JP2009042112A - Sensing device and sensing method using the same - Google Patents

Abstract

In a sensing device, a substance to be measured in a sample cell can be effectively captured on the surface of a plasmon active substrate or in the vicinity thereof, and highly sensitive sensing can be performed.
In a sensing device, a sample cell 10 filled with or flowing down with a fluid sample S containing a substance to be measured, and a sample contact surface 20s arranged so as to contact the sample S in the sample cell 10 are provided. Is irradiated with excitation light L0 to generate a plasmon active substrate 20 that generates a plasmon enhancement field on the sample contact surface 20s, an excitation light irradiation optical system 30 that irradiates the excitation light L0, and a sample contact surface 20s of the plasmon active substrate 20 The physical property detection system 45 for detecting the physical property of the sample S and the plasmon active substrate 20 collect the capture light L3 on the sample contact surface 20s, and the substance to be measured in the sample S on the sample contact surface 20s by the light capture effect. A light-capturing light irradiation optical system 50 is provided.
[Selection] Figure 1

Description

  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.

  In general, when performing measurement using the effect of a plasmon enhancement field caused by surface plasmons or localized plasmons, it is necessary to place a substance to be measured very close to the surface of a device (plasmon active substrate) that generates a plasmon enhancement field. This is because the plasmon enhancement field is generated only in the vicinity of the surface of the plasmon active substrate, and the effect of the enhancement field is reduced as the measured substance is separated from the surface of the plasmon active substrate. Therefore, when measuring the substance to be measured in the sample solution, the measurement accuracy can be improved by increasing the concentration of the substance to be measured on the surface of the plasmon active substrate.

  As a method for adsorbing the substance to be measured on the surface of the plasmon active substrate, for example, in the case of a surface plasmon resonance (SPR) measuring device, a ligand that specifically binds to the substance to be measured is immobilized on a metal film that is a plasmon active substrate. There is a method of binding a substance to be measured to a ligand, whereby the substance to be measured in the sample is fixed on the surface of the metal film to perform sensing.

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.
JP 2005-195397

  However, in the method of adsorbing a substance to be measured on the surface of a plasmon active substrate using a ligand, a sufficient amount of binding cannot be secured unless the reaction between the ligand and the substance to be measured is sufficiently waited. It is difficult to measure quickly. In addition, substances that bind to the ligand are limited, and the types of substances to be measured are limited.

  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. Furthermore, in the apparatus described in Patent Document 1, in practice, the concentration of the solution passing near the metal surface is merely increased, and it cannot be said that the adsorption effect at the actual measurement location is sufficient. In addition, when an applied voltage is applied, it is necessary to charge the substance to be measured, and there is a possibility that the reactivity may be changed by charging the charge. Furthermore, there is a possibility that the molecular characteristics may be changed by applying an applied voltage.

  The present invention has been made in view of the above circumstances, and a sensing device capable of effectively increasing the concentration of a substance to be measured on or near the surface of a plasmon active substrate, and a highly accurate measurement thereof, and It is an object to provide a sensing method using the.

The sensing device of the present invention includes a sample cell in which a fluid sample containing a substance to be measured is filled or flowed down;
A plasmon active substrate that is disposed so as to contact a sample in the sample cell and 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 sensing device comprising a physical property detection system for detecting a physical property of a sample on the sample contact surface of the plasmon active substrate,
A light-capturing light irradiation optical system is provided that collects trapped light on the sample contact surface of the plasmon active substrate and traps a substance to be measured in the sample on the sample contact surface due to a light trapping effect. Is.

  Here, the sample may be filled or flowed down in the entire sample cell, or may be filled or flowed down in a part of the sample cell.

  Here, “capturing the substance to be measured” means “to keep the substance to be measured in a region where the electric field is strong due to the electric field gradient”.

  The sensing device preferably includes a scanning unit that scans the collection position of the captured light within the sample cell. The scanning unit only needs to be able to move the collection position of the captured light relative to the sample cell. For example, the scanning unit may include a galvano mirror to scan the captured light itself. The sample cell may be provided with an XYZ stage that can move in three dimensions as a stage for arranging the sample cell, and the sample cell may be moved. Further, the means for scanning the trapped light itself and the one-dimensional and two-dimensional that move the sample cell Alternatively, a combination with a three-dimensional stage may be used.

  The physical property detection system includes a measurement light irradiation optical unit that irradiates measurement light onto the sample surface of the plasmon active substrate, reflected light of the measurement light on the sample contact surface, and / or scattered light generated on the sample contact surface. It is desirable to include a detection unit for detecting. Examples of the sensing device provided with such a physical property detection system include a surface plasmon resonance measurement device, a localized plasmon resonance measurement device, and an enhanced Raman spectroscopy device.

The plasmon active substrate is preferably made of a metal body having a concavo-convex structure smaller than the wavelength of the excitation light.
In this specification, “an uneven structure smaller than the wavelength of the measurement light” means that the average size and the average pitch of the protrusions and recesses forming the uneven structure are smaller than the wavelength of the measurement light. There may or may not be metal in the recess.

The main component of the metal body is preferably at least one metal selected from the group consisting of Au, Ag, Cu, Al, Pt, Ni, Ti, and alloys thereof.
In the present specification, the “main component” is defined as a component having a content of 90% by mass or more.

  The sample cell is preferably a capillary cell having one end in contact with the sample contact surface of the plasmon active substrate.

  It is preferable that the plasmon active substrate is provided with a surface modification that ion-bonds to the substance to be measured and / or a surface modification that covalently bonds to the substance to be measured on the sample contact surface.

  Furthermore, it is desirable that the substance to be measured is subjected to surface modification for increasing the size of the substance to be measured. The particle size that can be captured by light is several tens of nanometers, and particles having a size smaller than this are difficult to capture light. Therefore, it is desirable to perform surface modification when the size of the substance to be measured is small and it is difficult to capture light. Specifically, when the substance to be measured is particles of several tens of nm or less, polymethyl methacrylate (PMMA) latex fine particles, polystyrene spheres, glass beads, etc. may be modified to the substance to be measured.

In the sensing method of the present invention, a fluid sample containing a substance to be measured is brought into contact with a plasmon active substrate that is irradiated with excitation light on the sample contact surface to generate a plasmon enhancement field on the sample contact surface.
A sensing method for detecting physical properties of a substance to be measured on the sample contact surface of the plasmon active substrate in a state where the plasmon active substrate is irradiated with the excitation light to generate the plasmon enhancement field,
In a state where the sample is in contact with the plasmon active substrate, the captured substance is condensed on the sample contact surface of the plasmon active substrate to move the substance to be measured in the sample onto the sample contact surface. ,
The physical property of the substance to be measured is detected in a state where the substance to be measured is captured on the sample contact surface.

  In this sensing method, before condensing the capture light on the sample contact surface, the capture light is scanned in a direction gradually approaching the sample contact surface from a position away from the sample contact surface in the sample cell. Finally, the light may be condensed on the sample contact surface of the active substrate.

  The sensing device of the present invention includes a light-capturing light irradiation optical system that collects captured light on a sample contact surface of a plasmon active substrate and captures a substance to be measured in the sample on the sample contact surface due to a light capturing effect. Thereby, the substance to be measured in the sample can be light-captured at the collection position of the trapped light and fixed on the sample contact surface, so that the concentration of the substance to be measured on the sample contact surface can be increased. In the sensing device of the present invention, since a sufficient amount of a substance to be measured exists on the surface of the plasmon active substrate or in the vicinity thereof, the analysis can be performed reliably, so that highly accurate analysis can be stably performed. Can do. Further, unlike the case of electrophoresis as described in Patent Document 1, there is no destructive and non-contact with the substance to be measured without charging the substance to be measured or applying an applied voltage to the sample. Thus, since the concentration of the substance to be measured on the sample contact surface can be increased, analysis can be performed without changing the characteristics of the sample.

  Furthermore, when a scanning unit that scans the collection position of the trapped light within the sample cell is provided, more substance to be measured in the sample can be captured, and the substance to be measured is more efficiently captured on the sample contact surface. It can be captured and a highly accurate analysis can be performed.

  In the case where the plasmon active substrate has a surface modification that binds to the substance to be measured on the sample contact surface, the binding between the plasmon active substrate and the substance to be measured can be promoted by the light capturing effect, The binding time with the substance to be measured can be shortened, and highly sensitive analysis can be performed stably.

  According to the sensing method of the present invention, in a state where the sample is in contact with the plasmon active substrate, the captured light is condensed on the sample contact surface of the plasmon active substrate, whereby the substance to be measured in the sample is placed on the sample contact surface. Since the physical property of the substance to be measured is detected while the substance to be measured is captured on the sample contact surface, a sufficient amount of the substance to be measured exists on or near the surface of the sample contact. Since the analysis can be performed reliably, highly sensitive analysis can be performed stably.

  Light trapping (light trap) that collects laser light from a light source and irradiates it in the vicinity of the fine particles, using the light radiation pressure (light pressure) generated in the fine particles to capture and move the fine particles freely Or optical tweezers) technology is known. The principle of this light capture is as follows.

<Principle of light capture>
Although light has no mass, if one considers light as a particle (photon), one photon has a momentum given by h / λ (h: Planck's constant, L: wavelength of light). When light enters a medium with a different refractive index and is reflected or refracted at the interface between the media, the direction of travel of the light changes and the momentum also changes. Therefore, force acts on the interface as a reaction from the law of conservation of momentum. . When light is reflected or refracted on the surface of the minute substance, the momentum of the photon changes by Δp, so that the interface is pushed by Δp as a reaction. This can be considered that light has given force to the minute object, and this is called light pressure.

  The light pressure acting on the micrometer-sized fine particles is explained as follows. When laser light is focused on transparent fine particles with a higher refractive index than the surrounding medium under a microscope, the laser light incident on the fine particles is refracted on the surface of the fine particles, and the momentum of the photons changes and the light pressure acts on the fine particles. . The photons that have passed through the fine particles are refracted again when they exit the medium, and light pressure acts on the fine particles again. When the light pressure is calculated and integrated for all the light beams, a force F toward the focal point f of the laser light is obtained.

  Note that the light pressure generated when the laser light is focused on the fine particles having a low refractive index or the metal fine particles is generated in a direction away from the focal position of the laser light, so that light cannot be captured. When the refractive index of the fine particles is lower than that of the surrounding medium, the direction of change in the momentum of the photons is opposite to that of the fine particles having a high refractive index, and the total light pressure works in a direction away from the focal point f of the laser light. Also, in the case of fine particles that reflect laser light, such as metal fine particles, the force caused by the collision of photons acts to push the fine particles, and the force is much larger than the light pressure due to refraction, so the metal fine particles are blown off ( Masuhara Micro Conversion Project: See Microchemistry, Doujin (1993)).

On the other hand, the light pressure acting on nm-sized fine particles is explained as follows. When considering the light pressure acting on nanometer-sized substances such as molecules and colloidal particles, that is, nanoparticles that are sufficiently smaller than the wavelength of the laser beam used (about 1/10 of the wavelength), the Rayleigh approximation holds, and the nanoparticles are Rayleigh's theory of light scattering can be approximated as an induced dipole, and the light pressure acting on the dipole is expressed by the following equation (1) (YR Shen: Principle Of Nonlinear Optics (Pure & Applied Optics Series: 1- 349), Wiley-Interscience (1984)).

Here, E: electric field strength, B: magnetic flux density, a: polarizability, e _b : dielectric constant, r: nanoparticle radius, n _a : nanoparticle refractive index, n _b : medium refractive index. The first term on the right-hand side of equation (1) represents the gradient force, which is the electrostatic stress acting when the induced dipole is placed in an electric field with a non-uniform spatial distribution, and the refractive index of the nanoparticles. Is higher than the refractive index of the surrounding medium (n _a > n _b ), the polarizability α becomes a positive value according to equation (2), and the gradient force acts as a force to attract the nanoparticles to a place where the electric field strength is high It will be. When the refractive index of the nanoparticles is lower than the refractive index of the surrounding medium (n _a <n _b ), the polarizability α becomes a negative value according to equation (2), and the light pressure given by equation (1) is the electric field Since it works in the direction of a weak place, it cannot capture the nanoparticles. On the other hand, the second term is a scattering force, which is a force caused by light scattering by the nanoparticles, and is a force generated by temporal change of the traveling direction of light energy, that is, the pointing vector. Therefore, when transparent nanoparticles are irradiated with light having no electric field intensity distribution, only the scattering force works, and the light pressure works in the direction of pushing the nanoparticles in the light traveling direction.

When the laser beam is condensed with an objective lens under a microscope, the electric field strength at the condensing position is very large compared to the surroundings, and the gradient force is sufficiently large compared to the scattering force. Therefore, since the force acting on the nanoparticles only needs to consider the gradient force, equation (1) is expressed as the following equation (3). When the laser beam is focused by the objective lens and the refractive index of the nanoparticles is higher than the refractive index of the surrounding medium (n _a > n _b ), the gradient force is always the light intensity for the nanoparticles. Since it works toward a high place, that is, a collecting position, the nanoparticles are collected at the collecting position.

As described in item 2 above, metal fine particles contribute greatly to reflection and are not captured by light. However, when the metal fine particles are about the skin depth (particle diameter of several tens of nanometers), the gradient force becomes larger than the scattering force, and light is stably emitted. (K. Svoboda and SM Block, Opt. Lett. 19, 930 (1994), T. Sugiura, T. Okada, Y. Inoue, O. Nakamura, and S. Kawata, Opt. Lett. 22, 1663 (1997))
The light capturing technique using the principle of light capturing as described above is described in, for example, Japanese Patent Application Laid-Open No. 2005-7530, Japanese Patent Application Laid-Open No. 2005-80516, and Japanese Patent Application Laid-Open No. 2006-130454. Any known means may be used as a means for capturing light.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. The sensing device of the present invention can be applied to any device as long as it is a sensing device utilizing the effects of surface plasmons and / or localized plasmons. Specifically, it can be used not only for a surface plasmon resonance measuring apparatus and a localized plasmon resonance measuring apparatus, but also for an enhanced Raman spectroscopic apparatus that generates Raman scattering enhanced by a plasmon enhancement field and detects the Raman scattering. .

<First Embodiment>
First, a configuration of a Raman spectroscopic apparatus according to a first embodiment of the sensing apparatus of the present invention and a Raman spectroscopic method as a sensing method using the same will be described. FIG. 1 is an overall view of the apparatus, and FIGS. 2 and 3 are views showing a preferred example of a plasmon active substrate.

  The Raman spectroscopic device 1 of the present embodiment is disposed so as to contact the sample cell 10 and the sample S in the sample cell 10, and the plasmon active substrate that generates the plasmon enhancement field by irradiating the sample contact surface 20s with the excitation light L0. 20, an excitation light irradiation optical system 30 (measurement light irradiation optical unit 40) that irradiates a sample contact surface 20 s of the plasmon active substrate 20 with a light beam L that becomes excitation light L 0 and measurement light L 1, and detects Raman scattered light. A detection unit 43 and a light capturing light irradiation optical system 50 are provided. Here, the sample cell 10 is placed on a stage 15 that can move the sample cell 10 in the three-dimensional direction of xyz. The stage 15 corresponds to a scanning unit that moves the light collection position of the light capturing light.

  Here, the plasmon active substrate 20 is a Raman scattering device that emits Raman scattered light when irradiated with the excitation light L0 (light beam L). In addition, the measurement light irradiation optical unit 40 and the detection unit 43 constitute a physical property detection system 45.

  The excitation light irradiation optical system 30 (measurement light irradiation optical unit 40) is an optical system that irradiates the sample contact surface 20s with a light beam L (excitation light L0, measurement light L1) that is a specific single wavelength light. A light source 31 such as a laser, and a light guide system (not shown) such as a mirror and a lens for guiding light emitted from the light source 31 as necessary.

  The detection unit 43 receives detection light L2 including reflected light and scattered light generated on the sample contact surface 20s of the plasmon active substrate 20 by irradiation with the measurement light L1, and detects the Raman scattered light by dispersing the detection light L2. This is a spectroscopic detector for obtaining a Raman spectrum.

  The sample cell 10 is a rectangular cell or the like having a bottom plate 11 and a top plate 12 which are spaced apart from each other. The upper plate 12 is made of a material that is transparent to the captured light L3.

  The light capturing light irradiation optical system 50 irradiates the sample with the capturing light L3 from the upper plate 12 of the sample cell 10, condenses the capturing light L3 on the sample contact surface 20s of the substrate 20, and measures the substance to be measured in the sample S. Is captured on the sample contact surface 20s, and is disposed directly above the upper plate 12 of the sample cell 10 and a light source 51 such as a laser that outputs the captured light L3, and collects the captured light L3 on the sample contact surface 20s. A lens 52, a mirror 53 that reflects the captured light L3 output from the light source 51 to the objective lens 52, and a lens 54 that collimates the captured light L3 output from the light source 51 are provided.

  The substance to be measured for light capture is a fine particle having a diameter of μm to nm, for example, protein, peptide, amino acid, polymethyl methacrylate (PMMA) latex fine particles in an aqueous solution, toluene droplet, benzyl Alcohol droplets, cis-decalin droplets, paraffin droplets, silicon oil droplets, microcapsules, silica gel, polystyrene spheres, glass beads, titanium dioxide, Salmonella, calf thymus DNA, quantum semiconductors, and the like.

  As the captured light L3, it is preferable to use a light beam having a wavelength that does not absorb the substance to be measured. In particular, a continuous wave (CW) Nd: YAG laser having a wavelength of 1064 nm, which hardly absorbs many substances and obtains high power, is preferable as a light capturing light source.

  Specific examples of the light trapping effect by the trapped light irradiation will be given. When a polymethyl methacrylate (PMMA) latex microparticle with a particle size of 5 μm in an aqueous solution is focused on a 1064 nm laser beam with a spot diameter of about 500 nm using a 100 × objective lens (beam power 50 mW), The state in which the latex fine particles present are always in focus is maintained. Fine particles other than the focal point are in Brownian motion and cannot be kept in focus.

  The metal body 23 constituting the sample contact surface of the plasmon active substrate 20 may be a metal thin film having a flat surface that generates surface plasmons, but has a concavo-convex structure smaller than the wavelength of the measurement light L1 on the surface. Also good.

  With reference to FIG. 2 and FIG. 3, the suitable aspects 20A-20F of the plasmon active base | substrate 20 are demonstrated. 2A and 2B are perspective views, and FIG. 2C and FIGS. 3A to 3C are cross-sectional views.

A plasmon active substrate 20A shown in FIG. 2A is a device in which a plurality of metal particles 23a are fixed on a flat dielectric 22 in an array. In this example, the metal body 23 is a metal particle layer composed of a plurality of metal particles 23a.
The arrangement pattern of the metal particles 23a can be designed as appropriate, and is preferably substantially regular. In such a configuration, the individual metal particles 23 a are convex portions, and the average diameter and pitch of the metal particles 23 a are designed to be smaller than the wavelength of the measurement light L.

  A plasmon active substrate 20B shown in FIG. 2 (b) is a device in which a metal body 23 formed of a metal pattern layer in which fine metal wires 23b are formed in a lattice pattern on a flat dielectric 22 is formed. The pattern of the metal pattern layer can be designed as appropriate and is preferably substantially regular. In such a configuration, the average line width and pitch of the fine metal wires 23b are designed to be smaller than the wavelength of the measurement light L.

As shown in FIGS. 4A to 4C, the plasmon active substrate 20C shown in FIG. 2C is formed by anodizing a part of the anodized metal body (Al, etc.) 60 to oxidize the metal. This is a device obtained by growing a metal 23c by plating or the like into a plurality of fine holes 62a of a metal oxide body 62 formed as an object (Al ₂ O ₃ or the like) 62 in the process of anodization. In this device, the metal 23c is grown in a mushroom shape in the fine holes 62a of the metal oxide body 62 until the head protrudes from the surface of the metal oxide body 62 (see Japanese Patent Laid-Open No. 2005-172569). Since the fine holes 62a can be opened in a substantially regular pattern, the metals 23c can be arranged in a substantially regular pattern. 4A and 4B are perspective views, and FIG. 4C is a cross-sectional view.

  In the plasmon active substrate 20C shown in FIG. 2 (c), the electrode 21 is composed of a non-anodized portion (Al or the like) 61 of an anodized metal body, and the metal body 23 is formed in the process of anodization. The plurality of mushroom-like metals 23c grown in the plurality of fine holes 62a.

  In the example shown in FIG. 2C, the head of the mushroom-like metal 23c is in the form of particles, and when viewed from the surface of the device, the metal particle layer is formed on the surface of the dielectric 22. In such a configuration, the head portion of the mushroom-like metal 23c is a convex portion, and the average diameter and pitch thereof are designed to be smaller than the wavelength of the measurement light L.

  Note that the plasmon active substrate 20 may be composed of only the metal body 23 that is brought into contact with the sample S and causes surface enhanced Raman scattering.

  The plasmon active substrate 20D shown in FIG. 3A is anodized as shown in FIGS. 4A and 4B, and the metal oxide body 62 formed by anodic oxidation is removed, and anodized This is a device in which only the non-anodized portion 61 of the metal body is left (see Japanese Patent Laid-Open No. 2006-250924). In such a device, the metal body 23 is constituted by a non-anodized portion 61 having a plurality of dimple-like recesses 23d on the surface.

  A plasmon active substrate 20E shown in FIG. 3B is obtained by forming a metal layer 63 on the surface of the plasmon active substrate 20D along the uneven shape (see Japanese Patent Laid-Open No. 2006-250924).

  In the plasmon active substrate 20F shown in FIG. 3C, the metal layer 63 of the plasmon active substrate 20E is formed into particles by an annealing treatment, and metal particles 64 are formed on the non-anodized portion 61 of the anodized metal body. (See Japanese Patent Application No. 2006-198009 (unpublished at the time of filing this patent application)).

  In the plasmon active bases 20A to 20F shown in FIGS. 2 and 3, since the metal body 23 having a substantially regular concavo-convex structure is obtained, the enhanced Raman (SERS) effect can be obtained without variation over the entire surface of the device, which is preferable.

  The metal body 23 may be composed of a metal layer whose surface is roughened. Examples of the roughening method include an electrochemical method using oxidation reduction and the like. In addition, as the plasmon active substrate 20, a known device having the SERS effect can be used. For example, J.AM.CHEM.SOC. 2005, Vol. 127, 14992-14993, “Nanosphere arrays with controlled sub-10-nm gaps as surface-enhanced raman spectroscopy substrates”, CTAB (cetyltrimethylammonium A plasmon active substrate in which a plurality of Au particles surface-modified with bromide) is arranged is described. Japanese Unexamined Patent Publication No. 2005-233637 discloses a plasmon active substrate in which a gold nanorod thin film is formed on a substrate.

  In the Raman spectroscopic device 1 of the present embodiment, the sample cell 10 is filled or flowed with a fluid sample S containing a substance to be measured, and measurement is performed. The sample S only needs to have fluidity, and examples of the state thereof include liquid, gel, and sol.

  The plasmon active substrate 20 is preferably such that the sample contact surface 20 s is subjected to surface modification that ionically bonds to the substance to be measured or surface modification that covalently bonds. Such a configuration is preferable because the substance to be measured is firmly adsorbed to the sample contact surface 20s by ionic bonding or covalent bonding. In this case, by irradiating the capture light, the binding of the substance to be measured to such a ligand can be promoted, and the concentration of the substance to be measured on the sample contact surface 20s can be effectively increased. Can be done quickly.

  When the substance to be measured is at least one selected from the group consisting of a protein, a peptide, and an amino acid, a surface modification group having a charge opposite to that of the substance to be measured is used as a surface modification that ionically binds to the substance to be measured. And surface modifying groups such as carboxy group, sulfonic acid group, phosphoric acid group, amino group, quaternary ammonium group, imidazole group, guanidinium group, and derivative groups thereof. The sample contact surface 20s may have two or more of these surface modification groups.

When the substance to be measured is at least one selected from the group consisting of a protein, a peptide, and an amino acid, the surface modification that is covalently bonded to the substance to be measured includes a reactive ester group such as N-hydroxysuccinimidyl ester, Carbodiimide group, 1-hydroxybenzotriazole group, hydrazide group, thiol group, reactive disulfide group, maleimide group, aldehyde group, epoxide group, (meth) acrylate group, hydroxyl group, isocyanate group, isothiocyanate group, and derivatives thereof And surface modifying groups such as groups. The sample contact surface 20s may have two or more of these surface modification groups.
Among the exemplified surface modifying groups, a reactive ester group, a hydrazide group, a thiol group, and a reactive disulfide group are preferable.
In the above description, “reactivity” means having reactivity with the substance to be measured.

It is particularly preferable that the plasmon active substrate 20 has a surface that is ion-bonded to the substance to be measured and surface-modified that is covalently bonded to the sample contact surface 20s.
In this case, the surface modification that ion-bonds to the substance to be measured and the surface modification that covalently bonds to the substance to be measured may be simultaneously applied to the sample contact surface 20s, or these surface modifications may be performed sequentially. I do not care. Further, the surface modification positions of these surface modifications are not particularly limited, and these surface modifications may be bonded to each other, or these surface modifications may be bonded to the sample contact surface 20s independently of each other. Good.

  It is particularly preferable to subject the sample contact surface 20s to surface modification that ion-bonds to the substance to be measured, and to activate this surface modification by surface modification that covalently bonds to the substance to be measured. In this case, the surface modification that ionically bonds to the substance to be measured and the surface modification that covalently bonds to the substance to be measured are close to each other, and each substance to be measured is attached to the sample contact surface 20s by ionic bonding and covalent bonding. On the other hand, it is adsorbed firmly, which is preferable.

  For example, first, a carboxy group that ion-bonds with the substance to be measured is introduced into the sample contact surface 20s, and the introduced carboxy group is shared with the substance to be measured such as a reactive ester group, a hydrazide group, a thiol group, and a reactive disulfide group. It is preferable to activate by inducing the form of the functional group to be bound.

As a surface modification substance that has both a surface modification group that ionically bonds to the substance to be measured and a surface modification group that covalently bonds to the substance to be measured,
4,4-dithiodibutyric acid (DDA), 10-carboxy-1-decanethiol, 11-amino-1-undecanethiol, 7-carboxy-1-heptanethiol, 16-mercaptohexadecanoic acid, 11,11′- Molecules that form self-assembled films such as thiodiundecanoic acid;
Hydrogels such as agarose, dextran, carrageenan, alginic acid, starch, and cellulose, or derivatives thereof (eg, carboxymethyl derivatives);
Examples thereof include water-swellable organic polymers such as polyvinyl alcohol, polyacrylic acid, polyacrylamide, and polyethylene glycol.

  For example, when the substance to be measured is adenine, the surface modifying substance having both a surface modifying group that ionically bonds to the substance to be measured and a surface modifying group that covalently bonds to the substance to be measured includes 4,4-dithiodibutyric acid ( DDA), carboxymethyl dextran (CMD) and the like are preferably used.

  On the other hand, when the size of the substance to be measured is very small such as a diameter of 30 nm or less (if it is not spherical, the width of the widest portion is 30 nm or less), polarization in the substance to be measured is unlikely to occur. For this reason, there is a problem that light capture is difficult even when irradiated with light capture light, so that the target substance can be surface-modified in advance to cause polarization in the target substance. The diameter should be 60 nm or more (in the case of a non-spherical shape, the width of the widest part is 60 nm or more). Specifically, polymethyl methacrylate (PMMA) fine particles, polystyrene fine particles, silica fine particles and the like are modified on the substance to be measured.

  The modification method may be a general modification method. For example, the substance to be measured can be adsorbed nonspecifically by mixing the substance to be measured with the fine particle dispersion. Alternatively, the reactive functional group may be exposed by carrying out hydrolysis treatment for PMMA fine particles, surface oxidation for fine polystyrene particles, and silane coupling treatment for silica fine particles, and this may be strongly modified by the base point.

The spectroscopic analysis method by the Raman spectroscopic device 1 of the present embodiment is as follows.
In a state in which the sample S having fluidity including the substance to be measured is brought into contact with the plasmon active substrate 20 that is irradiated with the excitation light L0 on the sample contact surface 20s and generates a plasmon enhancement field on the sample contact surface 20s. By condensing the capture light L3 on the sample contact surface 20s of the substrate 20, the substance to be measured in the sample S is moved onto the sample contact surface 20s, which is the condensing position of the capture light L3, and the sample contact surface 20s is covered. In a state where the measurement substance is captured, the measurement light L1 (excitation light) is irradiated onto the sample contact surface 20s, and the Raman scattered light L2 generated along with the sample contact surface 20s is detected.

  Here, by irradiating the sample contact surface 20s with the excitation light L0 (measurement light L1), surface plasmons and localized plasmons are generated, and a plasmon enhancement field is formed at and near the sample contact surface 20s. Raman scattered light is enhanced by this plasmon enhancement field. Furthermore, since the substance to be measured is captured on the sample contact surface 20s by the captured light, the signal can be increased, the SN ratio can be increased, and highly accurate measurement can be performed.

  Furthermore, before collecting the capture light on the sample contact surface, the capture light condensing position (condensation spot) is gradually scanned from the position away from the sample contact surface in the sample cell in a direction approaching the sample contact surface. Finally, it is desirable to collect light on the active sample contact surface. The focused spot can be scanned by moving the sample cell in the horizontal direction (xy direction) and the vertical direction (z direction) by the xyz stage 15.

  In this way, by moving the condensing spot while capturing the substance to be measured in the sample cell, the substance to be measured spreading over the entire sample cell is more effectively captured on the sample contact surface of the substrate. The concentration of the substance to be measured on the surface can be increased more effectively than when the trapped light is simply collected on the sample contact surface of the substrate.

  Therefore, in the Raman spectroscopic device 1 of the present embodiment, the analysis can be reliably performed in a state where a sufficient amount of the substance to be measured exists on the surface of the plasmon active substrate 20 or in the vicinity thereof, and the SERS effect is also effectively obtained. Since it is obtained, highly sensitive analysis can be performed stably.

  In addition, when the plasmon active substrate 20 has a surface modification that binds to the substance to be measured on the sample contact surface 20s, the binding between the plasmon active substrate 20 and the substance to be measured is promoted by the light capturing effect. And the amount of the substance to be measured adsorbed on the surface of the plasmon active substrate 20 can be increased. Also in this case, highly sensitive analysis can be performed stably and quickly.

  The wavelengths that can be used as measurement light and excitation light are not limited. As specific examples, those having center wavelengths of 785 nm, 633 nm, 532 nm, and 355 nm can be used. For example, when measuring R6G (6-carboxyrhodamine), the power of the measurement light and the excitation light may be about 800 μW at the 785 nm wavelength, about 20 μW at the 633 nm wavelength, and about 500 nW at the 532 nm wavelength. The wavelength of the trapped light is preferably 1064 nm of infrared light with little absorption by the measurement substance, and the power is preferably about 50 mW.

When the measurement light / excitation light and the capture light have different wavelengths as in the present embodiment, it is preferable to use the capture light having a wavelength that does not inhibit the measurement. Although it is preferable to prevent the trapped light from entering the detector by arranging a filter or the like in front of the detector, it is still difficult to completely cut the incident of the trapped light even if the filter is disposed. . Incident light incident on the detector becomes a large noise for very weak Raman light, and accurate Raman spectroscopic detection becomes difficult. In Raman spectroscopy, in general, scattered light having a Raman shift of 4000 cm ⁻¹ or less is dispersed. Therefore, it is preferable that the measurement light and the capture light are separated from each other by 4000 cm ⁻¹ or more.

(Design change example of the first embodiment)
In the first embodiment, the case where the sample cell is a box-shaped cell has been described. However, as shown in FIG. 5, the sample cell 10 is a capillary cell having one end in contact with the sample contact surface 20 s of the plasmon active substrate 20. May be.
Even in such a configuration, measurement can be performed in the same manner as in the first embodiment, and the same effect can be obtained. Such a configuration is preferable because the amount of the sample is small and the influence of convection due to the Joule heat of the sample can be ignored.

Second Embodiment
With reference to FIG. 6, a configuration of a microscopic Raman spectroscopic device that is a sensing device according to a second embodiment of the present invention and a Raman spectroscopic method using the same will be described. FIG. 6 is an overall view of the apparatus. Here, the same reference numerals are given to the same components as those in the first embodiment.

  Similar to the first embodiment, the Raman spectroscopic device 2 of the present embodiment is arranged so as to contact the sample cell 10 and the sample S in the sample cell 10, and the sample contact surface 20s is irradiated with the excitation light L1 to cause plasmon. A plate-shaped plasmon active substrate 20 that generates an enhancement field, an irradiation optical system 70 that irradiates a sample contact surface 20s of the plasmon active substrate 20 with a light beam L, and a detection unit 43 that detects Raman scattered light. is there.

  In the present embodiment, the irradiation optical system 70 also serves as the excitation light irradiation optical unit 30, the measurement light irradiation optical unit 40, and the light capturing light irradiation optical system 50, and a light beam is applied from the upper plate 12 of the sample cell 10 to the sample. L is irradiated, the light beam L is condensed on the sample contact surface 20s of the substrate 20, and the substance to be measured in the sample S is captured on the sample contact surface 20s. That is, the light beam L emitted from the irradiation optical system 70 has all the roles as the excitation light L0, the measurement light L1, and the light capturing light L3. Here, the irradiation optical system 70 includes a light source 71 such as a laser that outputs a light beam, an objective lens 72 that is disposed immediately above the upper plate 12 of the sample cell 10 and focuses the light beam L on the sample contact surface 20s, A dichroic that reflects the light beam L output from the light source 71 toward the objective lens 72 and transmits the detection light L2 including reflected light and scattered light generated on the sample contact surface 20s of the plasmon active substrate 20 to the detection unit 43. A mirror 73 and a lens 74 that converts the light beam L output from the light source 71 into parallel light are provided.

  In the present embodiment, in order to perform microscopic observation of the sample S, the detection unit 43 is disposed above the objective lens 72. The objective lens 72 can be two-dimensionally moved relative to the sample cell 10 in the xy direction in the drawing. The objective lens 72 is movable relative to the sample cell 10 also in the z direction shown in the figure. Here, the sample cell 10 is moved relative to the objective lens 72 by the xyz stage 15 on which the sample cell is placed.

  Similarly to the first embodiment, the detection unit 43 receives detection light L2 generated on the sample contact surface 20s of the plasmon active substrate 20 by irradiation with the measurement light L1, and detects the Raman scattered light by dispersing the detection light L2. It is a spectroscopic detector for obtaining a Raman spectrum. In this embodiment, a microscopic image monitor of the sample S is also provided (not shown).

  The sample cell 10 is a box-shaped cell similar to that of the first embodiment, and a plasmon active substrate 20 similar to that of the first embodiment is fixed on the bottom plate 11 of the sample cell 10.

  As in this embodiment, if the light beam L emitted from the irradiation optical system 70 plays all the roles as the excitation light L0, the measurement light L1, and the capture light L3, that is, the substance to be measured at one wavelength. If it is the structure which can perform light capture of this, excitation of a plasmon, and a measurement, an apparatus structure will become simple and it can be set as a small apparatus. Here, the light beam L is sufficiently narrowed as compared with normal measurement light or excitation light so that light can be captured, the power density at the spot position is 50 mW or more, and a lens with a large numerical aperture It is necessary to collect the light.

  The spectroscopic analysis method is the same as that of the first embodiment except that the Raman spectroscopic measurement can be performed while performing microscopic observation of the sample S. As described above, the present invention can also be applied to the microscopic Raman spectroscopic apparatus 2, and the same effects as those of the first embodiment can be obtained.

(Modification of the second embodiment)
In the second embodiment, one light source 70 is used as the light source for the excitation light L0, the measurement light L1, and the captured light L3. However, in the microscopic Raman spectroscopic apparatus as in the first embodiment. Alternatively, a configuration of a microscopic Raman spectroscopic device 2 ′ as shown in FIG. 7 in which the light sources 30 and 40 for the excitation light L0 and the measurement light L1 and the light source 50 for the capture light L3 are separated.

  Here, in addition to the configuration of the second embodiment, the light capturing light irradiation optical system 50, the dichroic mirror 73 for inputting the laser light L and the light capturing light L3 from the light source 71 to the common objective lens 72, and A capture light cut filter (notch filter) 76 is provided in front of the detector 43.

  The captured light L3 output from the light source 51 is collimated by the lens 54, reflected by the dichroic mirrors 75 and 73, and irradiated on the sample contact surface 20s of the plasmon active substrate 20 through the objective lens 72. The captured light cut filter 76 is disposed so that reflected light, scattered light, etc. of the captured light L3 can be prevented from entering the detector 43 and only the Raman scattered light can be detected with high accuracy.

  Even in such a configuration, the measurement can be performed in the same manner as in the second embodiment, and similarly, the analysis can be reliably performed in a state where a sufficient amount of the substance to be measured exists on the surface of the plasmon active substrate 20 or in the vicinity thereof. Since the SERS effect can be effectively obtained, highly sensitive analysis can be stably performed.

<Third Embodiment>
With reference to FIG. 8, the surface plasmon resonance measuring apparatus 3 which is a sensing apparatus of 3rd Embodiment which concerns on this invention, and the measuring method using this are demonstrated. Here, the same reference numerals are given to the same components as those in the first embodiment.

  The surface plasmon resonance measurement apparatus 3 includes a light source 131 such as a semiconductor laser that emits a light beam L, a prism 110 that is made of a material that transmits the light beam L, and is disposed at a position where the light beam L is incident from one end surface. A metal film 23 made of, for example, gold, silver, or the like, formed on one surface 110a of the prism 110, and a sample that holds the sample S so that the liquid sample S is in contact with the metal film 23 from the opposite side of the prism 110. A holding unit (sample cell) 10d, a cylindrical lens 132 that focuses the light beam L emitted from the light source 131 in a divergent light state only in a plane perpendicular to the major axis of the prism 110 (a plane parallel to the paper), and the prism 110 And a photodetector 143 which is a detection unit for detecting the intensity of the light beam L totally reflected at the interface 110a between the metal film 20 and the metal film 20. Furthermore, a light capturing light irradiation optical system 50 similar to that of the first embodiment is provided.

  In the present embodiment, the metal film 23 formed on the one end surface 110 a of the prism 110 is the plasmon active substrate 20, and the physical property detection system 145 includes the prism 110, the light source 131, the cylindrical lens 132, and the photodetector 143. The excitation light irradiation optical system 130 is configured by the light source 131 and the cylindrical lens. The light beam L incident on the interface is excitation light L0 that excites plasmons and is also measurement light L1.

Since the light beam L is focused as described above by the action of the cylindrical lens 132, various incident angles θ with respect to the interface 110a are illustrated as an example of the minimum incident angle θ ₁ and the maximum incident angle θ _{2 in} the figure. The component which injects is included. The incident angle θ is an angle that is greater than the total reflection angle. Therefore, the light beam L is totally reflected at the interface 110a, and the reflected light beam L includes components reflected at various reflection angles.

  As the photodetector 143, for example, a CCD line sensor is used in which the light receiving portion extends in a direction in which all the light beams L reflected at various reflection angles can be received. Therefore, the light detection signal Sn output for each light receiving element of the light detector 143 indicates the intensity of the light beam L for each of the various reflection angles (that is, for each of various incident angles).

  Hereinafter, a sample analysis method using the surface plasmon resonance measuring apparatus 3 having the above configuration will be described. At the time of sample analysis, the light-capturing light irradiation optical system 50 collects the light-capturing light L3 on the metal film 23, whereby the substance to be measured in the sample S is applied to the metal film 23 due to the light-trapping effect of the trapping light L3. This is performed in a state where the light is captured by the condensing spot of the captured light. The light beam L focused as described above by the action of the cylindrical lens 115 is irradiated toward the metal film 23. The light beam L totally reflected at the interface 110 a between the metal film 23 and the prism 110 is detected by the photodetector 143.

  As described above, the light detection signal Sn output for each light receiving element of the light detector 143 indicates the intensity I of the totally reflected light beam L for each incident angle θ.

Here, the light incident at a specific incident angle θ _SP excites surface plasmons at the interface between the metal film 23 and the sample S, so that the reflected light intensity I sharply decreases for this light. The incident angle θ _SP can be obtained by using the light detection signal Sn output for each light receiving element of the photodetector means 143, and the substance to be measured in the sample S can be quantitatively analyzed based on the value of θ _SP. it can.

  Also in the present embodiment, the light capturing light irradiation optical system 50 captures the substance to be measured on the metal film 23 that is the plasmon active substrate and obtains the signal of the total reflection attenuation angle, so that the SN ratio is highly accurate. Can be obtained.

1 is an overall view of a Raman spectroscopic device according to a first embodiment of the present invention. (A)-(c) is a figure which shows the suitable example of a plasmon active base | substrate. (A)-(c) is a figure which shows the suitable example of a plasmon active base | substrate. (A)-(c) is a manufacturing process figure of the plasmon active substrate shown in FIG.2 (c). Example of design change of the Raman spectrometer shown in FIG. Overall view of a Raman spectroscopic device (microscopic Raman spectroscopic device) according to a second embodiment of the present invention. Example of design change of the Raman spectroscope in FIG. Overall view of a surface plasmon resonance measuring apparatus according to a third embodiment of the present invention.

Explanation of symbols

1, 2 Sensing device (Raman spectroscopy device)
3 Sensing device (surface plasmon resonance measuring device)
10a to 10d Sample cell 15 Scanning unit (xyz stage)
20, 20A-20F Plasmon active substrate 20s Sample contact surface 22 Dielectric 23 Metal body 30, 130 Excitation light irradiation optical system 40 Measurement light irradiation optical unit 43, 143 Detection unit 45 145 Physical property detection system 50 Light capture light irradiation optical system 52 Objective lens S Sample L Light beam L0 Excitation light L1 Measurement light L2 Detection light L3 Capture light

Claims (13)

A sample cell in which a fluid sample containing a substance to be measured is filled or flowed down;
A plasmon active substrate that is disposed so as to contact a sample in the sample cell and 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 sensing device comprising a physical property detection system for detecting a physical property of a sample on the sample contact surface of the plasmon active substrate,
A light-capturing light irradiation optical system is provided that collects trapped light on the sample contact surface of the plasmon active substrate and traps a substance to be measured in the sample on the sample contact surface due to a light trapping effect. Sensing device.   The sensing device according to claim 1, further comprising a scanning unit that scans the collection position of the captured light within the sample cell.   The physical property detection system is generated at a measurement light irradiation optical unit that irradiates measurement light onto the sample contact surface of the plasmon active substrate, and reflected light of the measurement light at the sample contact surface and / or the sample contact surface. The sensing device according to claim 1, further comprising a detection unit that detects scattered light.   The sensing device according to any one of claims 1 to 3, wherein the plasmon active substrate is made of a metal body having a concavo-convex structure smaller than a wavelength of the excitation light.   The main component of the metal body is at least one metal selected from the group consisting of Au, Ag, Cu, Al, Pt, Ni, Ti, and alloys thereof. Sensing device.   6. The sensing device according to claim 1, wherein the sample cell is a capillary cell having one end in contact with the sample contact surface of the plasmon active substrate.   The sensing device according to any one of claims 1 to 6, wherein the plasmon active substrate has a surface modification that ionically bonds to the substance to be measured on the sample contact surface. The substance to be measured is at least one selected from the group consisting of a protein, a peptide, and an amino acid;
At least one group selected from the group consisting of a carboxy group, a sulfonic acid group, a phosphoric acid group, an amino group, a quaternary ammonium group, an imidazole group, and a guanidinium group is used as the surface modification that ionically bonds to the substance to be measured. The sensing device according to claim 7, wherein:   The sensing device according to any one of claims 1 to 8, wherein the plasmon active substrate has a surface modification that is covalently bonded to the substance to be measured on the sample contact surface. The substance to be measured is at least one selected from the group consisting of a protein, a peptide, and an amino acid;
The surface modification covalently bonded to the substance to be measured is at least one group selected from the group consisting of a reactive ester group, a hydrazide group, a thiol group, and a reactive disulfide group. 9. The sensing device according to 9.   The sensing device according to claim 1, wherein the substance to be measured is subjected to surface modification for increasing the size of the substance to be measured. A sample having fluidity including a substance to be measured is brought into contact with a plasmon active substrate that is irradiated with excitation light on the sample contact surface to generate a plasmon enhancement field on the sample contact surface,
A sensing method for detecting physical properties of a substance to be measured on the sample contact surface of the plasmon active substrate in a state where the plasmon active substrate is irradiated with the excitation light to generate the plasmon enhancement field,
In a state where the sample is in contact with the plasmon active substrate, the captured substance is condensed on the sample contact surface of the plasmon active substrate to move the substance to be measured in the sample onto the sample contact surface. ,
A sensing method, wherein physical properties of the substance to be measured are detected in a state where the substance to be measured is captured on the sample contact surface.   Before condensing the capture light on the sample contact surface, the capture light is gradually scanned from a position away from the sample contact surface in the sample cell in a direction approaching the sample contact surface, and finally the sample contact is performed. The sensing method according to claim 12, wherein the light is condensed on a surface.

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