JP2002207040A - Carrier aggregate to which organism-related matter is immobilized - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a chip capable of easily discriminate which type of organism-related matter on the chip causes a hybridization reaction regardless of the location of the organism-related matter arranged on the chip to which the organism-related matter is immobilized. SOLUTION: The organism-related matter is immobilized to carriers. The carriers are supported at supports. This aggregate is made of a plurality of the supports. The carrier aggregate to which the organism-related matter is immobilized is prepared by making the characteristics of the carriers and/or the supports different from one another according to the type of the organism- related matter immobilized to the carriers.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an aggregate comprising a plurality of carriers on which biological substances such as nucleic acids are immobilized. This aggregate is useful as a material for testing and detecting a specific biological substance.

[0002]

2. Description of the Related Art In recent years, a new analysis method or methodology called a DNA microarray method (DNA chip method) has been developed which enables the collective expression analysis of a large number of genes using the revealed base sequence information. I am collecting. D used to analyze expression of many genes at once
The manufacture of NA chips involves immobilizing nucleic acids, which are a kind of biological substance, on a substrate such as glass coated with a nylon sheet or polylysine, etc. [Scie
nce 270, 467-470 (1995)], or a method of directly solid-phase synthesizing various short-chain nucleic acids on a substrate such as silicon to form a single chip on which various nucleic acids are arranged [US Patent 5, Four
45,934, 5,774,305] and a carrier on which various kinds of nucleic acids are fixed by bundling fibers having different nucleic acids immobilized thereon to form a fiber array and slicing the array in a direction crossing the fiber axis. A method for forming a thin slice (chip) [JP-A-2000-24560, JP-A-2000-24561] and the like are disclosed.

[0003]

However, in a conventional chip on which a bio-related substance (nucleic acid) is immobilized, the hybridization reaction generated between the bio-related substance placed on the chip and the bio-related substance in the sample is not sufficient. In order to determine which type of bio-related substance on the chip has caused a reaction, a bio-related substance or a support such as a fiber on which the bio-related substance is immobilized is accurately placed on the chip, and It is performed by grasping the type and location of the bio-related substance immobilized on the chip and comparing it with the position where the reaction is occurring on the chip, so that the bio-related substance or the support where the bio-related substance is immobilized on the chip It is necessary to arrange the body at a predetermined position with high precision, and it has the disadvantage that the manufacturing process becomes complicated and the demand for high placement accuracy increases the chip manufacturing cost. It was. Therefore, the present invention provides a chip that can easily determine which type of biological substance on a chip has caused a hybridization reaction, regardless of the position of the biological substance placed on the chip. Aim.

[0004]

The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and as a result, it is extremely effective to change the characteristics of a carrier, a support, and the like for each type of biological substance. This led to the completion of the present invention.

That is, the present invention is an aggregate comprising a plurality of carriers on which a biological substance is immobilized, wherein the characteristics of the carrier are different for each type of the immobilized biological substance. Bio-related substance-immobilized carrier aggregate.
Furthermore, the present invention relates to an aggregate comprising a plurality of supports each of which supports a carrier on which a biomaterial is immobilized, wherein the properties of the carrier and / or the support are immobilized on the carrier. A biologically relevant substance-immobilized carrier aggregate, wherein the aggregate is different for each type. The carrier-immobilized carrier aggregate has, for example, a carrier and / or a support having properties such as shape, size, length, light absorption characteristics (brightness), light absorption wavelength characteristics (color), and magnetized state. And at least one of the charged states. Specifically, a magnetic substance is included or dispersed in a carrier and / or a support. Further, the above-mentioned biomaterial-immobilized carrier aggregate is, for example, one in which the aggregate is two-dimensionally arranged, one in which the carrier or the support is a sphere, one in which a solid or hollow rod is used, and the like. is there. The above-mentioned biological substance is, for example, a nucleic acid.

The bio-related substance-immobilized carrier aggregate of the present invention is, for example, arranged on a plane to form a bio-related substance chip which is an example of the bio-related substance immobilized carrier assembly.
When a hybridization reaction occurs between a biological substance in a sample and a biological substance immobilized on a biological substance immobilizing carrier on a biological substance chip, a portion of the biological substance in which the hybridization reaction has occurred By detecting the characteristics of the immobilized carrier, it is easy to understand what the bio-related substance that caused the reaction is,
There is no need to arrange the biomaterial-immobilized carrier at a predetermined position with high precision.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
In order to obtain a biomaterial-immobilized carrier aggregate having different characteristics for each type of immobilized biomaterial as in the present invention, 1) immobilization of different types of biomaterial on carriers having different characteristics. Become 2) Immobilize a bio-related substance on a carrier, and then change the characteristics of the carrier depending on the type of the bio-related substance. 3) Carriers having different types of biological substances immobilized thereon are supported on supports having different properties. 4) A support capable of immobilizing a biological substance is supported on a support having different characteristics, and then, for each support having different characteristics,
Different kinds of bio-related substances are immobilized on a carrier supported on the support. 5) A support on which a different bio-related substance is immobilized is supported on a support, and then the characteristics of the support are changed depending on the type of the bio-related substance immobilized on the support. 6) A support capable of immobilizing a bio-related substance is supported on a support, then different types of bio-related substances are immobilized on the support supported by the support, and finally, the characteristics of the support are immobilized on the support. It changes depending on the type of bio-related substance. Such a method is mentioned. When using a support that supports the carrier, it is also possible to change the characteristics of the carrier together, or to change the characteristics of the carrier alone.

[0008] In the present invention, the biologically relevant substances include deoxyribonucleic acid (DNA) and ribonucleic acid (RN).
A), nucleic acids such as peptide nucleic acids (PNA) and oxypeptide nucleic acids (OPNA), or amino acids, proteins, carbohydrates (eg, polysaccharides), lipids, and the like.

Examples of the constituent material of the carrier for immobilizing the bio-related substance of the present invention include polyamide resins such as nylon 6, nylon 66 and aromatic polyamide, polyethylene terephthalate, polybutylene terephthalate, polylactic acid and polyglycolic acid. Polyester resin, acrylic resin, polyolefin resin such as polyethylene and polypropylene, polyvinyl alcohol resin, polyvinylidene chloride resin, polyvinyl chloride resin, polyurethane resin, phenol resin, polyvinylidene fluoride and polytetrafluoroethylene Fluorine resin, polyalkylene paraoxybenzoate resin, polystyrene resin, diacetate, triacetate, chitin, chitosan, etc. as a raw material, protein called promix Materials, glass, carbon materials, cotton, flax, ramie, plant material such as jute, wool, animal materials such as silk, such as asbestos mineral materials, such as various metal materials and the like.

These materials may be used as they are in an untreated state, but if necessary, may be introduced with a reactive functional group, or may be subjected to plasma treatment, radiation such as γ-ray, electron beam or the like. Processing may be performed.

The form of these materials is not particularly limited as long as the biological substance can be immobilized, and solid or gel-like materials can be used.

The support of the present invention is not particularly required to be capable of directly immobilizing a bio-related substance, and a carrier capable of immobilizing a bio-related substance as described above or a carrier on which a bio-related substance is immobilized is not physically supported. Or can be supported by chemical action,
There are no particular restrictions on the constituent materials of the material, as long as it can impart characteristics for discriminating the type of the immobilized bio-related substance described below, without adversely affecting the bio-related substance, and there are no particular restrictions on the constituent materials thereof. , A metal material, and other known materials can be used.

In the present invention, the properties of the carrier and the support for specifying the type of the biological substance immobilized on the carrier are given by the shape, size, length, light absorption property (brightness), Light absorption wavelength characteristics (color), one of magnetized state and charged state,
This can be done by giving a difference in strength or the like.

For example, when the size of the carrier for immobilizing a bio-related substance is 10 kinds and the light wavelength absorption characteristics, that is, when the color changes by 10 kinds, 100 discriminations can be made. If the body is dispersedly mixed or included and its magnetic intensity changes by 10 types, 1000 types can be determined.

In the case of magnetic strength, magnetism is used for carrier recognition by forming a magnetization band (magnetic code region) with an electromagnet (magnetic head) after applying magnetic particle powder to a carrier immobilized with a biological substance, etc. It is also possible. As the type of magnet, it is possible to use an alnico-based magnet, a ferrite-based magnet, a rare earth metal-based magnet, a bonded magnet, and the like, and as the magnetic particle powder, a gamma-type iron oxide or a barium-ferrite-based magnet is preferable. Can be used. It is possible to assign addresses up to ²ⁿ by the magnetization of n bit cells in the magnetic code area.
For reading, similarly, magnetic information can be converted into current and taken out using a magnetic head.

In the carrier-immobilized carrier aggregate according to the present invention, the carrier-immobilized carriers on which different organism-related substances are immobilized have different characteristics, so that the aggregates are regularly arranged. If it is not necessary, for example, if it is arranged two-dimensionally, that is, without overlapping on a plane, it is possible to obtain a planar assembly (a biological substance chip) of the biological substance immobilized body.

In the case of obtaining such a planar aggregate of the biomaterial-immobilized carriers, if the shape of the biomaterial immobilized carriers is spherical, they can be easily rolled and moved by inclination or vibration. By putting a large number of spherical bio-related substance-immobilized carriers into a jig with a recess with a flat bottom and a width that allows them to be dispersed without overlapping vertically, by vibrating the jig in a horizontal state, It is possible to easily obtain a dispersed state in which the spherical biomaterial-immobilized carrier does not overlap on a plane, and when it is physically fixed on a sheet or the like, a planar aggregate of the biomaterial-immobilized carrier can be obtained. it can.

When the carrier for immobilizing a biological substance is formed as a solid or hollow rod, a plurality of carriers for immobilizing the biological substance are arranged in parallel, and the reaction-curing After filling and hardening the liquid and fixing the arrangement state, it is sliced thinly in the direction intersecting the arrangement axis, so that a large number of thin pieces of the bio-related substance-fixed carrier aggregate (bio-related substance chips) can be easily prepared. It can also be manufactured.

In particular, when a nucleic acid is used as a biologically-related substance, a DNA microarray (DNA chip) that enables simultaneous expression analysis of many genes can be obtained.

[0020]

The present invention will be described below in detail with reference to examples. 1 to 6 are schematic diagrams showing one embodiment of the present invention. Fig. 1 shows the absorption wavelength characteristics (color) of light in the visible light region, in which the external shape in which different types of biological substances are fixed is cylindrical.
Are different from each other, and 9 types of biologically relevant substance-immobilized carriers 11
FIG. 3 is a diagram illustrating an example of a biologically-related substance-immobilized carrier aggregate that is three-dimensionally arranged and fixed with a resin 10. FIG. 2 shows nine types of biological-related substance-immobilized carriers 12 each having a cylindrical outer shape on which different types of biomolecules are immobilized and having different cylindrical diameters.
FIG. 3 is a diagram showing an example of a biologically relevant substance-immobilized carrier aggregate that is two-dimensionally arranged in 3 × 3 and is fixed with a resin 10. FIG.
Means that 9 types of spherical bio-related substance-immobilized carriers 13 having different diameters on which different types of biomolecules are immobilized are 3 ×
3 is a diagram illustrating an example of a biologically-related substance-immobilized carrier aggregate that is three-dimensionally arranged and fixed on a sheet with a resin 10. FIG. FIG. 4 shows a three-dimensional three-dimensional three-dimensional three-dimensional bio-related substance-immobilized carrier 14 in which different types of biomolecules are immobilized, and nine types of spherical bio-related substance-immobilized carriers 14 having different light absorption wavelength characteristics (colors) in the visible light range. FIG. 3 is a diagram showing an example of a biomaterial-immobilized carrier aggregate arranged and fixed on a sheet with a resin 10. FIG. 5 shows that a carrier 15 on which different types of biological substances are immobilized is fixed to the hollow portion of a hollow cylindrical support 16, and the light absorption wavelength characteristic (color) of the support in the visible light region. , Nine types of biologically-related substance-immobilized carriers, which are different from each other,
It is a figure which shows the example of the bio-related substance fixed support | assembly fixed by (1). FIG. 6 shows a carrier 15 on which different types of bio-related substances are immobilized in a hollow portion of a hollow cylindrical support 17 and nine types of bio-related substance immobilized carriers having different diameters of the support. FIG. 2 is a view showing an example of a biologically relevant substance-immobilized carrier aggregate which is two-dimensionally arranged in 3 × 3 and is fixed with a resin 10.

Next, with respect to the example of the carrier-immobilized body-immobilized substance shown in FIG. 4, the organism-related substance will be described as a nucleic acid. However, the technical scope of the present invention is not limited by these examples.

[0022]

Example 1 As carriers for immobilizing nucleic acids, white, red,
Glass beads coated with polylysine having a diameter of about 500 μm and colored in dark blue, light blue, green, yellow, purple, gray, and black were used, and the glass beads were used for unmodified oligonucleotides (probes 1 to 9). After being immersed in a petri dish filled with an aqueous solution (nucleic acid concentration of 10 μg / ml) each day and night, the aqueous solution of the oligonucleotide was removed, dried in air, and baked at 60 ° C. for 1 hour to obtain a white color on which oligonucleotide 1 was immobilized. Nucleic acid-immobilized carriers having different colors from nine types of carriers from the glass bead carrier (S1) to the black glass bead carrier (S9) on which oligonucleotide 9 was immobilized were obtained. These carriers (S1)-
(S9) is dropped into an array jig 21 having nine mortar-shaped hollows 22 having a diameter of 700 μm and having a Teflon coating 20 formed on the surface as illustrated in FIG. A 100-μm-thick PET film 32 coated with a 50-μm-thick hot-melt epoxy adhesive 31 ° C. is applied so that the adhesive-coated surface is in contact with the carrier, and a hot plate heated to 60 ° C. is pressed from the back surface. The carrier was transferred to a PET film, and the 9 nucleic acid-immobilized carrier aggregates were held in a heating atmosphere at 60 ° C. for 4 hours to cure the adhesive, and the bio-related substance having the structure shown in FIG. A related substance-immobilized film was obtained.

[0023]

Example 2 As carriers for immobilizing nucleic acids, white, red,
A nylon solid porous yarn having a diameter of about 300 μm and having been subjected to a hydrophilic treatment and colored dark blue, light blue, green, yellow, purple, gray, and black was used. The nylon solid porous yarn was immersed in a petri dish filled with an aqueous solution of unmodified oligonucleotides (probes 1 to 9) (nucleic acid concentration: 10 μg / ml) for one day, and then the aqueous solution of the oligonucleotide was removed. After baking at 60 ° C. for 1 hour, oligonucleotide 9 was removed from white nylon porous solid thread carrier (P1) on which oligonucleotide 1 was immobilized.
The linear nucleic acid-immobilized carrier in which the colors of the nine types of carrier up to the black nylon porous solid yarn (P9) on which is immobilized was different was obtained. These P1 to P9 linear nucleic acid-immobilized carriers were
× 3 state, arranged, filled with two-liquid room temperature-curable fixing reaction-curable resin between the arrayed body, and cured in that state to obtain a linear nucleic acid-immobilized body array block, This block is sliced into a thickness of about 500 μm at right angles to the arrangement direction of the linear nucleic acid-immobilized carriers, and a slice of the nucleic acid-immobilized carrier aggregate in which the nucleic acid-immobilized carriers (P1) to (P9) are arranged on a plane is obtained. Obtained.

[0024]

Example 3 A nylon film having a width of 3 mm and a thickness of 100 μm coated with gamma-type iron oxide as a magnetic powder on one surface was applied to a magnetic head (electromagnet) having a gap of 100 μm at the tip of the core to direct current. The magnetic code regions in which six 200 μm bit cells were arranged at intervals of 4 mm were formed on a nylon film, and addresses 1 to 12 were magnetically applied. (N
With the number of bit cells, addresses up to ²ⁿ can be assigned to each magnetic code area. This film is cut at intervals of 4 mm corresponding to the magnetic code area, and nylon chips (M1) having magnetizations having different addresses on one side, respectively.
(M12) was obtained and used as a carrier for immobilizing nucleic acids. Each of the nylon chips (M1) to (M12) is immersed in a petri dish filled with an aqueous solution of unmodified oligonucleotides (probes 1 to 12) (nucleic acid concentration: 10 μg / ml) for one day, and then the aqueous solution of the oligonucleotide is removed. After drying in air, baking was performed at 60 ° C. for 1 hour, and oligonucleotide 1 was immobilized from the nylon chip (M1) immobilized on the non-magnetized surface to the nylon on which oligonucleotide 12 was immobilized. Chips of 12 kinds of carriers up to the chip (M12) were obtained. These (M1) to (M1
By arranging 2) on an arrangement jig having a rectangular recess as illustrated in FIG. 10 with the magnetized surface facing down, an aggregate of nylon chips each having a magnetic address was obtained. .

[0025]

According to the present invention, the characteristics of a biomaterial-immobilized carrier at a portion where a reaction has occurred can be detected for a hybridization reaction generated between a biomaterial in a biomaterial-immobilized carrier assembly and a biomaterial in a sample. By doing so, the type of bio-related substance that has reacted can be easily grasped, eliminating the need to place the bio-related substance at a predetermined position with high precision as in the past, and it can be used for batch expression analysis of bio-related substances. A possible biomaterial chip can be easily manufactured at low cost.

[0026]

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a two-dimensional biomaterial-immobilized carrier in which different types of biomaterials are fixed and have a cylindrical outer shape and different light absorption wavelength characteristics (colors) in the visible light range. 4 shows an example of a placed bio-related substance-immobilized carrier aggregate.

FIG. 2 is a biologically-related substance-immobilized carrier assembly in which different types of biomolecules are immobilized, and a biologically-related substance-immobilized carrier having a cylindrical outer shape and a different cylindrical diameter is two-dimensionally arranged. Here is an example.

FIG. 3 shows an example of a biomaterial-immobilized carrier aggregate in which spherical biomaterial-immobilized carriers having different diameters to which different types of biomolecules are immobilized are two-dimensionally arranged.

FIG. 4 is a diagram showing a bio-related substance having two-dimensionally arranged spherical bio-related substance-immobilized carriers having different absorption wavelength characteristics (colors) of light in the visible light range in which different types of biomolecules are immobilized. 1 shows an example of a substance-immobilized carrier assembly.

FIG. 5 is a diagram showing a carrier in which different types of biological substances are immobilized in a hollow portion of a hollow cylindrical support;
An example of a biomaterial-immobilized carrier aggregate in which biomaterial-immobilized carriers having different absorption wavelength characteristics (colors) of light in a visible light region of a support are two-dimensionally arranged.

FIG. 6 is a diagram showing a carrier on which different types of bio-related substances are immobilized in a hollow portion of a hollow cylindrical support, and a bio-related substance immobilized carrier having a different support diameter. An example of a two-dimensionally arranged bio-related substance-immobilized carrier aggregate is shown.

FIG. 7 shows an arrangement jig used for producing a biomaterial-immobilized carrier aggregate.

FIG. 8 shows that different bio-related substances are immobilized.
2 shows a biomaterial-immobilized film on which spherical carriers of different colors are arranged.

FIG. 9 is an example of a diagram showing a magnetic code area.

FIG. 10 shows an example of a biomaterial-immobilized carrier aggregate in which rectangular carriers having different magnetities, on which different types of biomaterials are immobilized, are arranged.

[0027]

[Explanation of symbols]

 10: Resin 11: Cylindrical carrier having different colors 12: Cylindrical carrier having different diameters 13: Spherical carrier having different diameters 14: Spherical carrier having different colors 15: Biomaterial-immobilized carrier 16: Different Hollow cylindrical support having color 17: Hollow cylindrical support having different diameters 20: Teflon (registered trademark) coating 21: Arrangement jig 22: Indentation 31: Epoxy adhesive 32: PET film 41: Rectangular carrier 42: rectangular carrier 43: resin

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 31/20 G01N 31/22 121D 31/22 121 33/543 501D 33/543 501 33/553 33/553 33/566 33/566 37/00 102 37/00 102 C12N 15/00 A F term (reference) 2G042 AA01 BD12 BD20 CA10 CB03 DA08 FA11 FB05 HA07 2G053 AB01 BA08 BC20 CA02 DB20 4B024 AA19 CA01 CA11 HA12 HA19 4B029 AA21 BB20 4B063 QA01 QQ42 QR32 QR55 QS32

Claims (12)

[Claims] 1. An immobilization of a bio-related substance, comprising an aggregate of a plurality of carriers on which a bio-related substance is immobilized, wherein characteristics of the carrier are different for each type of the immobilized bio-related substance. Carrier aggregate. 2. An aggregate comprising a plurality of supports on which a carrier on which a biological substance is immobilized is supported, wherein the properties of the carrier and / or the support are immobilized on the carrier. A biologically relevant substance-immobilized carrier aggregate, which is different for each type. 3. The carrier according to claim 1, wherein the carrier has at least one of shape, size, length, light absorption characteristics (brightness), light absorption wavelength characteristics (color), magnetized state, and charged state. Bio-related substance-immobilized carrier aggregate. 4. The characteristic of the carrier and / or the support is shape,
The biomaterial-immobilized carrier assembly according to claim 2, wherein the aggregate is at least one of a size, a length, a light absorption characteristic (brightness), a light absorption wavelength characteristic (color), a magnetized state, and a charged state. 5. The carrier-immobilized carrier aggregate according to claim 3, wherein the carrier contains or disperses a magnetic substance. 6. The carrier-immobilized carrier aggregate according to claim 4, wherein a magnetic material is included or dispersed in the carrier and / or the support. 7. The biologically relevant substance-immobilized carrier aggregate according to any one of claims 1 to 6, wherein the aggregate is two-dimensionally arranged. 8. The carrier-immobilized carrier aggregate according to claim 1, wherein the carrier is a sphere. 9. The carrier-immobilized carrier aggregate according to claim 2, wherein the support is a sphere. 10. The carrier-immobilized carrier assembly according to claim 1, wherein the carrier is a solid or hollow rod. 11. The carrier-immobilized carrier aggregate according to claim 2, wherein the support is a solid or hollow rod. 12. The method according to claim 1, wherein the biological substance is a nucleic acid.
12. The carrier-immobilized carrier aggregate according to any one of items 11 to 11.