JP2006322905A - Interface chip-capillary connector and electrophoresis system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an interface chip-capillary connector capable of executing a precise measurement, without having to make the ability of separating a sample, and to provide an electrophoresis system that uses the connector. <P>SOLUTION: The interface chip-capillary connector is used for an apparatus, in which the sample in a capillary is irradiated with excitation light and/or probe light, in order to carry out a photothermal conversion spectroscopic analysis. At least a portion of the capillary, which is irradiated with the excitation light and/or the probe light, is fixed by a fixing member transparent to the excitation light and/or the probe light. A groove for embedding the capillary is formed on the fixing member, and a gap between the groove and the capillary is filled up with an adhesive agent. The adhesive agent has a refractive index equal to that of the portion of the capillary, being irradiated with the excitation light and/or the probe light, thereby enabling accurate analysis to be executed, without depending on the shape of the capillary. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an interface chip / capillary connector used for microanalysis and the like, and an electrophoresis system using the connector. In particular, the present invention relates to an electrophoresis system using photothermal conversion spectroscopy.

  Conventionally, photothermal conversion spectroscopy has been used as a method for analyzing a small amount of sample. In order to measure a small amount of sample, a highly sensitive detection method is indispensable, but by using a photothermal conversion spectroscopic analysis method using a thermal lens effect generated by light absorption of a sample in a liquid in a fine channel, Highly sensitive detection is possible (see, for example, Patent Document 1).

  In photothermal conversion spectroscopy, when a sample is focused and irradiated with light, the temperature of the solvent rises locally due to the thermal energy released due to the light absorption of the solute in the sample, and the refractive index changes. As a result, the analysis method uses the photothermal conversion effect that a thermal lens is formed.

  FIG. 7 is an explanatory diagram of the principle of the thermal lens. In FIG. 7, when a very small sample is condensed and irradiated with excitation light through an objective lens of a microscope, a photothermal conversion effect is induced. For many materials, the refractive index decreases with increasing temperature. Therefore, the sample irradiated with the excitation light is irradiated with light and the refractive index decreases more as the temperature rises closer to the center of light collection, and the refractive index decreases as the temperature rises closer to the periphery due to thermal diffusion. Is small. Optically, this refractive index distribution has the same effect as a concave lens, so this effect is called a thermal lens effect, and the magnitude of the effect, that is, the power of the concave lens is proportional to the light absorption of the sample. On the contrary, when the refractive index increases in proportion to the temperature, the same effect as the convex lens occurs.

  As described above, the photothermal conversion spectroscopic analysis method is suitable for detecting the concentration of a very small sample because it observes the diffusion of heat, that is, the refractive index change.

  For example, in a photothermal conversion spectroscopic analysis system using a plate-like member with a flow path, the plate-like member with a flow path is arranged below the objective lens of the microscope, and excitation light of a predetermined wavelength output from the excitation light source is The sample in the flow path for analysis of the plate member with flow path is condensed and irradiated by the objective lens of this microscope. A thermal lens is formed around the focused irradiation position of the focused irradiation.

  On the other hand, detection light (probe light) output from the detection light source and having a wavelength different from that of the excitation light is incident on the microscope, and is focused and irradiated on the thermal lens formed on the sample by the excitation light by the objective lens of the microscope. Diverges or condenses through the light. Light emitted from the sample after diverging or condensing becomes signal light, and the signal light is detected by a detector through only a condensing lens and a filter, or a filter. The intensity of the signal light detected by this detector corresponds to the thermal lens formed on the sample.

  As described above, in the above-described photothermal conversion spectroscopic analysis apparatus, the thermal lens is formed at the focused irradiation position (hereinafter referred to as “focal position”) of the excitation light, and the change in the refractive index of the formed thermal lens is: It is detected by detection light having a wavelength different from that of excitation light.

  In photothermal conversion spectroscopy, measurement is performed based on a slight change in the amount of light, so if an event that causes a change in the amount of light (for example, the sample position deviates from the focused irradiation position) occurs, Measurement is not possible. Therefore, it is important to accurately place the sample at the focused irradiation position.

  In addition, when the surface of the member having the flow path for flowing the sample is not flat with respect to the excitation light and the detection light, for example, in the case of a curved surface, refraction occurs at the curved surface portion as shown in FIG. Since some of the light does not enter the detector, the amount of light detected decreases, and accurate measurement cannot be performed. Therefore, in the photothermal conversion spectroscopic analysis system, the measurement is generally performed by flowing a sample through a fine channel provided in a plate-like member having a flat surface. In FIG. 9, the solid line indicates the optical path of the detection light when there is no thermal lens, and the broken line indicates the optical path of the detection light when the thermal lens is generated.

  A capillary electrophoresis system is known as an electrophoresis system for analyzing a very small amount of protein, nucleic acid, or the like. Since capillaries have a circular cross section, it has been difficult to use photothermal conversion spectroscopy as a detection part of an electrophoresis system. However, using an interface chip makes it easy to perform measurement using photothermal conversion spectroscopy. (See, for example, Patent Document 2)

  As shown in FIG. 8, the interface chip is a plate-like member provided with a flow path, and holes for inserting capillary end portions are processed at both ends of the plate-like member. In FIG. 8, since the capillary end and the flow path are provided inside the interface chip, they are indicated by broken lines. By inserting the end of the capillary into this hole and connecting it to the interface chip, the capillary channel and the fine channel provided in the plate member are communicated. The interface chip and the capillary end are fixed with an adhesive. By irradiating excitation light and detection light to the channel portion of the interface chip, the sample can be measured.

Japanese Patent Laid-Open No. 10-232210 JP 2002-296233 A

  However, the interface chip / capillary connector 3 has the following problems.

  When connecting the capillary and the interface chip, the positional accuracy of the hole processing of the interface chip insertion portion for communicating the flow paths with each other is required. This is because the inner diameter of a capillary generally used for capillary electrophoresis analysis is 100 μm or less, and in order to communicate with each other, it is necessary that at least a processing error is 100 μm or less. When the mutual flow path position is shifted due to the processing position error, the pressure loss at the connecting portion increases, the sample volume introduced into the capillary varies, and the analysis results vary.

  In addition, if the cross-sectional shape of the flow path of the capillary and the cross-sectional shape and size of the channel of the interface chip do not match at the connecting portion, the sample flow may be disturbed at the connecting portion, which may adversely affect the sample separation performance. However, it is very difficult to make the channel cross-sectional shape of the capillary and the channel cross-sectional shape of the interface chip exactly the same shape and size.

An object of the present invention is to provide an interface chip / capillary connector capable of measuring with high accuracy without degrading sample separation ability, and an electrophoresis system using the connector.

  In order to solve the above problems, an interface chip / capillary assembly according to the present invention is an interface chip / capillary used in an apparatus for analyzing a sample by irradiating a sample inside the capillary with excitation light and / or probe light. A capillary connected body, wherein at least a portion of the capillary that irradiates the excitation light and / or probe light is fixed by a fixing member that is transparent to the excitation light and / or probe light. A groove for embedding a capillary is formed in the fixing member. The fixing member may be composed of a plurality of fixing member pieces, and the capillaries may be fixed by at least two fixing member pieces having a portion having a thickness equal to the outer diameter of the capillary. The capillary and the fixing member are fixed by a filler, and the filler has a refractive index equal to the refractive index of the capillary portion irradiated with the excitation light and / or probe light. The interface chip / capillary assembly of the present invention is used for photothermal conversion spectroscopy.

Furthermore, the present invention is an electrophoresis system using the above-described interface chip / capillary assembly, wherein the sample inside the capillary is separated by electrophoresis.

  According to the present invention, since the capillary is embedded and fixed in the fixing member, the sample position is not shifted from the focused irradiation position of the excitation light and / or probe light, and accurate measurement can be performed. Further, since the sample flows only in the capillary, the separation performance of electrophoresis does not decrease, and the sample volume introduced into the capillary does not vary.

  According to the present invention, the capillary is embedded in the fixing member, and the gap between the capillary and the fixing member is filled with a filler having a refractive index equal to the refractive index of the capillary portion irradiated with excitation light and / or probe light. Therefore, the excitation light and the detection light are not refracted at the curved surface portion, and accurate measurement can be performed regardless of the capillary shape.

  According to the present invention, since the two sides of the capillary are sandwiched and fixed by two or more fixing member pieces having a portion having a thickness equal to the outer diameter of the capillary, the capillary can be fixed securely and accurate measurement can be performed. Can do.

According to the present invention, since the shape of the fixing member other than the capillary fixing portion is arbitrary, the interface chip / capillary assembly of the present invention can be applied to various electrophoresis systems.

  Hereinafter, the configuration of an interface chip / capillary assembly and an electrophoresis system according to an embodiment of the present invention will be described with reference to the drawings.

  The interface chip is configured by the structure illustrated in FIG. First, for example, a groove having a width of 400 μm and a depth of 400 μm is processed on the surface of a substrate such as transparent quartz glass to obtain a fixed member piece. The grooves may be processed by chemical or physical processing such as wet etching, dry etching, machining by a dicing saw, end milling, or the like.

  A capillary (cross-sectional shape is circular, diameter: 375 μm) is placed in the processed groove, an adhesive as a filler is filled in the gap of the groove, and a cover substrate such as quartz glass is placed on the top and bottom substrates. The capillaries are integrated (FIG. 2) to form an interface chip / capillary assembly.

  The fixing member, the capillary portion irradiated with the excitation light and / or the probe light, and the filler need to be a material transparent to the excitation light and / or the probe light used for sample measurement. Further, the refractive index of the fixing member, the capillary portion irradiated with the excitation light and / or the probe light, and the filler are substantially the same. In addition to the adhesive, an organic solvent such as matching oil or a liquid such as an aqueous solution may be used as the filler as long as it has the same refractive index as the fixing member and the capillary portion that irradiates excitation light and / or probe light. Is possible. As long as the material of the fixing member has the same refractive index as that of the capillary portion that irradiates the excitation light and / or the probe light, borosilicate glass or soda lime glass close to the refractive index of quartz glass other than quartz glass Glass materials such as aluminoborosilicate glass can also be used. Resin materials such as methyl methacrylate, polymethyl methacrylate, vinyl acetate resin, and acrylic resin can also be used. As shown in FIG. 3, the capillary peels the film at an arbitrary position in advance, and the film peeling part is arranged on the substrate. This film peeling part becomes a measurement site. Depending on the purpose of analysis, the material of the capillary may be other than glass.

  As shown in FIG. 4, semicircular grooves can be processed on the surfaces of two quartz glass substrates, and capillaries can be arranged in the grooves. When the capillaries and the groove shapes are substantially the same and are in close contact with each other, the surface of the member that allows the sample to flow with respect to the excitation light and the detection light becomes a flat surface, enabling high-precision measurement. Even in this case, the measurement accuracy becomes higher if a filler such as an adhesive is filled.

  As shown in FIG. 5, it is also possible to fix the capillary with a plurality of fixing member pieces. A capillary is placed on a glass substrate, the capillary is sandwiched between two glass substrates each having a portion having a thickness equal to the outer diameter of the capillary, and a cover glass substrate is further covered from above. The gap between the capillary and the fixing member piece may be filled with an adhesive and fixed, or the fixing member piece may be provided with a concave and convex fitting portion without using an adhesive, and fixed.

  There are two types of capillary tubes having a circular cross section and a square cross section. In the case of a square tube, the capillary tube is hardly affected by the refraction of excitation light and detection light on the surface. However, in the case of the photothermal conversion spectroscopic analysis method, the detected light amount changes greatly due to a slight deviation of the sample position from the focused irradiation position. Therefore, in order to ensure the position accuracy, it is necessary to use a fixing member. preferable.

  As shown in FIG. 6, the interface chip / capillary assembly shown in FIG. 1 is incorporated into an electrophoresis apparatus and an analysis apparatus to form an electrophoresis system. The analyzer is an apparatus using photothermal conversion spectroscopy.

  In addition to electrophoresis, the interface chip / capillary conjugate of the present invention can be effectively used in other separation methods for analysis of separated sample substances such as chromatography.

(Example)
Using the electrophoresis system shown in FIG. 6, separation and high-sensitivity detection of four types of amino acids (glycine, methionine, alanine, and proline) were performed. The interface chip / capillary connector shown in FIG. 1 was used. A fluid sample containing four kinds of amino acids was introduced into a capillary and separated by capillary electrophoresis. The amino acid was detected by photothermal conversion spectroscopy by irradiating excitation light and detection light to the part where the capillary film of the interface chip / capillary assembly was peeled off. Excitation light etc. were made into the following conditions.

Sample: glycine, methionine, alanine, proline (4x10-5M)
Excitation light: wavelength 532 nm, output 50 mW
Excitation light: wavelength 633 nm, output 10 mW
Capillary effective length: 40cm

  As a result of the measurement, a resolution of 100,000 theoretical plates of the target substance was obtained, and detection with high sensitivity was confirmed. As a result of repeating the measurement under the same conditions 20 times, the CV (variation coefficient) value was within 5%, and high reproducibility was obtained.

(Comparative example)
Amino acids were separated and detected in the same manner as in Example except that the interface chip / capillary conjugate shown in FIG. 8 was used. As a result of repeating the measurement under the same conditions 20 times, the CV (coefficient of variation) value was about 20%, and the reproducibility was poor.

FIG. 1 is an exploded perspective view showing a schematic configuration of an interface chip / capillary assembly according to an embodiment of the present invention. FIG. 2 is a perspective view showing a schematic configuration of the interface chip / capillary connector according to the embodiment of the present invention. FIG. 3 is a cross-sectional view showing a schematic configuration of the interface chip / capillary assembly according to the embodiment of the present invention. FIG. 4 is a cross-sectional view showing a schematic configuration of an interface chip / capillary assembly according to another embodiment of the present invention. FIG. 5 is an exploded perspective view showing a schematic configuration of an interface chip / capillary assembly according to still another embodiment of the present invention. FIG. 6 is a schematic diagram showing a schematic configuration of the electrophoresis system according to the embodiment of the present invention. FIG. 7 is an explanatory diagram of the principle of the thermal lens. FIG. 8 is a partial top view showing a conventional interface chip / capillary connector. FIG. 9 is an explanatory diagram of a thermal lens. (A) is a schematic diagram when the focal point is in the center of the capillary, and (B) is a schematic diagram when the focal point is shifted from the center of the capillary.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fixed member 1a, 1b, 1c Fixed member piece 2 Capillary 3 Filler 4 Fine flow path 5 Thermal lens 6 Objective lens 7 Photodiode

Claims (6)

An interface chip / capillary assembly used in an apparatus for analyzing the sample by irradiating the sample inside the capillary with excitation light and / or probe light,
An interface chip / capillary coupling body, wherein at least a portion of the capillary that irradiates the excitation light and / or probe light is fixed by a fixing member that is transparent to the excitation light and / or probe light.
The interface chip / capillary coupling body according to claim 1, wherein a groove for embedding a capillary is formed in the fixing member.
The said fixing member is comprised from the several fixing member piece, The said capillary is fixed by the at least 2 piece fixing member piece provided with the part which has a thickness equal to the outer diameter of the said capillary. 2. The interface chip / capillary assembly according to 1.
The capillary and the fixing member are fixed by a filler, and the filler has a refractive index equal to a refractive index of the capillary portion irradiated with the excitation light and / or probe light. Item 4. The interface chip / capillary assembly according to any one of Items 1 to 3.
5. The interface chip / capillary assembly according to claim 1, wherein the analysis is performed by photothermal conversion spectroscopy.
6. The electrophoresis system having an interface chip / capillary coupling body according to claim 1, wherein a sample in the capillary is separated by electrophoresis.

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