characteristics corresponding to them. For example, in the case of fluorescence, sensing can be performed with a simple optical system by using a filter matched to the fluorescence and using a cooled CCD, a photomultiplier tube (PMT), or the like as a detector.
　また、上記実施形態では、試料接触面にプラズモンを励起する励起光が、試料が裁置されている試料接触面全体を同時に照射可能な態様について説明したが、少なくとも２つの複数の測定点を同時照射する態様とするだけでも本発明の効果を得ることができる。その際は、全ての測定点において吸着工程を実施した後に測定工程を実施してもよいし、１回の吸着工程の後に測定工程を実施し、測定工程終了後に励起光照射領域をずらして該領域の吸着工程及び測定工程を実施してもよい。 In the above embodiment, the description has been given of the mode in which the excitation light for exciting the plasmon on the sample contact surface can simultaneously irradiate the entire sample contact surface on which the sample is placed. However, at least two measurement points are simultaneously applied. The effect of the present invention can be obtained simply by irradiating. In that case, the measurement process may be performed after the adsorption process is performed at all measurement points, or the measurement process is performed after one adsorption process, and the excitation light irradiation region is shifted after the measurement process is completed. A region adsorption step and a measurement step may be performed.
　また、上記実施形態では、プラズモン活性基体の試料接触面とは逆側（プラズモン活性基体の裏面）から励起光及び測定光を入射し、ラマン散乱光（信号光）を検出する態様について説明したが、試料接触面側から励起光、測定光の入射及び／又はラマン散乱光の検出を行う態様としてもよい。
　 Moreover, although the said embodiment demonstrated the aspect which injects excitation light and measurement light from the opposite side (back surface of a plasmon active base | substrate) with respect to the sample contact surface of a plasmon active base | substrate, and detects a Raman scattered light (signal light). Further, it is possible to adopt an aspect in which excitation light, measurement light incidence and / or Raman scattering light is detected from the sample contact surface side.

Claims (10)

  1. 　被測定物質を含む試料が接触される試料接触面を有し、該試料接触面に励起光が照射されることにより該試料接触面にプラズモン増強場を生じるプラズモン活性基体と、
    　前記励起光を照射する励起光照射光学系と、
    　前記試料に前記励起光を含む測定光を照射する測定光照射光学系と、
    　前記測定光の照射により前記試料接触面上の試料から発せられ、且つ、該照射により前記試料接触面に生じたプラズモン増強場により増強された信号光の物理特性を検出する物理特性検出系とを備えてなり、
    　前記測定光を前記試料の複数の測定点に照射して、該複数の測定点における前記物理特性を検出するものであり、
    　前記励起光照射光学系は、前記試料接触面の少なくとも２つの前記測定点を含む領域を同時に照射可能な前記励起光を照射するものであることを特徴とするセンシング装置。     A plasmon active substrate having a sample contact surface with which a sample containing a substance to be measured is brought into contact, and generating a plasmon enhancement field on the sample contact surface by irradiating the sample contact surface with excitation light;
    An excitation light irradiation optical system for irradiating the excitation light;
    A measurement light irradiation optical system for irradiating the sample with measurement light including the excitation light;
    A physical property detection system for detecting the physical property of the signal light emitted from the sample on the sample contact surface by the irradiation of the measurement light and enhanced by the plasmon enhancement field generated on the sample contact surface by the irradiation; Prepared
    Irradiating a plurality of measurement points of the sample with the measurement light to detect the physical characteristics at the plurality of measurement points;
    The excitation light irradiation optical system irradiates the excitation light capable of simultaneously irradiating a region including at least two measurement points on the sample contact surface.
  2. 　前記励起光照射光学系は、前記試料接触面の全ての前記測定点を含む領域を同時に照射可能な前記励起光を照射するものであることを特徴とする請求項１に記載のセンシング装置。 The sensing device according to claim 1, wherein the excitation light irradiation optical system irradiates the excitation light that can simultaneously irradiate a region including all the measurement points on the sample contact surface.
  3. 　前記試料接触面における前記励起光の強度が略均一であることを特徴とする請求項１又は２に記載のセンシング装置。 The sensing device according to claim 1 or 2, wherein the intensity of the excitation light on the sample contact surface is substantially uniform.
  4. 　前記励起光及び前記測定光が直線偏光であり、前記励起光の偏光方向と前記測定光の偏光方向とが略同一であることを特徴とする請求項１～３のいずれかに記載のセンシング装置。 4. The sensing device according to claim 1, wherein the excitation light and the measurement light are linearly polarized light, and a polarization direction of the excitation light and a polarization direction of the measurement light are substantially the same. .
  5. 　前記励起光及び前記測定光が円偏光であることを特徴とする請求項１～３のいずれかに記載のセンシング装置。 The sensing device according to any one of claims 1 to 3, wherein the excitation light and the measurement light are circularly polarized light.
  6. 　前記試料から発せられる信号光が、蛍光、ラマン散乱光、コヒーレントアンチストークス散乱光、第二次高調波光、第三次高調波光のいずれかであることを特徴とする請求項１～５のいずれかに記載のセンシング装置。 6. The signal light emitted from the sample is any one of fluorescence, Raman scattered light, coherent anti-Stokes scattered light, second harmonic light, and third harmonic light. The sensing device described in 1.
  7. 　前記励起光の波長が、７００ｎｍ～１６００ｎｍであることを特徴とする請求項１～６のいずれかに記載のセンシング装置。 The sensing device according to any one of claims 1 to 6, wherein the excitation light has a wavelength of 700 nm to 1600 nm.
  8. 　励起光が照射されることによりプラズモン増強場を生じる試料接触面に被測定物質を含む試料を接触させる試料接触工程と、
    　該試料に前記励起光を照射して、該励起光照射部分の前記試料接触面にプラズモン増強場を生じさせて、該プラズモン増強場により前記被測定物質の少なくとも一部を前記試料接触面に吸着させる吸着工程と、
    　該吸着工程後に、前記試料接触面上の該試料に前記励起光を含む測定光を照射して、該照射により前記試料接触面にプラズモン増強場を生じさせるとともに、前記試料から発せられる信号光の物理特性を検出する測定工程とを有するセンシング方法であって、
    　前記測定工程は、前記試料の複数の測定点に対して該測定点毎に実施されるものであり、
    　前記吸着工程において、少なくとも２つの前記測定点を含む領域に前記励起光を同時に照射することを特徴とするセンシング方法。     A sample contact step in which a sample containing a substance to be measured is brought into contact with a sample contact surface that generates a plasmon enhancement field when irradiated with excitation light;
    Irradiating the sample with the excitation light to generate a plasmon enhancement field on the sample contact surface of the excitation light irradiation portion, and at least a part of the substance to be measured is adsorbed to the sample contact surface by the plasmon enhancement field An adsorption process,
    After the adsorption step, the sample on the sample contact surface is irradiated with measurement light including the excitation light to generate a plasmon enhancement field on the sample contact surface by the irradiation, and signal light emitted from the sample A sensing method having a measurement process for detecting physical properties,
    The measurement step is performed for each of the plurality of measurement points of the sample,
    In the adsorption step, a region including at least two measurement points is irradiated with the excitation light simultaneously.
  9. 　前記吸着工程において、全ての前記測定点を含む領域に前記励起光を同時に照射することを特徴とする請求項８に記載のセンシング方法。 The sensing method according to claim 8, wherein, in the adsorption step, the excitation light is simultaneously irradiated to a region including all the measurement points.
  10. 　　前記吸着工程において、前記プラズモン増強場により前記被測定物質の少なくとも一部を前記試料接触面上に配向させて吸着させることを特徴とする請求項８又は９に記載のセンシング方法。 The sensing method according to claim 8 or 9, wherein in the adsorption step, at least a part of the substance to be measured is oriented and adsorbed on the sample contact surface by the plasmon enhancement field.
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