JP2009139168A - Capillary unit and living body analyzer using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a capillary unit providing high detection sensitivity when the fluorescence of a sample is measured, and also to provide a living body analyzer using it. <P>SOLUTION: In the capillary unit having: a capillary tube to be filled with the sample; and a measuring part for measuring the sample in the capillary tube, the measuring part has an opening part opened at its one end and a mirror arranged so as to reflect the light emitted from the sample charged in the capillary tube within the measuring part to condense it. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a capillary electrophoresis apparatus, and more particularly, to a capillary unit used in a capillary electrophoresis apparatus and a biological analyzer using the same.

  A plate-type gel electrophoresis apparatus using fluorescence detection has been used as a method for analyzing the molecular weight of DNA or protein. However, in recent years, a capillary gel electrophoresis apparatus in which a capillary is filled with a gel or a fluid polymer and used as an analysis tube has been used.

FIG. 6 shows a conventional capillary electrophoresis apparatus. 61 is a flat plate, 62 is a capillary, 63 is a capillary unit, 64 is an optical unit, 65 is a light source, 66 is a light receiving unit, and 67 is a drive unit. A conventional capillary electrophoresis apparatus fills a capillary with an electrolyte and puts both ends into an electrolytic cell. If a sample is put into one end of the capillary and voltage is applied to both ends of the capillary, the sample moves toward the other end while being separated according to the mobility of each component constituting the sample. For this reason, the sample can be analyzed by installing an optical unit in the middle of the capillary tube and detecting the passage time of each component. Absorbance measurement is often used for this detection, and a capillary unit 63 in which a capillary is embedded in a flat plate is installed so as to be sandwiched by an optical U-shaped unit 64 (one end is a light receiving portion and the opposite side is a light source). Yes. Since the side of the capillary unit is masked with a material that does not transmit light, the sample can be analyzed by applying a voltage to the capillary. Before performing analysis, with the capillary filled with a buffer solution, the drive unit detects the transmitted light that has passed through the capillary by irradiating parallel light from the light source, while the drive unit detects the direction perpendicular to the optical axis from the light source. The optical unit is moved to a position where the intensity of the transmitted light is maximized, and the position of the light source and the center of the capillary are aligned (for example, see Patent Document 1).
JP-A-5-52806

  However, in the conventional configuration, when the fluorescence of the sample is measured, the fluorescence emitted from the sample in the capillary tube is emitted radially, so that all of the fluorescence cannot be received by the light receiving unit, and the detection sensitivity There was a problem that it was not possible to raise it.

 The present invention solves the above-described conventional problems, and an object of the present invention is to provide a capillary unit capable of realizing high detection sensitivity when measuring fluorescence of a sample, and a biological analyzer using the same.

  In order to solve the above-described conventional problems, a capillary unit of the present invention and a biological analyzer using the same are provided in a capillary unit having a capillary for filling a sample and a measurement unit for measuring the sample in the capillary. The measuring unit has an opening that is open at one end and has a mirror that is arranged to reflect and collect light emitted from a sample filled in a capillary tube in the measuring unit. It is.

  Furthermore, the capillary unit of the present invention and a biological analyzer using the same include a detection unit arranged to penetrate the measurement unit when the capillary unit according to claim 2 is mounted. It is what.

  According to the capillary unit of the present invention and the bioanalyzer using the same, the fluorescence emitted from the sample inside the capillary can be efficiently collected on the detection unit, so that the fluorescence of the weak sample can be detected with high sensitivity. Can do. Further, there is no need for a complicated alignment mechanism between the caliper tube T and the optical unit as in the conventional absorption measurement.

  Embodiments of a capillary unit and an optical system positioning method according to the present invention will be described below in detail with reference to the drawings.

(Embodiment 1)
FIG. 1 is a perspective view showing a configuration of a detection unit according to Embodiment 1 of the present invention. Fig.1 (a) shows the perspective view which shows the structure of the detection part of the capillary block in Embodiment 1 of this invention. The capillary block 11 is made of a resin material such as Polymethyl Methacrylate (hereinafter referred to as PMMA), and includes a first capillary 12, a detection window 13 for measurement, and a mirror block 16. The first capillary 12 is a glass tube having an outer diameter of 0.3 mm and an inner diameter of 0.1 mm, the surface of which is coated with polyimide, and is embedded in a through-hole having a diameter of 0.6 mm in the capillary block 11 using an adhesive. . A sample or buffer is injected into the first capillary 12.

  The size of the detection window 13 is 6 mm × 6 mm. The capillary block 11 is recessed in the shape of the detection window 13, and the first capillary 12 from which the coating has been removed is exposed to form a detection portion. The mirror block 16 is disposed inside the detection unit so as to surround substantially the half circumference of the first capillary 12. The material used for the mirror block 16 may be a metal material or a resin material that can reflect light generated from the sample. Al is suitable as the metal material. In addition, as shown in the figure, a concave mirror 15 and a positioning reference surface 14 are provided. The concave mirror 15 needs to be able to reflect the excitation light for obtaining the fluorescence of the sample inside the first capillary 12 and the fluorescence from the sample. Therefore, the concave mirror 15 is processed so that the surface of the metal material used for the mirror block 16 is polished and the surface becomes a mirror surface. Alternatively, when the mirror block 16 is made of a resin material, a metal material thin film such as Al may be formed on the surface of the concave mirror 15. The concave mirror 15 is a circular arc with a radius of 1 mm centered above the first capillary 12 and has a shape with a focal point 0.3 mm above the holding reference surface 111 on the fiber side. In this example, a cylindrical mirror was produced.

  FIG.1 (b) shows the perspective view which shows the structure of the detection part of the optical unit in Embodiment 1 of this invention. The optical unit 17 is arranged in a biological analyzer (not shown) so as to be connected to the measurement unit of the capillary block 11 through a detection window when the capillary unit of the present invention is attached to the biological analyzer.

  The optical unit 17 (6 mm × 6 mm) includes a light receiving optical fiber 18, a first pumping optical fiber 19, a second pumping optical fiber 110, a holding reference surface 111, and a guide 112. The light receiving optical fiber 18 (diameter 0.5 mm) is installed at the upper center of the optical unit 17 and receives the fluorescence directly incident from the sample and the fluorescence reflected by the concave mirror 15 and the excitation light incident thereon. At this time, utilizing the difference in wavelength between the fluorescence and the excitation light, the excitation light incident on the light receiving optical fiber 18 is removed by a band-pass filter (not shown) inside the apparatus.

  The first pumping optical fiber 19 (diameter 0.5 mm) and the second pumping optical fiber 110 (diameter 0.5 mm) are installed on both sides of the light receiving optical fiber 18. Is irradiated in the direction perpendicular to the holding reference plane 111. The first pumping optical fiber 19 and the second pumping optical fiber 110 have the same characteristics. Here, a configuration in which two pumping optical fibers are provided is shown, but if a high output fiber is used, the number of fibers may be one. Alternatively, three or more fibers may be used to obtain a sufficient amount of light.

  The holding reference surface 111 is installed on the upper surface of the optical unit 17 and functions as a stopper of the capillary block 11 by contacting the positioning reference surface 14. Note that the holding reference surface 111 may use a protrusion from the side surface of the optical unit. The guide 112 is installed on the upper surface of the optical unit 17 and positions the first capillary 12.

  FIG.1 (c) shows the perspective view which shows the structure of the guide of the optical unit in Embodiment 1 of this invention. In FIG. 1C, the guide 112 includes a chamfered portion 113 and a capillary holding portion 114. When the optical unit 17 and the capillary block 11 are combined by chamfering a part of the optical unit by chamfering an angle of 20 ° to 70 ° with respect to the holding reference surface 111, the chamfered portion 113 is the first capillary 12. Can be guided to the capillary holder 114. The capillary holding part 114 is installed so as to form a groove shape with respect to the holding reference surface 111, and the width of the groove is the same as the outer diameter (diameter 0.3 mm) of the first capillary 12. The capillary holding part 114 holds the first capillary 12 guided by the chamfered part 113 while the optical unit 17 and the capillary block 11 are combined.

  Next, the optical system and the capillary positioning method will be described with reference to FIGS. The first capillary 12 is a glass tube having an outer diameter of 0.3 mm and an inner diameter of 0.1 mm, the surface of which is coated with polyimide, and is embedded in a through-hole having a diameter of 0.6 mm in the capillary block 11 using an adhesive. Therefore, the assembly error between the first capillary 12 and the capillary block is ± 0.15 mm. In order to make the center of the first capillary 12 coincide with the focal point of the concave mirror block, the target position of the first capillary 12 is horizontal when the center of the through hole having a diameter of 0.6 of the capillary block is the origin. 0mm, vertical direction must be + 0.15mm. The dimensions of the guide 112 are determined so as to guide the first capillary 12 to this target position. Here, the horizontal direction is a direction parallel to the positioning reference plane 14. The vertical direction is a direction perpendicular to the horizontal direction and the longitudinal direction of the first capillary 12. 2 and 3, the horizontal direction and the vertical direction are indicated by arrows.

  Next, horizontal positioning will be described with reference to FIG. FIG. 2 shows a cross-sectional view of the capillary block and the detection unit of the optical unit in the first embodiment of the present invention observed from the longitudinal direction of the first capillary. FIG. 2A shows a sectional view before the combination of the capillary block and the optical unit. FIG. 2B shows a cross-sectional view after the combination of the capillary block and the optical unit.

When the capillary block 11 and the optical unit 17 are combined, the optical unit 17 is inserted into the detection window 13. At that time, the first capillary 12 is guided to the capillary holding portion 114 by the chamfered portion 113 and is held by the capillary holding portion 114, whereby the center of the through hole having a diameter of 0.6 of the capillary block is set as the origin and the horizontal position which is the target position. It can be positioned at a position of 0 mm in the direction. Thereafter, the positioning reference surface 14 of the capillary block 11 and the holding reference surface 111 of the optical unit 17 abut each other, thereby positioning the optical unit 17 and the mirror block 16 in the vertical direction.
Next, the positioning in the vertical direction will be described with reference to FIG. FIG. 3 is a cross-sectional view of the capillary block and the detection unit of the optical unit according to Embodiment 1 of the present invention observed from a direction perpendicular to the longitudinal direction of the first capillary. Fig.3 (a) shows sectional drawing before the combination of the capillary block and optical unit in Embodiment 1 of this invention. FIG. 3B shows a cross-sectional view after the combination of the capillary block and the optical unit in the first embodiment of the present invention.

  When the capillary block 11 and the optical unit 17 are combined, the guide 112 of the optical unit 17 moves the first capillary 12 in the vertical direction with the center of the through hole having the diameter 0.6 of the capillary block as the target position as the origin. Push it up to the 15mm position. The concave mirror 15 may reflect light having a wavelength other than the wavelengths of excitation light and fluorescence. The shape of the concave mirror 15 is a circular arc with a radius of 1 mm centered above the first capillary 12 and has a focal point of 0.3 mm closer to the fiber side than the holding reference surface 111. By installing the concave mirror 15, the sample in the first capillary 12 can be efficiently irradiated with excitation light, and the fluorescence from the sample can be efficiently collected on the light receiving optical fiber 110. The positioning reference surface 14 is provided below the first capillary 12 and functions as a stopper of the optical unit 17 by contacting the holding reference surface 111 of the optical unit 17.

  By combining the capillary block 11 and the optical unit 17 in this way, the first capillary 12 can be positioned, and therefore a capillary drive unit that does not require positioning and does not require correction time can be provided. .

  FIG. 4 shows the configuration of the capillary unit in the first embodiment of the present invention. FIG. 4A is a perspective view of the capillary unit according to Embodiment 1 of the present invention. The capillary unit 417 includes a first buffer solution holding block 41, a second buffer solution holding block 42, a capillary block 11, a sample injection block 43, and a gel injection block 44. When performing electrophoresis, the capillary unit 417 is set in the apparatus with these five blocks connected.

  In the first buffer solution holding block 41 and the second buffer solution holding block 42, a first injection port 45 (diameter 1 mm) and a second injection port 46 (diameter 1 mm) for injecting a buffer solution on the upper surface, respectively. ) As well as a first air vent hole 47 (diameter 1 mm) and a second air vent hole 48 (diameter 1 mm). Therefore, when the buffer solution is injected from the first injection port 45 and the second injection port 46, the air in the first buffer solution holding block 41 and the second buffer solution holding block 42 becomes the first air vent hole 47. And the second air vent hole 48 allows the injection without resistance.

  A sample injection port 411 (diameter 1 mm) and a fourth air vent hole 412 (diameter 1 mm) for injecting a sample are provided in the upper part of the sample injection block 43. Therefore, when the sample is injected from the sample injection port 411, the air in the sample injection block 43 passes through the fourth air vent hole 412, so that the sample can be injected without resistance.

  In the upper part of the gel injection block 44, a gel injection port 49 (diameter 1 mm) and a third air vent hole 410 (diameter 1 mm) for injecting the electrophoresis medium are provided. Therefore, when the electrophoresis medium is injected from the gel injection port 49, the air in the gel injection block 44 escapes from the third air vent hole 410, and can be injected without resistance.

  FIG. 4B shows a cross-sectional view of each block of the capillary unit according to Embodiment 1 of the present invention. In FIG. 4B, a first electrode 414 and a second electrode 415 are provided in the first buffer solution holding block 41 and the second buffer solution holding block 42 so as to be immersed in the buffer solution. ing. Part of the first electrode 414 and the second electrode 415 protrudes from the first buffer solution holding block 41 and the second buffer solution holding block 42, respectively. A second capillary 416 is embedded on the side of the second buffer holding block 42 that is joined to the sample injection block 43. A osmotic membrane 413 is installed between the first buffer solution holding block 41 and the gel injection block 44. Therefore, the buffer solution injected from the first inlet 45 is not mixed with the electrophoresis medium, and the electric field generated by the first electrode 414 in the first buffer solution holding block 41 is passed through the buffer solution and the electrophoresis medium. It can be generated in the capillary block 11. The left and right ends of the sample injection block 43 are connected by a through hole, and are connected in such a manner that both ends are sandwiched between the capillary block 11 and the second buffer solution holding block 42.

  FIG. 4 (c) shows the capillary unit after block connection in the first embodiment of the present invention. In FIG. 4C, a buffer unit 417 is obtained by connecting the buffer holding blocks 41 and 42 to the sample injection block 43 and the capillary block 11.

  FIG. 5 shows a configuration diagram of the capillary unit and the capillary unit holding part (apparatus side) in the first embodiment of the present invention. FIG. 5A shows a cross-sectional view of the capillary unit and device before assembly in the first embodiment of the present invention. FIG.5 (b) shows sectional drawing of the capillary unit and apparatus after the assembly in Embodiment 1 of this invention.

  As shown in FIG. 5B, the capillary unit 417 is held by the capillary unit holding unit 51. The capillary unit holder 51 is provided with a third electrode 52 and a fourth electrode 53. When the capillary unit 417 is held by the capillary unit holding unit 51, the first electrode 414 and the second electrode 415 are in contact with the third electrode 52 and the fourth electrode 53. The capillary unit holder 51 is provided with the optical unit 17. When the capillary unit 417 is held by the capillary unit holding part 51, the optical unit 17 is inserted into the detection window 13.

  Using the capillary unit shown in FIG. 5, a capillary block 11 in which no mirror block is installed (comparative example) and an installation (example) are prepared. The same sample as in the comparative example and the example was electrophoresed under the same conditions, and the fluorescence of the detected sample was measured. A 1 μM FITC-labeled sample is filled in the first capillary 12 as a sample, and then the excitation light from the fluorescence microscope is supplied through the first excitation optical fiber 41 and the second excitation optical fiber 42 to the first capillary 12. The FITC-labeled sample in the capillary 12 was irradiated. Thereafter, an image of the fluorescence emitted from the FITC-labeled sample incident on the light receiving optical fiber 18 was obtained with a fluorescence microscope. Based on the image, the fluorescence intensity was evaluated with a digital value of 8 bits (256 gradations). In addition, in order to measure background noise in Comparative Examples and Examples, similar experiments were performed using a buffer solution. A value obtained by dividing the signal (1 μM FITC-labeled sample) by noise (buffer) was defined as S / N. The results are shown in Table 1.

  From the experimental results of the comparative example and the example shown in Table 1, the fluorescence intensity from the sample is improved by about 2.7 times (62/24) by installing the mirror block. That is, it shows that the fluorescence detection sensitivity is increased by about 2.7 times. Further, when the S / N ratios of the example and the comparative example are compared, it can be seen that the example is improved by about 1.2 times compared to the comparative example. From the above results, it is shown that the fluorescence of the sample emitted from the periphery of the capillary tube in the detection unit can be detected more efficiently by using the concave mirror of the present invention.

  A capillary unit according to the present invention and a biological analyzer using the same have a high-sensitivity capillary unit with a simple configuration and easy operation by condensing light from a capillary with a mirror block, and capillary electrophoresis It is useful as a device.

(A) Perspective view showing the configuration of the detection unit of the capillary block (b) Perspective view showing the configuration of the detection unit of the optical unit (c) Perspective view showing the configuration of the guide of the optical unit Sectional drawing which observed the detection part of the capillary block and optical unit in Embodiment 1 of this invention from the longitudinal direction of the 1st capillary Sectional drawing which observed the detection part of the capillary block and optical unit in Embodiment 1 of this invention from the orthogonal | vertical direction with respect to the longitudinal direction of the 1st capillary (A) Perspective view of capillary unit in Embodiment 1 of the present invention (b) Cross-sectional view of each block of capillary unit in Embodiment 1 of the present invention (c) After block connection in Embodiment 1 of the present invention Cross section of capillary unit Configuration diagram of capillary unit and capillary unit holding unit (apparatus side) in Embodiment 1 of the present invention Perspective view of a conventional capillary electrophoresis apparatus

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Capillary block 12 1st capillary 13 Detection window 14 Positioning reference surface 15 Concave mirror 16 Mirror block 17 Optical unit 18 Optical fiber for light reception 19 First optical fiber for excitation 110 Second optical fiber for excitation 111 Holding reference surface 112 Guide 113 Chamfered portion 114 Capillary holding portion 41 First buffer solution holding block 42 Second buffer solution holding block 43 Sample injection block 44 Gel injection block 45 First injection port 46 Second injection port 47 First air vent hole 48 Second air vent 49 Gel inlet 410 Third air vent 411 Sample inlet 412 Fourth air vent 413 Osmosis membrane 414 First electrode 415 Second electrode 416 Second capillary 417 Capillary unit 51 Capillary unit Holding unit 52 3rd Electrode 53 fourth electrode 61 flat 62 capillary 63 capillaries unit 64 the optical unit 65 light source 66 light receiving unit 67 drive unit

Claims (9)

In a capillary unit having a capillary for filling a sample and a measurement unit for measuring the sample in the capillary,
The measurement unit is a capillary unit having an opening having one end opened and a mirror arranged to reflect and collect light emitted from a sample filled in a capillary tube in the measurement unit. The capillary unit according to claim 1, wherein the mirror is disposed on a mirror block, and the mirror block is disposed in the measurement unit. The capillary unit according to claim 1, wherein the condensing by the mirror is arranged so as to focus on the measurement unit. The capillary unit according to claim 1, wherein the mirror has a concave shape. The capillary unit according to claim 2, wherein the mirror block is provided with positioning reference surfaces at both ends of the mirror. A bioanalyzer comprising a detection unit arranged to penetrate the measurement unit when the capillary unit according to claim 2 is attached. The bioanalyzer according to claim 6, wherein the detection unit includes a holding reference surface for connecting to a positioning reference surface of the mirror block and a guide unit for holding the capillary tube. The bioanalyzer according to claim 7, wherein the detection unit is provided with a light source for applying excitation light to the sample in the capillary and a light receiving unit for detecting fluorescence emitted from the sample on the holding reference plane. The bioanalyzer according to claim 7, wherein the guide unit has a detection unit installed so that light emitted from the sample is reflected by the mirror and collected on the light receiving unit.

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