JP2008164584A - Raman spectrometer and raman spectral method using same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable Raman spectrum analysis with high sensitivity by effectively drawing an object substance to be measured in a sample cell to the surface of a Raman scattering device in the vicinity thereof in a Raman spectrometer. <P>SOLUTION: The Raman spectrometer 1 includes a sample cell 10; a scattering device 20 arranged so as to be in contact with a sample S in the sample cell 10, for irradiating measuring light L1 to a sample contacting surface 20s and thereby generating Raman scattering light; a measurement light irradiation optical system 30 for irradiating the measuring light L1 to the sample contacting surface 20s of the Raman scattering device 20; and a detecting unit 40 for detecting the Raman scattering light. The device 1 performs measurement by filling or flowing down the sample S including the object substance that has electrophoresis or is previously adjusted so as to have electrophoresis, in the sample cell 10; and includes a voltage applying unit 50 for electrophoresing the object substance in the sample S to the sample contacting surface 20s of the device 20, via the sample cell 10. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an apparatus and method for detecting Raman scattered light by bringing a sample containing a substance to be measured into contact with a Raman scattering device that is irradiated with measurement light to generate Raman scattered light.

  The Raman spectroscopic analysis is an analysis method for obtaining a spectrum of Raman scattered light (Raman spectrum) by dispersing scattered light obtained by irradiating a substance with single wavelength light, and is used for identification of the substance. Although Raman scattered light is weak light, it is known that the intensity of Raman scattered light is enhanced when light is irradiated in a state where a substance is in contact with a metal body, particularly a metal body having fine irregularities on the surface. ing. This effect is referred to as the surface enhanced Raman scattering (SERS) effect.

  In general, when performing a Raman spectroscopic analysis using a Raman scattering device having the SERS effect, it is necessary to perform an analysis in a state where a substance to be measured in the sample is located at or near the surface of the Raman scattering device. It is particularly preferable to perform the analysis in a state where the substance to be measured is adsorbed on the surface of the Raman scattering device. This is because the SERS effect decreases as the substance to be measured moves away from the Raman scattering device.

  For example, in the field of surface plasmon sensors, a ligand that specifically binds to a substance to be measured is immobilized on a metal film where surface plasmon is generated in advance, and the substance to be measured in the sample is immobilized on the surface of the metal film to perform sensing. To be done. However, in such a method, a sufficient amount of binding cannot be secured unless the reaction between the ligand and the substance to be measured is sufficiently waited, and it is difficult to carry out the measurement quickly. In addition, substances that bind to the ligand are limited, and the types of substances to be measured are limited.

  On the other hand, electrophoresis is known as a method for separating a substance to be measured when performing a microanalysis of a sample containing a plurality of substances to be measured such as proteins, peptides, and amino acids. Among the electrophoresis methods, the capillary electrophoresis method is preferable because the amount of the sample is very small and the influence of convection due to the Joule heat of the sample can be ignored.

  Patent Documents 1 and 2 disclose an apparatus that can simultaneously perform sample separation by capillary electrophoresis and Raman spectroscopic analysis of the separated sample.

  In Patent Document 1, a fiber-like SERS active microelectrode is inserted into a capillary for electrophoresis of a substance to be measured, so that the separated substance to be measured is layered around the SERS active microelectrode by capillary electrophoresis. An apparatus for performing Raman spectroscopic analysis of a substance to be measured separated by irradiating measurement light onto a SERS active microelectrode that can be collected has been disclosed (see paragraph 0008 and FIG. 1 and the like).

Patent Document 2 discloses an apparatus for depositing a plurality of substances to be measured separated by capillary electrophoresis at different locations on the SERS active substrate and performing Raman spectroscopic analysis of the precipitate (claims 7 and 4). Etc.).
Japanese Patent Laid-Open No. 9-281076 Special Table 2003-511666

In the apparatuses described in Patent Documents 1 and 2, the substance to be measured can be collected on the Raman scattering device having the SERS effect.
However, in the apparatus described in Patent Document 1, it is necessary to process the Raman scattering device into a fiber shape that can be inserted into a capillary for electrophoresis, and the processing of the Raman scattering device is very complicated.
In the apparatus described in Patent Document 2, since it is necessary to deposit a plurality of substances to be measured separated by capillary electrophoresis on a Raman scattering device having the SERS effect, it is troublesome to deposit each substance to be measured. And takes time. In addition, since a plurality of substances to be measured separated by capillary electrophoresis need to be deposited at different locations of the Raman scattering device, the deposition operation requires as much work and time as the number of substances to be measured.

  Further, in a general Raman spectroscopic apparatus, the sample cell has a non-capillary shape, but a method for collecting the substance to be measured in the non-capillary sample cell on the Raman scattering device has not been proposed.

  The present invention has been made in view of the above circumstances, and regardless of the shape of the sample cell, the substance to be measured in the sample cell is effectively attracted to the surface of the Raman scattering device or in the vicinity thereof, and high-sensitivity Raman spectroscopy is achieved. It is an object of the present invention to provide a Raman spectroscopic device that can perform analysis, has a simple apparatus configuration, and can perform analysis easily and quickly, and a Raman spectroscopic method using the same.

The Raman spectrometer of the present invention is
A sample cell;
A Raman scattering device disposed so as to contact the sample in the sample cell, and the sample contact surface being irradiated with measurement light to generate Raman scattered light;
A measurement light irradiation optical system for irradiating the sample contact surface of the Raman scattering device with the measurement light;
In a Raman spectroscopic device provided with a detecting means for detecting the Raman scattered light,
The sample cell has an electrophoretic property, or a fluid sample containing a substance to be measured that has been adjusted in advance to have an electrophoretic property is filled or flowed down, and measurement is performed.
Voltage application means is provided for applying a voltage to the sample cell to the sample cell so that the substance to be measured in the sample is electrophoresed on the sample contact surface side of the Raman scattering device. It is what.

"The substance to be measured has electrophoretic properties" means that the substance to be measured is in a charged state and has a property that can be moved by an electric field.
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.

  For reference, the field is different from that of Raman spectrometers, but in the field of surface plasmon sensors, a voltage is applied to a sample in a non-capillary sample cell, and the surface plasmon It is disclosed that a substance to be measured is detected by electrophoresis on the generated metal film side (Claim 1 and FIG. 1, etc. of JP-A-9-304339).

The Raman scattering device includes an electrode, a dielectric sequentially formed on the electrode, and a metal body that is brought into contact with the sample and causes surface enhanced Raman scattering.
It is preferable that the voltage application unit includes the electrode that is a part of the Raman scattering device and a counter electrode that is disposed to face the Raman scattering device via the sample.

The Raman scattering device is made of a metal body that is brought into contact with the sample to cause surface-enhanced Raman scattering, and functions as an electrode itself.
The voltage application unit may include an electrode made of the Raman scattering device and a counter electrode disposed to face the Raman scattering device through the sample.

  In the present specification, the “counter electrode” may be front facing or diagonally facing the Raman scattering device. In addition, the position of the counter electrode is not particularly limited as long as a sample exists between the Raman scattering device and the counter electrode. Therefore, the counter electrode may be attached to the sample cell without being arranged in the sample, or may be arranged in the sample.

It is preferable that the metal body that causes surface-enhanced Raman scattering has a concavo-convex structure that is smaller than the wavelength of the measurement 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 Raman scattering device and the other end in contact with the counter electrode.

  In the Raman scattering device, it is preferable that the sample contact surface is subjected to surface modification that is ionically bonded to the substance to be measured and / or surface modification that is covalently bonded to the substance to be measured.

  According to a first Raman spectroscopy method of the present invention, a sample is brought into contact with a Raman scattering device that is irradiated with measurement light to generate Raman scattered light, and the Raman scattered light is detected in the Raman spectroscopy method in which the Raman scattered light is detected. A sample having fluidity or fluidity containing a substance to be measured that has been adjusted in advance to have electrophoretic properties is prepared, and a voltage is applied to the sample while the sample is in contact with the Raman scattering device. Applying the measurement, the substance to be measured in the sample is attracted to the sample contact surface side of the Raman scattering device by electrophoresis, and the Raman scattered light is detected in a state in which the substance to be measured is attracted. It is a feature.

According to a second Raman spectroscopy method of the present invention, in the Raman spectroscopy method in which a sample is brought into contact with a Raman scattering device that is irradiated with measurement light to generate Raman scattered light, and the Raman scattered light is detected. Preparing a fluid sample containing a substance to be measured having amphotericity, adjusting the pH of the sample, adjusting the substance to be measured to a positively charged state or a negatively charged state,
With the sample in contact with the Raman scattering device, a voltage is applied to the sample, and the substance to be measured in the sample is attracted to the sample contact surface side of the Raman scattering device by electrophoresis, The Raman scattered light is detected in a state in which the substance to be measured is attracted.

  In the first and second Raman spectroscopic methods of the present invention, the Raman scattering device is obtained by subjecting the sample contact surface to surface modification that is covalently or ionically bonded to the substance to be measured. The Raman scattered light may be detected by covalently or ionically bonding the substance to be measured attracted by the electrophoresis. In this method, it is preferable that the surface modification and the substance to be measured are covalently bonded or ionically bonded, and then the voltage application is stopped to detect the Raman scattered light.

In addition, it is preferable to remove impurities before the detection of the Raman scattered light after the surface modification and the substance to be measured are covalently bonded or ionically bonded.
Here, the “impurity” means a sample obtained by removing a sample to be measured which has been covalently bonded or ionically bonded to the surface modification from the sample brought into contact with the Raman scattering device.

  The Raman spectroscopic apparatus of the present invention has a configuration including voltage applying means for applying a voltage to a sample in a sample cell and causing a substance to be measured in the sample to be electrophoresed on the sample contact surface side of the Raman scattering device. is doing.

  According to the Raman spectroscopic apparatus of the present invention, a sample having a fluidity including an analyte or a fluid to be measured that has been adjusted in advance to have an electrophoretic property is used. It can be drawn toward the sample contact surface side of the Raman scattering device.

  In the Raman spectroscopic device of the present invention, the effect of attracting the substance to be measured to the surface of the Raman scattering device or its vicinity can be obtained regardless of the shape of the sample cell. In the Raman spectroscopic apparatus of the present invention, the analysis can be performed reliably in a state where a sufficient amount of the substance to be measured exists on the surface of the Raman scattering device or in the vicinity thereof, and the surface enhanced Raman scattering effect can be effectively obtained. Therefore, highly sensitive analysis can be performed stably. The Raman spectroscopic device of the present invention is a device that has a simple device configuration and can perform analysis easily and quickly.

  In the Raman spectroscopic apparatus of the present invention, the amount of the substance to be measured that is attracted to the surface of the Raman scattering device or the vicinity thereof can be adjusted by adjusting the applied voltage as necessary.

  When the Raman scattering device has a surface modification that binds to the substance to be measured on the sample contact surface, the above-described pulling effect can promote the binding between the Raman scattering device and the substance to be measured. The amount of the substance to be measured adsorbed on the surface can be increased. In this case, since the detection of Raman scattered light can be carried out after removing the sample that causes noise in the measurement, highly sensitive analysis can be carried out stably.

“First Embodiment”
A configuration of a Raman spectroscopic device according to a first embodiment of the present invention and a Raman spectroscopic method using the same will be described with reference to the drawings. FIG. 1 is an overall view of the apparatus, and FIGS. 2 and 3 are views showing a suitable example of a Raman scattering device.

  The Raman spectroscopic device 1 according to the present embodiment is arranged so as to be in contact with the sample cell 10 and the sample S in the sample cell 10, and is irradiated with the measurement light L1 on the sample contact surface 20s to generate a Raman scattered light. A Raman scattering device 20, a measurement light irradiation optical system 30 that irradiates the sample contact surface 20 s of the Raman scattering device 20 with the measurement light L 1, and a detection means 40 that detects Raman scattering light are provided.

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

  The detection means 40 receives detection light L2 including reflected light and scattered light generated on the sample contact surface 20s of the Raman scattering device 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 an upper plate 12 that are spaced apart from each other. The sample cell 10 is made of an insulating material. The upper and lower sides of the sample cell 10 are determined for convenience, and the upper and lower sides of the sample cell 10 can be designed as appropriate.

  In the present embodiment, the sample cell 10 has a voltage applying means 50 that applies a voltage to the sample S and causes the substance to be measured in the sample S to be electrophoresed on the sample contact surface 20 s side of the Raman scattering device 20. Is provided.

  In the present embodiment, the Raman scattering device 20 is a device including an electrode 21, a dielectric 22 sequentially formed on the electrode 21, and a metal body 23 that is brought into contact with the sample S and generates surface enhanced Raman scattering (SERS). It is. In the Raman scattering device 20, the electrode 21 is fitted into the bottom plate 11 of the sample cell 10 so that the surface of the metal body 23 that is the sample contact surface 20 s contacts the sample S in the sample cell 10, and is fixed to the sample cell 10. ing. About the fixation aspect to the sample cell 10 of the Raman scattering device 20, it can design suitably.

  A counter electrode 51 is disposed on the upper plate 12 of the sample cell 10 so as to be opposed to the Raman scattering device 20 in front of the sample S. A power source 52 and wiring 53 for applying a voltage to the electrode 21 and the counter electrode 51 that form a part of the Raman scattering device 20 are disposed outside the sample cell 10.

  In the present embodiment, the voltage application means 50 is configured by the electrode 21, the counter electrode 51, the power supply 52, and the wiring 53 that form part of the Raman scattering device 20.

  Since the enhancement effect of the Raman scattered light is large, the metal body 23 forming the Raman scattering device 20 preferably has an uneven structure smaller than the wavelength of the measurement light L1.

  With reference to FIG.2 and FIG.3, the suitable aspects 20A-20F of the Raman scattering device 20 are demonstrated. 2 (a) and 2 (b) are perspective views, and FIG. 2 (c) and FIGS. 3 (a) to 3 (c) are cross-sectional views.

A Raman scattering device 20A shown in FIG. 2A is a device in which a plurality of metal particles 23a are fixed in an array on a laminated body of a flat electrode 21 and a flat dielectric 22. 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 Raman scattering device 20B shown in FIG. 2B has a metal body 23 composed of a metal pattern layer in which fine metal wires 23b are formed in a lattice pattern on a laminate of a flat electrode 21 and a flat dielectric 22. Is a device 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.

The Raman scattering device 20C shown in FIG. 2 (c) is formed by anodizing a part of the anodized metal body (Al, etc.) 60 and oxidizing the metal as shown in FIGS. 4 (a) to 4 (c). 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 Raman scattering device 20C shown in FIG. 2 (c), the electrode 21 is made of a non-anodized portion (Al, etc.) 61 of an anodized metal body, and the dielectric 22 anodizes a part of the anodized metal body 60. And the metal body 23 is composed of a plurality of mushroom-like metals 23c grown in the plurality of fine holes 62a of the metal oxide body 62 formed in the process of anodization. .

  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.

  The Raman scattering device 20 may be composed only of the metal body 23 that is brought into contact with the sample S and causes surface-enhanced Raman scattering.

  The Raman scattering device 20D shown in FIG. 3A performs anodic oxidation as shown in FIGS. 4A and 4B, removes the metal oxide body 62 formed by anodic oxidation, and performs anodic oxidation. 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 Raman scattering device 20E shown in FIG. 3B is obtained by forming a metal layer 63 on the surface of the Raman scattering device 20D along the uneven shape (see JP-A-2006-250924).

  In the Raman scattering device 20F shown in FIG. 3C, the metal layer 63 of the Raman scattering device 20E is made into particles by 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 Raman scattering devices 20A to 20F shown in FIG. 2 and FIG. 3, since the metal body 23 having a substantially regular concavo-convex structure is obtained, the 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 Raman scattering device 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 Raman scattering device in which a plurality of Au particles whose surfaces are modified with bromide) is arranged is described. Japanese Unexamined Patent Publication No. 2005-233637 discloses a Raman scattering device 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 sample S having electrophoretic properties or fluidity including a substance to be measured that has been adjusted in advance to have electrophoretic properties. Measurements are made. The sample S only needs to have fluidity, and examples of the state thereof include liquid, gel, and sol.

  In the present embodiment, a voltage is applied to the sample S in a state where the sample S is in contact with the Raman scattering device 20, and the substance to be measured in the sample S is electrically supplied to the sample contact surface 20 s side of the Raman scattering device 20. The detection of the Raman scattered light can be carried out in a state where the substance to be measured is attracted to the sample contact surface 20s side of the Raman scattering device 20 after the electrophoresis.

  If the substance to be measured is an amphoteric substance such as protein, peptide, amino acid, etc., the pH of the sample S can be adjusted to adjust the substance to be measured to a positively charged state or a negatively charged state. If the electrode 21 is an anode, the substance to be measured is adjusted to a negatively charged state, and if the electrode 21 is a cathode, the substance to be measured is adjusted to a positively charged state to measure the sample on the sample contact surface 20s side of the Raman scattering device 20. Can attract material.

  In the Raman spectroscopic device 1 according to the present embodiment, it is also possible to measure the sample S including a plurality of substances to be measured. In this case, the sample can be separated using the difference in the electrophoresis speed, and the measurement can be performed in the order in which the sample is attracted to the sample contact surface 20 s of the Raman scattering device 20. When carrying out such measurement, it is preferable to take out the Raman scattering device 20 and clean the sample contact surface 20s every time measurement of one kind of substance to be measured is completed.

  The Raman scattering device 20 is preferably a device in which the sample contact surface 20s is subjected to a surface modification that ionically bonds to a substance to be measured or a 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 bonds or covalent bonds. In such a case, the concentration of the substance to be measured on the sample contact surface 20s becomes high, which is preferable.

  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 modifying group having a charge opposite to that of the substance to be measured is used as the surface modification that ionically binds to the substance to be measured. And those having a surface modifying group such as a carboxy group, a sulfonic acid group, a phosphoric acid group, an amino group, a quaternary ammonium group, an imidazole group, a guanidinium group, and a derivative group 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 those having a surface modifying group such as a group. 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 Raman scattering device 20 has a sample contact surface 20 s that has been subjected to surface modification that ionically bonds to the substance to be measured and surface modification that covalently bonds.
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 ionically bonds with 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.

  When the sample contact surface 20s as described above is subjected to surface modification that is ion-bonded or covalently bonded to the measured substance as described above, the measured substance is chemically attached to the sample contact surface 20s. Because it is adsorbed by bonding, highly sensitive measurement can be performed stably even if surface scattering and the substance to be measured are covalently bonded or ionically bonded and then detection of Raman scattered light is detected after voltage application is stopped. can do.

  If the voltage application can be stopped as described above, the Raman scattered light can be detected after removing the sample liquid which becomes noise before detecting the Raman scattered light. When detecting Raman scattered light, if the measurement is performed in the sample liquid, the Raman scattering peak of the substance or solvent that is not the measurement target in the sample liquid must be overlapped with the Raman scattering peak of the measured substance. In this case, since the Raman scattered light other than these substances to be measured becomes noise, the S / N ratio is lowered. Therefore, it is more stable to perform highly sensitive detection by detecting the Raman scattered light in a state where the sample liquid is removed after the surface modification and the substance to be measured are covalently or ionically bonded. Is possible and preferable.

  The removal of the sample liquid may be performed after the substance to be measured is bonded to the sample contact surface 20s and before the voltage application is stopped, or after the stop.

  The method for detecting the Raman scattered light after removing the sample liquid is not particularly limited as long as it does not break the bond between the substance to be measured and the surface modification. For example, after removing the sample liquid, detection of Raman scattered light may be performed in a state where there is no liquid in the sample cell 10, or after removing the solution, the sample cell 10 and the sample contact surface 20s are washed once or more. Then, detection of Raman scattered light may be performed.

  Examples of the cleaning method include ultrasonic cleaning and a method of cleaning using a solvent that is inactive to Raman and has no reactivity with the substance to be measured. When performing ultrasonic cleaning, care must be taken so that the bond between the substance to be measured and the surface modification is not broken. Examples of the solvent which is inactive to Raman and has no reactivity with the substance to be measured include pure water. Here, “Raman inactivity” means that the Raman scattering peak does not overlap with the Raman scattering peak of the substance to be measured.

  The detection of the Raman scattered light after washing may be performed in a state where there is no liquid in the sample cell 10, or a solvent that is not Raman-active and has no reactivity with the substance to be measured is injected into the sample cell 10. You may implement after.

The Raman spectroscopic device 1 of the present embodiment is configured as described above.
The Raman spectroscopic apparatus 1 according to the present embodiment applies a voltage to the sample in the sample cell 10 to cause the substance to be measured in the sample to be electrophoresed on the sample contact surface 20 s side of the Raman scattering device 20. It has the composition provided with.

  According to the Raman spectroscopic device 1 of the present embodiment, a sample S having electrophoretic properties or fluidity including a substance to be measured that has been adjusted in advance so as to have electrophoretic properties is used. The substance to be measured can be drawn toward the sample contact surface 20 s side of the Raman scattering device 20.

  In the Raman spectroscopic apparatus 1 of the present embodiment, an effect of attracting the substance to be measured to the surface of the Raman scattering device 20 or the vicinity thereof is obtained regardless of the shape of the sample cell 10. In the Raman spectroscopic apparatus 1 of the present embodiment, analysis can be performed reliably in a state where a sufficient amount of a substance to be measured exists on the surface of the Raman scattering device 20 or in the vicinity thereof, and the SERS effect can also be obtained effectively. Therefore, highly sensitive analysis can be performed stably.

  In the Raman spectroscopic apparatus 1 of the present embodiment, the amount of the substance to be measured that is attracted to the surface of the Raman scattering device 20 or the vicinity thereof can be adjusted by adjusting the applied voltage as necessary.

  When the Raman scattering device 20 has a surface modification that binds to the substance to be measured on the sample contact surface 20s, the above-described pulling effect can promote the binding between the Raman scattering device 20 and the substance to be measured. The amount of the substance to be measured adsorbed on the surface of the Raman scattering device 20 can be increased. Also in this case, highly sensitive analysis can be performed stably.

In the apparatus described in Patent Document 1 listed in the section of “Background Art”, it is necessary to process the Raman scattering device into a fiber shape that can be inserted into a capillary for electrophoresis. In the apparatus 1 of this embodiment, There is no need to process the Raman scattering device into a special shape.
In Patent Documents 1 and 2, in order to perform sample separation by ordinary capillary electrophoresis, two containers filled with a buffer solution are prepared, and one end and the other end of the capillary are immersed in these containers, respectively. After the buffer solution is filled therein, it is necessary to perform electrophoresis by injecting a sample into one end of the capillary and applying a voltage between the two containers filled with the buffer solution. In the Raman spectroscopic device 1 of this embodiment, a buffer solution and a container for filling the buffer solution are not required, and the sample S may be filled or flowed down in the sample cell 10 and a voltage may be applied to the sample S.
Therefore, the Raman spectroscopic device 1 of the present embodiment is a device that has a simple device configuration and can perform analysis easily and quickly.

(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, one end of the sample cell 10 is in contact with the sample contact surface 20s of the Raman scattering device 20, and the other end is a counter electrode. A capillary cell in contact with 51 may be used.
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”
A configuration of a Raman spectroscopic device according to a second embodiment of the present invention and a Raman spectroscopic method using the same will be described with reference to the drawings. FIG. 6 is an overall view of the apparatus. The apparatus of this embodiment is a microscopic Raman spectroscopic apparatus, and the same reference numerals are given to the same components as those of the first embodiment.

  Similar to the first embodiment, the Raman spectroscopic device 2 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 sample contact surface 20s is irradiated with the measurement light L1 to cause Raman. A plate-shaped Raman scattering device 20 that generates scattered light, a measurement light irradiation optical system 30 that irradiates the sample contact surface 20s of the Raman scattering device 20 with the measurement light L1, and a detection means 40 that detects the Raman scattered light. Is.

  In the present embodiment, in order to perform microscopic observation of the sample S, an objective lens 71 is disposed on the sample cell 10. The objective lens 71 is two-dimensionally movable relative to the sample cell 10 in the xy direction shown in the drawing. The objective lens 71 is movable relative to the sample cell 10 also in the z direction shown in the figure.

  In the present embodiment, the measurement light irradiation optical system 30 includes a light source 31 such as a laser and a light separation element 32. The light separation element 32 guides the measurement light L1 emitted from the light source 31 to the objective lens 71 and the sample cell 10 side, and generates detection light L2 including reflected light and scattered light generated on the sample contact surface 20s of the Raman scattering device 20. It is an optical element that leads to the detection means 40. The measurement light irradiation optical system 30 may include a light guide system (not shown) such as other mirrors and lenses in the optical path of the measurement light L1 as necessary.

  As in the first embodiment, the detection means 40 receives the detection light L2 generated on the sample contact surface 20s of the Raman scattering device 20 by irradiation with the measurement light L1, and spectrally detects the detection light L2 to detect the Raman scattered light. 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 Raman scattering device 20 similar to that of the first embodiment is fitted into the bottom plate 11 of the sample cell 10.

  Also in the present embodiment, the sample cell 10 has a voltage applying means 50 that applies a voltage to the sample S and causes the substance to be measured in the sample S to be electrophoresed on the sample contact surface 20 s side of the Raman scattering device 20. Is provided. However, in the present embodiment, the counter electrode 51 is disposed obliquely opposite the Raman scattering device 20 in the sample cell 10 so as to open an optical path between the objective lens 71 and the Raman scattering device 20. .

  The Raman spectroscopic device 2 of the present embodiment is configured as described above. The measurement is the same as that of the first embodiment except that the Raman spectroscopic analysis can be performed while performing the microscopic observation of the sample S. 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.

  The technology of the present invention can be applied to a Raman spectroscopic device that obtains a Raman spectrum by spectroscopically analyzing scattered light obtained by irradiating a substance with single wavelength light, and identifies the substance.

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 Raman scattering device. (A)-(c) is a figure which shows the suitable example of a Raman scattering device. (A)-(c) is a manufacturing process figure of the Raman scattering device 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.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 2 Raman spectroscopy apparatus 10 Sample cell 20, 20A-20C Raman scattering device 20s Sample contact surface 21 Electrode 22 Dielectric body 23 Metal body 20D-20F Raman scattering device which itself functions as an electrode 30 Measurement light irradiation optical system 40 Detection means 50 Voltage application means 51 Counter electrode S Sample L1 Measurement light L2 Detection light

Claims (15)

A sample cell;
A Raman scattering device disposed so as to contact the sample in the sample cell, and the sample contact surface being irradiated with measurement light to generate Raman scattered light;
A measurement light irradiation optical system for irradiating the sample contact surface of the Raman scattering device with the measurement light;
In a Raman spectroscopic device provided with a detecting means for detecting the Raman scattered light,
The sample cell has an electrophoretic property, or a fluid sample containing a substance to be measured that has been adjusted in advance to have an electrophoretic property is filled or flowed down, and measurement is performed.
Voltage application means is provided for applying a voltage to the sample cell to the sample cell so that the substance to be measured in the sample is electrophoresed on the sample contact surface side of the Raman scattering device. A Raman spectrometer. The Raman scattering device includes an electrode, a dielectric sequentially formed on the electrode, and a metal body that is brought into contact with the sample and causes surface enhanced Raman scattering.
The voltage application means includes the electrode that is a part of the Raman scattering device, and a counter electrode that is arranged to face the Raman scattering device via the sample. Item 11. The Raman spectroscopic device according to Item 1. The Raman scattering device is made of a metal body that is brought into contact with the sample to cause surface-enhanced Raman scattering, and functions as an electrode itself.
The said voltage application means is provided with the electrode which consists of the said Raman scattering device, and the counter electrode arrange | positioned facing the said Raman scattering device through the said sample, The Claim 1 characterized by the above-mentioned. Raman spectrometer.   The Raman spectroscopic apparatus according to claim 2, wherein the metal body has an uneven structure that is smaller than a wavelength of the measurement 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. The Raman spectroscopic apparatus according to any one of the above.   6. The Raman cell according to claim 2, wherein the sample cell is a capillary cell having one end in contact with the sample contact surface of the Raman scattering device and the other end in contact with the counter electrode. Spectrometer.   The Raman spectroscopic apparatus according to claim 1, wherein the Raman scattering device is a surface of the sample contact surface that is surface-modified to ionically bond to the substance to be measured. 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 Raman spectroscopic apparatus according to claim 7, wherein:   The Raman spectroscopic apparatus according to claim 1, wherein the Raman scattering device 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 has 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. The Raman spectroscopic device according to claim 9. In a Raman spectroscopy method in which a sample is brought into contact with a Raman scattering device that is irradiated with measurement light to generate Raman scattered light, and the Raman scattered light is detected.
As the sample, prepare a sample having electrophoretic properties or fluidity containing a substance to be measured that has been adjusted in advance to have electrophoretic properties,
With the sample in contact with the Raman scattering device, a voltage is applied to the sample, and the substance to be measured in the sample is attracted to the sample contact surface side of the Raman scattering device by electrophoresis,
A Raman spectroscopy method, wherein the Raman scattered light is detected in a state in which the substance to be measured is attracted. In a Raman spectroscopy method in which a sample is brought into contact with a Raman scattering device that is irradiated with measurement light to generate Raman scattered light, and the Raman scattered light is detected.
As the sample, a fluid sample containing a substance to be measured that is electrically amphoteric is prepared,
Adjusting the pH of the sample to adjust the substance to be measured to a positively charged state or a negatively charged state;
With the sample in contact with the Raman scattering device, a voltage is applied to the sample, and the substance to be measured in the sample is attracted to the sample contact surface side of the Raman scattering device by electrophoresis,
A Raman spectroscopy method, wherein the Raman scattered light is detected in a state in which the substance to be measured is attracted. The Raman scattering device has been subjected to a surface modification that covalently or ionically bonds with the substance to be measured on the sample contact surface,
The Raman spectroscopic method according to claim 11 or 12, wherein the Raman scattered light is detected by covalently or ionically bonding the surface modification and the substance to be measured attracted by the electrophoresis.   The Raman spectroscopic method according to claim 13, wherein the voltage application is stopped after the surface modification and the substance to be measured are covalently bonded or ionically bonded before the detection of the Raman scattered light. .   The Raman spectroscopic method according to claim 14, wherein after the surface modification and the substance to be measured are covalently bonded or ionically bonded, impurities are removed before the detection of the Raman scattered light is performed.

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