JP3462339B2 - Capillary electrophoresis detector - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a capillary electrophoresis detector used for detecting proteins, peptides, DNA (nucleic acid), carbohydrates, neurotransmitters, and the like, and more particularly,
The present invention relates to a detector for capillary electrophoresis suitable for analyzing a small amount of sample.

[0002]

2. Description of the Related Art Proteins, peptides, DNAs (nucleic acids) in living organisms are used to clarify the functions of living organisms at the molecular level.
Analysis of carbohydrates, substances related to neurotransmission, etc. is in progress.
A gel electrophoresis using polyacrylamide as a support is used as a powerful means. But,
When gel electrophoresis is carried out, it is not easy to obtain a high resolution because there are an extremely large number of manual steps such as gel preparation, staining, and quantification. Therefore, an attempt was made to directly detect a molecule that migrates in a buffer solution without using a gel as a support and omit the analysis procedure, but there was a problem that convection occurs in the solution due to the generated Joule heat. .

On the other hand, if electrophoresis is performed using a capillary with an inner diameter of 100 μm or less, convection of the buffer solution can be ignored, and electroosmotic flow is generated, but the velocity distribution in the tube is uniform. Therefore, the separation performance is not impaired. Further, in this capillary electrophoresis method, since Joule heat can be easily dissipated and a high voltage can be applied, a high resolution can be obtained in a short time. In addition, the absolute volume of the sample is as small as nl (10 ^-9 l) due to the thin capillary,
It is noted that it has the advantage of being suitable for microanalysis, and is expected to be applied to microanalysis such as neurotransmitters. Various detection methods such as a visible ultraviolet absorption method, a fluorescence method, a mass spectrometry method, and a Raman spectroscopy method are used as the detection method.
For general information on these capillary electrophoresis, see, for example, "Bunseki", No. 4, pp. 281-289 (1994) (Bun
seki, No.8, pp.281-289 (1994)), "Capillary Electrophoresis / Basic and Actual", Susumu Honda / Shigeru Terabe, Kodansha Scientific (1995).

As described above, the capillary electrophoresis method is in principle suitable for minute amount analysis, but has a problem with the detector. For example, the visible-ultraviolet absorption method, which is widely used at present as a detector for capillary electrophoresis, has a wide range of application, but it is pointed out that the detection sensitivity is insufficient due to the short optical path length. On the other hand, it has been attempted to improve the detection sensitivity by using the laser excitation fluorescence method.
(For example, "Chemistry and Industry", Vol. 47, pp. 1551 to 1554 (1994
(Kagaku To Kogyou, 47, pp.1551-1554)) requires a derivatizing agent that reacts with the target substance with high efficiency and has a large molar absorption coefficient at the laser oscillation wavelength. It is not easy, and there is a problem that the derivatization reduces the separation efficiency. In addition, the procedure becomes complicated, and there is a concern that noise may be generated due to mixing of impurities and that reproducibility may be deteriorated due to variations in reaction time and temperature. On the other hand, "Analytical Chemistry" magazine, 61st for electrochemical detection
Volume, 292A-303A (1989) (Analytical Chemistry, 61 ,
As described in pp.292A- 303A ), a highly sensitive detection is realized by a method called an end-capillary type in which a detector is placed near the outlet of a capillary column. This method enabled the detection of tens of amol (10 ^-18 mol) of catecholamines and neurotransmitters such as serotonin without derivatization. However, there are problems that the sample that can be detected is limited to the chemical species that react with the electrode and that the identification ability of the chemical species is lower than that of the ultraviolet absorption method. When using mass spectrometry as a detector for capillary electrophoresis,
Since it requires a large absolute amount of sample, it is not suitable for minute amount analysis and has a drawback that the device becomes bulky.

On the other hand, a vibrational spectroscopic detector has also been devised in order to further improve the separation efficiency of capillary electrophoresis. One of them is Raman spectroscopy. Raman spectroscopy is an analytical method for identifying and quantifying chemical species by utilizing the phenomenon that when incident light hits a molecule, it is modulated into light that reflects the energy state peculiar to the molecule. Since Raman spectroscopy can obtain the structural information of the molecule, it has an excellent ability to identify chemical species as compared with UV-visible absorption and fluorescence spectroscopy. Detectors using these Raman spectroscopy are disclosed, for example, in "Applied Spectroscopy", Vol. 42, pp. 515-518 (1988) (Ap.
plied Spectroscopy, 42, pp.515-518 (1988)). However, the sensitivity of Raman spectroscopy is inherently low, making it unsuitable for the analysis of very small sample volumes. However, the US patent US005306403A (Apr.26 , 1994)
Among them, Vo-Dinh et al. Attempt to detect Raman with high sensitivity in electrophoresis. Among them, in order to overcome the generally low sensitivity of Raman spectroscopy, they devised to attach a silver substrate having a surface-enhanced Raman scattering (hereinafter abbreviated as SERS) effect to the inner wall of the column. There is. However, as described above, the gel electrophoresis method has an extremely large absolute amount of the sample as compared with the capillary electrophoresis method, and is not suitable for the analysis of a very small amount of sample. Moreover, their method does not fully utilize the SERS effect. That is, 1) Raman spectroscopy using the SERS effect is originally SE
This is one of the surface analysis methods used to detect samples in the immediate vicinity of the RS substrate, and when the SERS substrate is placed on a large column used for general electrophoresis, most of the sample is used for detection. Not detected, detection efficiency is very poor, 2)
By using the SERS substrate as an electrode that can control the electrode potential, it is possible to increase the SERS effect by changing the adsorption amount of the sample and the surface condition of the electrode, but this is not the case in on-capillary detection such as them. It is possible.

[0006] Throughout these SERS, "Surface", Vol. 30, 528-545 (1992) (Hyoumen, 30, pp. 5).
28-545 (1992)). Furthermore, since they use a single channel type as a detector for Raman spectroscopy, it is difficult to obtain a spectrum on-line in terms of measurement time, and the ability to identify chemical species is low. In addition, Morris et al. "Applied Spectroscopy"
Journal, Vol. 47, pp. 855-857 (1993) (Applied Spectrosco
py, 47, pp.855-857), we have prepared a micro SERS active electrode that can directly detect a sample without combining it with a separation method such as gel electrophoresis or capillary electrophoresis. They report that the detector uses a microscopic Raman spectroscope using an objective lens, so that there is almost no loss when the electrodes are made small. In addition, this method makes full use of the SERS effect, and seems to be effective for a small amount of sample. However, since it is not combined with a separation method such as electrophoresis, it is obvious that its identification is difficult in a biological sample in which a plurality of chemical species are mixed.

[0007]

The capillary electrophoresis apparatus can be an effective component separation method for minute amounts of samples such as proteins, peptides, DNAs (nucleic acids), and substances related to neurotransmission, but it has hitherto been a problem. The detection method of (1) has a problem in the range of applicable samples, simplicity, and the like. The purpose of the present invention is to
It is an object of the present invention to provide a high-sensitivity detector effective for detecting an extremely small amount of sample by solving the problems of the above-mentioned conventional techniques.

[0008]

In order to solve the problem] was to achieve the above purpose
Therefore, in the present invention, surface enhanced Raman scattering activity is induced.
And a Raman spectroscope,
The electrode potential of enhanced Raman scattering active microelectrode can be changed.
I will In this case, the surface-enhanced Raman scattering activity is very small.
The shape of the electrodes may be fiber-like. Also, the surface increase
Microelectrode and Raman spectroscope for inducing strong Raman scattering activity
And is used as a detector of the Raman spectroscope.
By using the multi-channel type, online
Make sure you get the koutor. That is, the above object can be solved by the following means. That is, Raman spectroscopy using the SERS effect is used as a detector for capillary electrophoresis. This method is one of the surface analysis methods and can detect a sample in the vicinity of the electrode with high sensitivity. In addition to being advantageous for measurement in an aqueous solution, measurement through a capillary is possible. Therefore, this effect can be utilized by thinning a very small amount of sample separated by capillary electrophoresis. In particular,
The components separated by capillary electrophoresis are allowed to flow in a thin layer around the fiber-shaped microelectrode.
Furthermore, in order to effectively use the SERS effect, a small SERS
The electrodes are arranged in an end-capillary type placed near the outlet of the capillary column.

Here, as the material of the minute SERS electrode,
Gold, silver, copper, as well as nickel, palladium, platinum, which are best known for obtaining SERS activity,
Metals such as titanium and mercury and semiconductors such as nickel oxide and titanium oxide can be used, but they are made into a fiber shape so that they can be inserted into a capillary. For materials that are difficult to form into fibers, carbon fibers can be coated with a material that can obtain SERS activity by a vacuum thin film forming technique such as sputtering. Further, the portion not inserted into the capillary is sealed in a glass tube or the like to facilitate handling and form a microelectrode. The SERS effect can be fully utilized by adding roughness of about 100 nm from the atomic order to the electrode surface, so 1) electrochemical dissolution or redox treatment is applied to microelectrodes. 2) Rub with an abrasive such as alumina, 3) use a milling device that introduces a gas such as argon into a vacuum etching device, 4) perform a treatment such as cutting a diffraction grating on the surface, or 5 ) SE is applied to the fibrous material that has been treated as in 1) to 4) by vacuum thin film forming technology such as sputtering.
A micro SERS active electrode is obtained by coating a material capable of obtaining RS activity.

On the other hand, the Raman spectroscopic measurement apparatus measures with a micro Raman spectroscopic apparatus using an objective lens or the like in order to increase the efficiency with respect to the microelectrode, or in a microscopic area through a thin optical fiber type probe. Excitation and detection are performed. Further, by providing the optical system with the total reflection arrangement, it is possible to obtain an enhancing effect without roughness (particularly referred to as surface plasmon polariton enhanced Raman scattering). Moreover, in order to enhance the ability to identify chemical species, a multi-channel detector is used as a detector for Raman spectroscopy, and it is possible to obtain spectra online.

Further, by arranging the fibrous probe in the capillary, the separated minute amount of the component is thinned, and Raman spectroscopy using the SERS effect can be effectively used as the surface analysis. . Further, the end-capillary type can control the electrode potential, change the adsorption amount of the sample and the surface condition of the electrode, and enhance the SERS effect. Therefore, the sensitivity can be greatly increased as compared with the method of Vo-Dinh et al. Described in the section of the related art. Furthermore, the excellent identification ability of chemical species, which is a characteristic feature of Raman spectroscopy, can further increase the separation efficiency of components.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The contents of the present invention will be specifically described below with reference to examples of the embodiments of the present invention. In addition,
The present invention is not limited to the examples of the embodiments below.

[0013]

Embodiment 1 A silver wire (made by Furuuchi Chemical) having an outer diameter of 50 μm and a length of 2 cm was spot-welded to a gold lead wire (made by Furuuchi Chemical) having an outer diameter of 0.5 mm and a length of 7 cm. Insert the wire with lead wire into a tapered soft glass capillary (inner diameter 0.5 mm, outer diameter 1 mm, taper length 1 cm, total length 5 cm) and set so that silver wire protrudes from the taper portion by about 1 cm. The taper portion was inserted into an electric furnace, the temperature was gradually raised (20 ° C./minute), and the temperature was maintained at 650 ° C. for 5 minutes to seal the taper portion. Next, the silver wire at the tip was cut while leaving 1 to 2 mm from the tip of the capillary, the tip on the side opposite to the tapered portion of the capillary was sealed with epoxy resin, and the gold lead wire was fixed. Next, attach the above-mentioned capillary, platinum wire, and silver / silver chloride reference electrode prepared separately to a micromanipulator (manufactured by Nippon Micronics) and dip it in a Petri dish containing 0.1 M potassium nitrate aqueous solution under a microscope. Then
The silver wire was connected as a working electrode and the platinum wire as a counter electrode to a potentiostat (manufactured by Fuso Seisakusho). A potential of +0.3 V was applied to the silver wire for 10 seconds with respect to the reference electrode to melt and sharpen the tip. As a result, a fine SERS active silver electrode with a tip diameter of about 10 μm and a length of about 500 μm was obtained.

Next, the silver electrode was set in the capillary electrophoresis apparatus shown in FIG. In the electrophoretic device, one end of the capillary 1 is immersed in the buffer bayer 2, and the other end is covered with a porous glass 4 on the outside of an uncoated capillary part having a crack 3 as shown in FIG. Both ends of the quality glass were covered with an epoxy resin 5, fixed on a slide glass 6, and further connected to a detector through a cell 7 which was immersed in an electrolytic solution and had a liquid junction between the running solution of the capillary and the electrolytic solution. . Capillary for electrophoresis 1
The fused silica (made by GL Sciences) having an inner diameter of 25 μm and a length of 50 cm was used. Further, the voltage for migration was applied between the buffer bayer 2 and the liquid junction cell 7 by a constant voltage current supply device (HCZ-30PN-M manufactured by Matsusada Precision) 8 as shown in FIG.

Here, the detector has the structure shown in FIG. 1, the end of the capillary 1 was connected to a glass cell containing a reference electrode 9, and the silver electrode was inserted into the capillary from the end of the capillary. On the other hand, a Raman spectroscopic light source 12 such as a laser is condensed on the silver electrode 11 by a condensing system (made by Renishaw) 13, and scattered light is condensed by a condensing system (made by Renishaw) 14, and the Raman spectroscopic multi It was measured with a channel detector (manufactured by Renishaw) 15. Sample injection is performed by placing the sample bayer next to the buffer bayer, immersing the end of the capillary, raising the sample bayer to a height of 10 cm for 30 seconds, immediately returning it to its original height, and returning the tip of the capillary to the buffer bayer. It was The injection amount at this time was about 5 nanoliters. As the running solution, a 50 mM phosphate buffer solution (pH 6.8) containing 50 mM sodium dodecyl sulfate was used.

As a sample, a mixed solution of 1 μM glycine and α-alanine was injected, and the voltage for electrophoresis was set to 10 kV and electrophoresis was performed. Potential of silver electrode is potentiostat
(Manufactured by Fuso Seisakusho Co., Ltd.) and controlled to 0 to -0.8 V. An Argon ion laser (NEC, 514.5 nm, 50 mW) was used as a light source for Raman spectroscopy. CO around 1380 to 1410 cm ^-1
_When the intensity of the ₂ ^- Raman band was measured continuously, glycine was used 5 minutes after the start of electrophoresis, and CO ₂ of α-alanine was detected 10 minutes later.
^- peak due to the Raman band was observed. The typical Raman spectra obtained respectively are shown in FIG. When the potential of the silver electrode is changed, the Raman intensity also changes, -0.6
The highest intensity was obtained at V. SER a similar experiment
Raman intensity change was below the lower limit of measurement, although the measurement was performed using a platinum electrode with no S activity.

[0017]

[Second Embodiment] The same microelectrode as in the first embodiment is used, and an experiment is performed under the same conditions using a mixed solution of several μM of α-alanine and β-alanine (mixing ratio is unknown) as a sample. I went. The Raman band to be measured is the first embodiment.
CO ₂ Raman band and CC near 840-860 cm ^-1
H ₃ where the intensity of the Raman bands were measured simultaneously continuously, CO ₂ by α- alanine and β- alanine both in running after 10 minutes ^- a peak due to Raman band was observed, C
The relative intensity of the -CH ₃ Raman band was about half that of α-alanine alone, which indicated that α-alanine and β-alanine were contained in the same amount. The typical Raman spectra obtained respectively are shown in FIG.

[0018]

[Third Embodiment] In the same manner as the first embodiment, a gold wire is used instead of the silver wire to fabricate a microelectrode having a sharpened tip, and in order to enhance the SERS effect, 0.1 M
+1.2 to -0.5 with respect to the reference electrode in the potassium chloride solution of
The potential was applied periodically up to V several times to obtain a fine SERS activated gold electrode. Using a mixed solution of 1 μM arginine, tryptophan, and tyrosine as a sample, an experiment was conducted under the same conditions as in the first embodiment, and as a light source for Raman spectroscopy,
A helium neon laser (NEC, 632.8 nm, 25 mW ) was used. 1380～1410Cm CO ₂ in the vicinity of ^{-1 -} where the intensity of the Raman bands were continuously measured, by arginine in running after 2 minutes with a tryptophan in 3 minutes, the peak with a tyrosine were observed respectively in 4 minutes.

[0019]

As described above, in the capillary electrophoresis apparatus, by using the microelectrode having SERS activity as the detector, the sensitivity can be remarkably increased as compared with the conventional method. Furthermore, since a spectrum can be obtained, a sample that cannot be separated by capillary electrophoresis can be separated spectroscopically, and the separation performance can be improved. The above is
It has high utility value not only in the field of analytical chemistry but also in fields such as neuroscience, basic medicine, and biochemistry.

[Brief description of drawings]

FIG. 1 is a schematic enlarged view of a detector using a minute SERS active electrode.

FIG. 2 is a device diagram of a detector for capillary electrophoresis.

FIG. 3 is an enlarged view of a liquid junction method portion in FIG.

FIG. 4 shows an example of Raman spectrum in the first embodiment.
(a: glycine, b: α-alanine).

FIG. 5 shows an example of Raman spectrum in the second embodiment.
(a: α-alanine, b: β-alanine).

[Explanation of symbols]

1 ... Capillary (for electrophoresis), 2 ... Buffer bayer, 3 ... Crack, 4 ... Porous glass, 5 ... Epoxy resin, 6 ... Slide glass, 7 ... Liquid junction cell, 8 ... Constant voltage current supply device, 9 ... Reference electrode, 10 ... Glass cell (for detection), 11
… Small SERS active electrode, 12… Raman spectroscopy light source, 13… Focusing system (for Raman spectroscopy excitation), 14… Focusing system (for Raman spectroscopy detection), 15… Raman spectroscopy multi-channel detector, 16…
Lead wire, 17 ... Capillary (for fixing electrodes), 18 ... Conductive epoxy resin.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI G01N 27/26 335E (72) Inventor Masao Morita 3-19-3 Nishishinjuku, Shinjuku-ku, Tokyo Nihon Telegraph and Telephone Corporation ( 56) References US Pat. No. 5306403 (US, A) CHAN-TUH CHEN, MIC HAEL D.M. MORRIS, Ramen Spectroscopic Detection System for Capillary Electrophoresis, APPLIED S SPECTROSCOPY, 1988, VOL. 42 NO. 3,515-518 (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 27/447 G01J 3/44 G01N 21/64 G01N 21/65 G01N 21/69 JISST file (JOIS)

Claims (3)

(57) [Claims] 1. A surface-enhanced llama comprising a microelectrode for inducing surface-enhanced Raman scattering activity and a Raman spectroscope.
So that the electrode potential of the scattering-active microelectrode can be changed
A detector for capillary electrophoresis characterized by the following. 2. The detector for capillary electrophoresis according to claim 1, wherein the surface-enhanced Raman scattering active microelectrode has a fiber shape. 3. A microelectrode that induces surface-enhanced Raman scattering activity.
It is characterized in that it is composed of a pole and a Raman spectroscope, and that a spectrum is obtained online by using a multichannel type as a detector of the Raman spectroscope .
Capillary electrophoresis detector.