JP2005345186A - Capillary bead array, and method of detecting and measuring biopolymer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simplify a bead arraying method, and to enhance bead kind discrimination ability and sensitivity therefor, in a biopolymer detection technique using capillaries and beads represented by a capillary bead array. <P>SOLUTION: A bead restraint field 32 of a cavity portion expansion-widened in the capillary is provided in the each capillary 31 to stored the plurality of beads. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a technique for realizing simplification of a method for arranging beads and making it easy to identify the type of beads in a probe array technique and the like related to detection and measurement of biopolymers. It also includes improving the efficiency of reading after the reaction.

  In the detection, measurement and analysis techniques of biopolymers (all of which are hereinafter referred to as “detection”), various methods have been proposed, and many have already been commercialized and put into practical use. In such a case, a known probe biopolymer is immobilized on a substrate and a substrate, a sample biopolymer labeled with a fluorescent substance is reacted with the probe biopolymer immobilized on the substrate or the substrate, There is a technique for detecting a biopolymer by measuring the binding amount of a probe biopolymer and a sample biopolymer based on the fluorescence intensity derived from the substrate or a labeled fluorescent substance on the substrate. As a substrate or a substrate to be selected, it is roughly constituted by a flat plate and a bead, and attracts attention as a microarray technology and a bead utilization technology, respectively.

  First, a DNA microarray is exemplified as a plate using a flat plate as a substrate or a substrate. A DNA microarray is a probe in which probe DNAs are arranged and fixed as spots mainly on a surface-modified glass substrate. There are two main types of DNA microarrays: a method of synthesizing probe DNA on a substrate by applying semiconductor technology, and a method of arranging and immobilizing already synthesized oligo DNA or cDNA as droplets on a substrate. is there.

  Further, as a method using beads as a substrate or a substrate, there is a method using a flow cytometry technique. In this method, while the DNA microarray identifies the type of probe immobilized using position information, the type of probe is assigned by assigning an ID by using two or more fluorescent dyes for each bead. Is being identified.

  Microarray technology represented by DNA microarrays is widely used mainly for research purposes because it has a high degree of integration and a huge amount of information can be obtained by a single operation. However, it has been a problem that a high level of technology is required in the step of immobilizing the probe, the production cost per sheet is high, and the binding force to the substrate or the substrate is relatively weak in immobilizing the probe. On the other hand, in the bead technology, although advanced technology is required for bead ID conversion, it is excellent in that the bond between the probe and the substrate or the substrate is relatively strong, and the bead synthesis can be performed at a relatively low cost. Each has advantages and disadvantages. In the above example, methods using DNA were introduced, but techniques for replacing these with peptides, proteins, or cells are also progressing.

  Therefore, as disclosed in Patent Document 2 below, a capillary bead array technology has been devised as a form in which microarray technology and bead utilization technology are fused. The present technology has a great feature in that the step of immobilizing the probe on the substrate or the substrate and the step of arranging the probes on the substrate are separated. That is, the probe is immobilized on the beads in advance, and the beads are arranged in a groove or a capillary on the substrate. The type of probe can be determined from the position information of the beads, and the reaction is performed by passing the sample solution through a groove or a capillary. This not only solves many of the problems of microarray technology and bead utilization technology, but it can also be expected to improve the reactivity between the probe and the sample, and it can be built relatively easily for automation of equipment. Can be considered.

  In Patent Document 2, in order to confirm the position of the beads in a capillary bead array in which probe beads in which probes for detecting biomolecules are bound to capillaries formed on a soft resin or the like, for each fixed number of probe beads, A method is disclosed in which marker beads having a different color or size from probe beads are arranged. At this time, the probe may or may not be bound to the marker bead. Using the position of the marker bead as a marker, the order of the probe bead, that is, the type of the probe bound to the bead can be known. This method is effective when the position of the bead is visually confirmed mainly using an optical microscope or the like.

  On the other hand, in the capillary bead array technology, it is possible to apply a fluorescence detection method as a highly sensitive detection method of biomolecules. When the fluorescence detection method is applied, a sample solution containing a fluorescently labeled target biomolecule is introduced into the capillary. The biomolecule is captured by a probe bead to which a probe having complementarity with the biomolecule is bound. Only the probe beads that capture the fluorescently labeled target biomolecule emit fluorescence. Therefore, when fluorescence detection of capillary probe beads is performed with a fluorescence reader, only beads that have captured fluorescently labeled biomolecules are detected. Since probe beads that do not emit fluorescence are not detected, it is very difficult to accurately know the position and number of these probe beads. This means that the order of the probe beads that emit fluorescence, that is, the type of the probe bound to the probe beads cannot be accurately known.

  As described above, when the fluorescence detection method is applied to the capillary bead array, it is very difficult to confirm the order of the probe beads, that is, the type of the probe, only by the bead position confirmation method disclosed in Patent Document 2. It was. Therefore, there is a need for a method that can accurately identify the type of probe in a probe bead array using a fluorescence reader.

  In addition, when a sample solution is introduced into a capillary bead array, the concentration of the sample solution decreases rapidly as the reaction with the beads progresses, making it impossible to sufficiently react with all the tens to hundreds of beads. There was a problem. Although this problem can be solved by using a large amount of sample solution, reducing the number of samples is an economically demanded problem.

JP-A-11-243997 JP 2000-346842 A

  Capillary bead arrays are characterized by good reactivity between the sample solution and the beads, and are suitable for full automation by performing fluorescence measurement, but when beads are arranged on a linear capillary, the beads are ordered. It was difficult to arrange well and it was also difficult to identify the type of beads.

  An object of the present invention is to simplify the work of arranging beads in a capillary and improve the ability to identify beads in a capillary array technology in which microarray technology and bead utilization technology are fused.

  This invention discovered that the said subject was solved by providing the confinement field which stores a bead in a capillary, and came to this invention.

  That is, first, the present invention is an invention of a capillary bead array, and is provided with a bead restraining field in the middle of a capillary, which is a widened cavity portion in the capillary and can store a plurality of beads. Features. As a result, a certain amount of beads can be uniformly reacted with a small amount of sample solution without any special arrangement work. The planar shape of the bead restraining field is not particularly limited, and examples thereof include a disk shape, an ellipse shape, a diamond shape, a rectangular shape, and a polygonal shape. It is also preferable to intentionally provide baffle plates and protrusions in the bead restraining field to vortex the sample solution and promote the reaction with the beads.

  Here, the bead-restraining field serves as both a reaction field and a fluorescence measurement field. After the reaction, an operation such as washing is performed as desired, and then excitation light is irradiated to measure the fluorescence emitted by the beads. Can do.

  The bead restraining field in the capillary is preferably arranged with the IN side of the sample solution inclined downward from the OUT side.

  The number of capillaries constituting the capillary bead array need not be one, and it is preferable that there are two or more capillaries in order to react a large amount of sample solution or to see many kinds of reactions. When a plurality of capillaries are arranged concentrically, a compact capillary bead array can be produced.

  Second, the present invention is a biopolymer detection and measurement method using the above-described capillary bead array. A small amount of sample solution can be uniformly reacted with a large number of beads by putting the sample solution from the IN side of the bead restraint field and taking it out from the OUT side of the bead restraint field.

  The biopolymer detection and measurement means is not particularly limited, but a flow cytometer using fluorescence is preferably used. That is, the biopolymer is detected and measured by irradiating the beads with excitation light and measuring the fluorescence emitted by the beads.

  Here, it is preferable that only the fluorescence can be measured and the S / N ratio can be improved by making the optical axis of the excitation light irradiated to the beads not coincide with the optical axis of the fluorescence to be measured. In particular, the fluorescence intensity per unit reading area can be improved by tilting the optical axis of the fluorescence to be measured with respect to the restraining scene of the capillary bead array.

  Thirdly, the present invention is an invention of a biopolymer detection and measurement apparatus, comprising means for placing the capillary bead array, a pouring / extracting means for introducing / extracting a sample solution, and beads in a bead restraining field. Irradiation means for irradiating excitation light and means for measuring fluorescence emitted from the beads.

  According to the present invention, the work of arranging beads in the capillary can be simplified or omitted. Further, a certain amount of beads can be uniformly reacted with a small amount of sample solution. Furthermore, the discrimination ability was also improved by limiting the discrimination range of a large number of beads. Furthermore, by tilting the fluorescence reading surface, the speed and sensitivity related to detection and measurement could be improved.

  As shown in FIG. 1, the conventional capillary bead array is one in which a sample solution is allowed to flow through a linear capillary and reacts with the surface of the beads arranged in the capillary. In this method, it was difficult to arrange beads in a capillary in order. Furthermore, even when there is an error in the bead insertion order, it is difficult to discriminate it, and it has been strongly desired to solve these problems in the practical application of the industry. Based on such a background, the form in the case of implementing this invention is concretely demonstrated with reference to drawings below.

  FIG. 2 shows a capillary bead array of Example 1 of the present invention. A specific implementation method will be illustrated with reference to FIG. The sample solution is sent by placing a peristaltic pump on the IN side. At this time, the OUT side of the bead restraining field is arranged vertically above the IN side of the bead restraining field, thereby preventing bubbles from entering the capillary and the restraining field. The sample solution is fed from the IN side toward the OUT side, flows through the capillary from the IN side, fills the bead restraining field with the solution, and then is discharged from the OUT side.

  FIG. 3 shows a capillary bead array of Example 2 of the present invention. A specific implementation method will be described with reference to FIG. FIG. 4 is a cross-sectional view taken along line A-A ′ in FIG. 3. The sample solution is sent by placing a peristaltic pump on the IN side. As shown in FIG. 4, a gradient is provided between the IN side and the bead restraining field to prevent mixing of bubbles and the like. The sample solution is fed from the IN side toward the OUT side, flows through the capillary from the IN side, fills the restraint field with the solution, and then is discharged from the OUT side.

  In any of the above cases, the beads may be identified for each region. That is, when dyed beads are used, it is only necessary to prepare a dye color number equal to or greater than the number of types of beads existing in the region, thereby simplifying the identification work. Further, by irradiating the excitation light source from the upper vertical side and reading from the same direction or the direction perpendicular to the reading surface, the background signal can be reduced and the S / N ratio can be improved, and the detection and measurement sensitivity can be improved. be able to. Further, in FIG. 3, the constraint fields are arranged on the same circle center, and reading time can be shortened by performing reading by rotation about the same circle center.

  FIG. 5 shows the relationship between the optical axis of excitation light and the optical axis of fluorescence. Here, by making the optical axis of the excitation light irradiated to the beads not coincide with the optical axis of the fluorescence to be measured, only the fluorescence can be measured, and the S / N ratio can be improved. In particular, by tilting the optical axis of the fluorescence to be measured with respect to the constrained scene of the capillary bead array, it is possible to improve the luminous flux density by measuring the fluorescence emission of the area of S originally by S ′, which is a smaller area. Can be expected to improve sensitivity.

  According to the present invention, in a biopolymer detection technique using capillaries and beads typified by a probe array, the manufacturing cost can be reduced, and the time for detection and measurement can be shortened and the sensitivity can be improved. Thereby, a bead array using capillaries typified by a probe array can be provided to the market as a versatile device with low cost and high detection and measurement speed.

It is the schematic which shows the structure of the conventional bead array. 1 is a schematic diagram of a structure in Example 1. FIG. 6 is a schematic diagram of a structure in Example 2. FIG. 6 is a schematic cross-sectional view of a structure in Example 2. FIG. The relationship between the optical axis of excitation light and the optical axis of fluorescence in a restraint field is shown.

Explanation of symbols

11 ... capillary, 12 ... bead, 21 ... capillary, 22 ... restraint field, 23 ... bead, 31 ... capillary, 32 ... restraint field, 33 ... bead, 51 ... capillary, 52 ... restraint field, 53 ... beads.

Claims (10)

  A capillary bead array used for detection and measurement of biopolymers, wherein a bead restraining field is provided in the middle of the capillary, which is a widened cavity portion in the capillary and can store a plurality of beads. Characteristic capillary bead array.   2. The capillary bead array according to claim 1, wherein the bead restraining field in the capillary is arranged with the IN side of the sample solution inclined downward from the OUT side.   The capillary bead array according to claim 1 or 2, wherein the beads are stored in a single particle layer so as not to overlap each other in the bead restraining field.   The capillary bead array according to any one of claims 1 to 3, wherein the number of capillaries is two or more.   The capillary bead array according to any one of claims 1 to 4, wherein a plurality of capillaries are arranged concentrically.   A method for detecting and measuring a biopolymer, wherein the capillary bead array according to any one of claims 1 to 5 is used, and a sample solution is put in from the IN side of the bead restraint field and is taken out from the OUT side of the bead restraint field. .   The method for detecting and measuring a biopolymer according to claim 6, wherein the beads are irradiated with excitation light, and fluorescence emitted from the beads is measured.   8. The method for detecting and measuring a biopolymer according to claim 7, wherein the optical axis of excitation light applied to the beads does not coincide with the optical axis of fluorescence to be measured.   The method for detecting and measuring a biopolymer according to claim 7 or 8, wherein the optical axis of fluorescence to be measured is inclined with respect to the surface of the bead restraining field of the capillary bead array.   A means for placing the capillary bead array according to any one of claims 1 to 5, a pouring means for introducing / extracting a sample solution, an irradiation means for irradiating the beads in the bead binding field with excitation light, and a bead A biopolymer detection and measurement apparatus having means for measuring emitted fluorescence.

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