JP2002357605A - Fiber alignment body containing selective binding substance immobilized fiber and fiber bundle of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fiber alignment body containing a selective binding substance immobilized fiber and a fiber bundle of the same. SOLUTION: This fiber alignment body contains the selective binding substance immobilized fiber and the fiber bundle of the same. The selective binding substance immobilized fiber can be highly efficiently and reproducibly provided and the fibers are assembled into the fiber alignment body so that the selective binding substance immobilized fiber alignment body with the selective binding substances aligned arbitrarily highly densely and precisely can be highly reproducibly and efficiently provided. The use of optical fibers can facilitate the detection of the efficiently bound specimen through the fibers. The attachment of marks to the respective fibers allows the identification of each fiber bound with the specimen irrelevant to the position in the alignment body.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a fiber on which a substance that selectively binds to a test substance (herein, a “selective binding substance”) is immobilized, and a fiber array comprising a bundle of the fiber.

[0002]

2. Description of the Related Art Research on the analysis of genetic information of various organisms has begun. Many genes, including human genes, their base sequences, the proteins encoded by the gene sequences, and the secondary production of these proteins. Information on the sugar chains to be obtained is being revealed rapidly. The functions of macromolecules such as genes, proteins, and sugar chains whose sequences have been revealed can be examined by various methods. The main ones are Northern hybridization for nucleic acids,
Alternatively, the relationship between various genes and their biological function expression can be examined by utilizing the complementarity between various nucleic acids / nucleic acids such as Southern hybridization. Regarding proteins, the functions and expression of proteins can be examined using a protein-protein reaction, as typified by Western hybridization.

In recent years, a DNA microarray method (DNA chip method) has been used as a technique for analyzing the expression of many genes at once.
A new analytical method or methodology, called, has been developed and is attracting attention. These methods are in principle the same as the conventional methods in that they are nucleic acid detection and quantification methods based on a nucleic acid / nucleic acid hybridization reaction. It can also be applied to the detection and quantification of proteins and sugar chains based on chain / protein hybridization. These techniques employ a large number of D-types on a planar substrate piece called a microarray or chip.
A major feature is that those in which NA fragments, proteins, and sugar chains are aligned and fixed at high density are used. As a specific use of the microarray method, for example, a sample in which the expressed gene or the like of a research target cell is labeled with a fluorescent dye or the like is hybridized on a piece of a flat substrate, and nucleic acids (DNA or RNA) complementary to each other are bound to each other. A method of labeling the portion with a fluorescent dye or the like and then reading the portion at high speed with a high-resolution analyzer, or a method of detecting a response such as a current value based on an electrochemical reaction. Thus, the amount of each gene in the sample can be quickly estimated.

[0004] Techniques for immobilizing nucleic acids on a substrate include, as in the Northern method described above, a method of immobilizing nucleic acids on a nylon sheet or the like at a high density, and a technique of immobilizing a nucleic acid on a substrate such as glass to further increase the density. A method of coating and immobilizing poly-L-lysine, aminosilane or the like on the surface has been developed.

However, the method of spotting and immobilizing nucleic acids on a substrate, such as glass, on which a solid surface is chemically or physically modified, is difficult to reproduce because the spot density and the amount of nucleic acids that can be immobilized per spot are small. The point is pointed out. Further, it is difficult to greatly reduce the cost per chip due to the expensive manufacturing apparatus and the multi-stage manufacturing process. In addition, there is a point that the nucleic acid needs to be relied on the spotted position on the substrate, which is inconvenient in terms of arrangement and distinction on the substrate.

[0006] Microarrays using proteins and sugar chains have the same problems as microarrays using these nucleic acids.

As a method not using a flat substrate, a method using minute beads is known. However, in terms of a technique for distinguishing various kinds of minute beads in which individual nucleic acids, proteins, sugar chains and the like are immobilized, It is incomplete, and it is difficult to produce a compound in which the specified compounds are aligned with the specified sequence criteria with good reproducibility.

On the other hand, attempts have been made to immobilize nucleic acids in hollow fibers (JP-A-2000-245461). In this technology, individual nucleic acids are distinguished by fixing a certain kind of nucleic acid in one hollow fiber to create a correspondence with one nucleic acid and one fiber. But,
This technique also does not overcome the problem of the technique of spotting nucleic acids on a substrate in that, finally, hollow fibers are bundled and individual nucleic acids are recognized based on their positional relationship.

[0009]

Under these circumstances, the polymer can be immobilized at a predetermined concentration, can be arranged in a measurable form at a high density with good reproducibility, and furthermore, the individual polymer can be used. The establishment of a new systematic methodology that can be applied to inexpensive mass production that can be recognized without relying on the position sequence is strongly required for polymer analysis that is expected to increase in the future, and the present invention is to solve It is a task to do.

[0010] More specifically, the problem to be solved by the present invention is that a small amount of spotting or two-dimensional dispensing on a two-dimensional carrier such as a nylon sheet or a glass substrate is more difficult than a method for producing a polymer array. Individual samples can be distinguished and recognized without depending on the relationship, any arrangement can be assembled, the density can be freely changed according to the method of use, and the reaction between the easily combined sample and other samples can be easily detected It is an object of the present invention to establish a method for producing an array in which a selective binding substance is immobilized. Then, an object of the present invention is to provide a fiber array comprising a fiber on which a selective binding substance is immobilized and a bundle of the fiber.

[0011]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the selective binding substance alignment process and the immobilization process are performed on the same two-dimensional carrier. The method of immobilizing the polymer is modified as a one-dimensional structure on a fiber that can be distinguished by the support, magnetism, bar code, color, shape, etc. (on a single fiber) It has been found that by independently performing the above method, a fiber bundle capable of distinguishing the fibers to which the individual samples are bonded regardless of the position as the three-dimensional structure can be produced.

Further, by using a light-transmitting substrate or an electrically conductive substrate for the fiber, a fiber or a fiber array capable of directly detecting a substance reacted with a selective binding substance immobilized on the fiber is produced. The inventors have found what can be done, and have completed the present invention.

That is, the present invention provides a fiber array comprising a fiber having a selective binding substance immobilized thereon or a bundle of the fiber.

[0014]

DETAILED DESCRIPTION OF THE INVENTION In the present invention, synthetic fibers, semi-synthetic fibers, regenerated fibers, inorganic fibers such as inorganic fibers, natural fibers, Conjugate fibers and the like.

As typical examples of synthetic fibers, nylon 6,
Nylon 66, various polyamide fibers such as aromatic polyamide, polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid and other polyester fibers, polyacrylonitrile and other acrylic fibers, polyethylene and polypropylene Various fibers of polyolefin, various fibers of polyvinyl alcohol, various fibers of polyvinylidene chloride, various fibers of polyvinyl chloride, various fibers of polyurethane, phenolic fiber, fluorine based on polyvinylidene fluoride, polytetrafluoroethylene, etc. Fibers and various kinds of polyalkylene paraoxybenzoate fibers are exemplified. Fibers other than those for clothing, for example, optical fibers mainly composed of a transparent amorphous polymer such as polymethyl methacrylate or polystyrene can also be used. In particular, a plastic optical fiber whose core is made of a material such as polymethyl methacrylate, polystyrene, or polycarbonate, and whose cladding is made of a material having a lower refractive index is preferable. It may be a so-called core-sheath type optical fiber or a refractive index distribution type optical fiber. Further, the plastic optical fiber may be coated. The coating material is not particularly limited, but polyethylene,
Thermoplastic resins such as PVC, urethane, and fluororesin, or various rubber tubes are used.

Representative examples of semi-synthetic fibers include various cellulose-based fibers based on diacetate, triacetate, chitin, chitosan, and the like, and various protein-based fibers called promix. Typical examples of the regenerated fiber include various cellulosic regenerated fibers (rayon, cupra, polynosic, etc.) obtained by a viscose method, a copper-ammonia method, or an organic solvent method.

Representative examples of inorganic fibers include glass fibers,
Examples thereof include carbon fibers, metal fibers such as Au, Ag, Cu, and Al. In particular, a light-transmitting glass optical fiber is preferable. As in the case of the plastic optical fiber, a so-called core-sheath type optical fiber or a gradient index type optical fiber may be used. Further, the optical fiber made of glass may be coated. Further, a composite optical fiber of glass and plastic may be used.

Representative examples of natural fibers include vegetable fibers such as cotton, flax, ramie, jute, animal fibers such as wool and silk, and mineral fibers such as asbestos. The form of the fiber used in the present invention is not particularly limited. Also,
It may be a monofilament or a multifilament. Further, a spun yarn obtained by spinning short fibers may be used. When multifilament or spun yarn fibers are used, voids between single fibers can be used for fixing the selective binding substance.

The fiber used in the present invention may be used as it is without any treatment, but may be a fiber into which a reactive functional group is introduced, if necessary.
Fibers that have been subjected to radiation treatment, such as wire or electron beam, may be used. When the selective binding substance is immobilized on these fibers, various chemical or physical interactions between the fiber and the selective binding substance, that is, the functional groups of the fiber and the selective binding substance Can be achieved by a known method utilizing a chemical or physical interaction between

When the same selective binding substance is immobilized, several real fibers can be treated at once. The immobilization position may be the entire fiber, but is preferably at the fiber end in order to reduce the amount of the sample. In order to distinguish between fibers having different selective binding substances immobilized thereon, the fibers having different samples immobilized thereon can be distinguished by immobilizing the fibers on a support. The supports may be individually independent or individually bonded.
Alternatively, fibers having a changed shape can be used.

The fibers can be colored, and the fibers of different samples can be distinguished by the wavelength of the color. A method of detecting light emitted to the outside of the fiber, or passing light into the fiber and guiding the light passing through the fiber to a photodetector may be used to distinguish the light.

The distinction may be made by using a fiber on which a special character symbol is recorded. For example, a barcode or the like may be recorded on the fiber or in the fiber, and the fiber may be read to distinguish fibers of different samples.

The fiber may contain a conductive material such as metal, carbon, or conductive polymer or a magnetic material, or the surface of the fiber may be coated with a material such as a metal by means of sputtering, vapor deposition, plating, CVD or the like. It can also be made conductive. The material included in the second to fourth periods of the 3A to 5B groups of the periodic table, or a mixture thereof, may be coated on the surface of the fiber using the above-described means. In this manner, fibers of different samples may be distinguished electrically or magnetically.

The fibers used in the present invention preferably have a small thickness. In a preferred embodiment of the present invention, the thickness of one fiber is preferably 1 mm or less. In the case of a monofilament, for example, a commercially available fishing line has a thickness of 50 to 900 μm. Furthermore, according to recent spinning technology, 1d
tex (for polyethylene terephthalate, a diameter of about 8
μm), and a finer fiber (ultrafine fiber or ultrafine fiber) can be produced (diameter: 1 to 10 μm). Also when an optical fiber is used, the filament is preferably 3 to 1000 μm. 5
More preferably, the thickness is about 0 to 1000 μm, and particularly, from the viewpoint of handleability and the like, a plastic optical fiber, a glass optical fiber, and a composite optical fiber of glass and resin having a thickness of about 50 to 500 μm are preferable. Also, a so-called image conduit in which very thin optical fibers of 2 to 50 μm are bundled can be preferably used. By immobilizing another selective binding substance on each of the image conduits and collecting the image conduits, the measurement can be performed. Further, when the coupling state is detected by light, by using this image conduit, the converged light does not diverge inside the optical fiber, and the detection light can be efficiently sent to the sample. The number of optical fibers contained in the image conduit is preferably 2 to 50,000. If the number is more than 50,000, the outer diameter of the image conduit becomes too large, which may cause difficulty in handling.

When an optical fiber is used among the above-mentioned fibers, a label that emits light or generates a dye, such as a fluorescent label, is used as a label to be added to a test substance, which will be described in detail later. The binding of the test substance can be measured by light passing through the optical fiber and coming out to the other end side, and in this case, it is preferable because it is possible to detect which fiber is shining. This is also advantageous for automation. When conductive fibers are used, the binding of the test substance can be measured as an electric signal at the other end of the fiber. This is preferable because it can be detected, and is advantageous for automation of the device. Further, when conductive fibers are used, the bonding can be promoted by means for applying an electric field or current. In particular, when the hybridization of nucleic acids is promoted, it is preferable that the fibers on which the nucleic acids are fixed are substantially used as anodes. The arrangement of the cathode is not particularly limited as long as it does not directly touch the fiber. The type of electric field to be applied is particularly preferably DC, but AC may be used as a condition that the fiber substantially becomes an anode. That is, even when an AC electric field is applied, the time for applying the positive voltage and the negative voltage of the fiber is
The method may be such that the modulus of the positive voltage is increased, or the absolute value of the AC positive voltage and the absolute value of the negative voltage is increased in the modulus of the positive voltage. The magnitude of the applied voltage is not particularly limited.
V or more and 5000 V or less are preferable. When the applied voltage is 0.
If it is less than 1 V, the effect of applying a voltage may not be sufficiently obtained, and if it is more than 5000 V, handling may be difficult. There is no particular limitation on whether a current flows when a voltage is applied. If the cathode is arranged so as not to touch the hybridization solution, no current flows, but if the cathode is arranged so as to touch the hybridization solution, current flows through the solution. When a current flows, the magnitude is preferably 1 to 2000 mA.

Here, the term “selective binding substance” means a substance which can selectively or directly bind to a test substance, and typical examples thereof include nucleic acids, proteins, saccharides and other substances. Antigenic compounds. Nucleic acid is DN
A or RNA may be used. Since a single-stranded nucleic acid having a specific base sequence selectively hybridizes and binds to a single-stranded nucleic acid having a base sequence complementary to the base sequence or a part thereof, the term "selective binding" as used in the present invention is used. Substance ".
In addition, examples of the protein include an antibody, an antigen-binding fragment of an antibody such as a Fab fragment and an F (ab ′) ₂ fragment, and various antigens. Antibodies and their antigen-binding fragments selectively bind to the corresponding antigen,
Since an antigen selectively binds to a corresponding antibody, it corresponds to a “selective binding substance”. The saccharide is preferably a polysaccharide, and includes various antigens. In addition, substances having antigenicity other than proteins and saccharides can be immobilized. Particularly preferred "selective binding substances" are nucleic acids, antibodies and antigens. The selective binding substance used in the present invention may be a commercially available substance or a substance obtained from living cells or the like.

The preparation of DNA or RNA from living cells involves
Known methods, such as DNA extraction, are described by Blin et al. (Blin et al., Nucleic Ac).
ids Res. 3: 2303 (1976)), and for extraction of RNA, see Fabaloro.
(Favaloro et al., Methods Enzymol. 65: 718)
(1980)). The nucleic acid to be immobilized further includes a linear or circular plasmid DNA.
Or a chromosomal DNA, a DNA fragment obtained by cleaving them with a restriction enzyme or chemically, a DNA synthesized by an enzyme or the like in a test tube, or a chemically synthesized oligonucleotide can also be used.

Normally, one kind of selective binding substance is immobilized on one fiber. For example, when it is desired to bind a plurality of kinds of genes having mutations to the same immobilized region, It is also possible to immobilize a plurality of types of selective binding substances on one fiber.

The selective binding substances immobilized on the plurality of fibers may be different types of selective binding substances or the same selective binding substances. Further, among the plurality of fibers, one type of selective binding substance may be immobilized on some of the fibers, and the other type of selective binding substance may be immobilized on another part of the plurality of fibers. it can. The type and order of the selective binding substances are not limited by the positions of the fibers in the fiber array. It is also effective to immobilize the same selective binding substance on a plurality of fibers to increase the measurement sensitivity.

The selective binding substance can be fixed on the support fiber by a known method. When the unmodified selective binding substance is fixed to the fiber, the fiber can be fixed by baking or ultraviolet irradiation after the selective binding substance and the fiber act. In an embodiment to be described later, DN
A is fixed to a polymethyl methacrylate optical fiber fiber. When immobilizing a selective binding substance modified with an amino group on a fiber, glutaraldehyde or 1-
Crosslinking agents such as ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) can be used to bind to the functional groups of the fibers. The temperature at which the sample containing the selective binding substance is allowed to act on the fiber is preferably 5 ° C to 95 ° C,
C. to 65 C. is more preferred. Processing time is usually 5 minutes to 24
Time, preferably 1 hour or more.

In the present invention, the selective binding substance may be immobilized on the fiber as it is, or a derivative obtained by chemically modifying the selective binding substance, or a nucleic acid modified as necessary may be immobilized. Good. Chemical modifications of nucleic acids include amination,
Biotinylation, digoxigenation and the like are known [Cur
rent Protocols In Molecular Biology, Ed .; Frederic
k M. Ausubel et al. (1990), De-isotope experiment protocol (1) DIG hybridization (Shujunsha)], and these modifications can be adopted in the present invention. As an example, introduction of an amino group into a nucleic acid will be described. The bonding position between the aliphatic hydrocarbon chain having an amino group and the single-stranded nucleic acid is not particularly limited.
(Phosphodiester binding site or base site). This single-stranded nucleic acid derivative can be prepared according to the method described in Japanese Patent Publication No. 3-74239, US Pat. No. 4,667,025, US Pat. No. 4,789,737 and the like. In addition to this method, for example, a commercially available reagent for introducing an amino group [for example, Amino Link II (trade name); PE Biosystems Japan, Amino Modifiers (trade name); It can be prepared according to a well-known method (Nucleic Acids Res., 11 (18), 6513- (1983)) for introducing an aliphatic hydrocarbon chain having an amino group into the 5 ′ terminal phosphoric acid.

After the selective binding substance is immobilized on the fiber with the selective binding substance obtained by the above-described method, an appropriate treatment can be performed. For example, by performing a heat treatment, an alkali treatment, a surfactant treatment, or the like, the fixed selective binding substance can be modified. Or,
When using a selective binding substance obtained from a biological material such as a cell or a bacterial body, unnecessary cell components and the like may be removed. Then, the fibers after the treatment can be used as a material for detecting the selective binding substance. Note that these processes may be performed separately or simultaneously. Further, it may be appropriately performed before fixing the sample containing the selective binding substance to the fiber.

The fiber of the present invention on which the selective binding substance is immobilized can be used to detect a specific analyte in the sample by interacting with the test substance using the immobilized selective binding substance as a probe. Can be. The two types of test samples can be labeled (as distinguished) as described below, and the differences can be compared.

For the detection of the corresponding selective binding substance in the test sample, which selectively binds to the selective binding substance, known means capable of specifically recognizing the binding can be used. For example, a label such as a fluorescent substance, a luminescent substance, or a radioisotope is bound to the corresponding selective binding substance in the sample, and after the selective binding reaction and washing, the label can be detected. There is no particular limitation on the type of the label and the method of introducing the label, and various conventionally known means can be used. In particular, in the case where the fiber has optical transparency, a sample labeled at the fiber end is reacted, and the other end is detected by a detector, it is preferable to use a fluorescent substance or a luminescent substance for the label. Fluorescent substances and luminescent substances used for immunoassays and measurement of nucleic acid hybridization are well known in the art, and various kinds are commercially available, so use these commercially available fluorescent substances and luminescent substances. be able to. Further, after or simultaneously with the binding reaction between the selective binding substance immobilized on the fiber and the corresponding selective binding substance in the test sample, the labeled binding substance is selectively bound to the corresponding selective binding substance. It is also possible by reacting the free measurement substance thus obtained, washing, and then measuring the label of the measurement substance bound to the fiber via the corresponding selective binding substance and the selective binding substance. For example, when a nucleic acid having a specific base sequence is immobilized on a fiber as a selective binding substance, and the corresponding selective binding substance is a nucleic acid containing a region complementary to the nucleic acid, the nucleic acid is a corresponding selective binding substance. Among them, a nucleic acid complementary to a region other than the region complementary to the selective binding substance can be labeled and used as a measurement substance. Further, when an antigen is immobilized on a fiber as a selective binding substance, and when the corresponding selective binding substance is an antibody that reacts with the antigen and the antibody, a labeled second antibody that reacts with the antigen and the antibody is used for measurement. Can be used as a substance.
Also in these cases, it is preferable to immobilize the selective binding substance on one end region of the fiber, use a fluorescent substance or a luminescent substance for the label, and measure the reaction result from the other end side.

When the fiber has electrical conductivity, it is preferable to use a method for detecting a response such as a current value based on an electrochemical reaction. In this case, the sample and the sample immobilized on the electrode fiber are allowed to react under a material that promotes and suppresses the reaction between the sample and the sample, and all or a part of the material is combined with the sample and the sample. The presence or absence of the binding between the sample and the analyte and the degree of the binding can be detected by measuring the value of the current flowing through the electrode, that is, the fiber after the reaction and contained therein.

The test substance to be subjected to the measurement method using the fiber or the fiber array of the present invention includes a nucleic acid to be measured,
Examples include, but are not limited to, genes such as pathogenic bacteria and viruses, genes causing genetic diseases and parts thereof, various biological components having antigenicity, antibodies against pathogenic bacteria and viruses, and the like. . Examples of samples containing these test substances include body fluids such as blood, serum, plasma, urine, stool, cerebrospinal fluid, saliva, various tissue fluids, various foods and drinks, and dilutions thereof. It is not limited to these. The nucleic acid to be a test substance may be a nucleic acid extracted from blood or cells by a conventional method, or may be a nucleic acid amplified by a nucleic acid amplification method such as PCR using the nucleic acid as a template. In the latter case, the measurement sensitivity can be greatly improved. When a nucleic acid amplification product is used as a test substance, the amplified nucleic acid can be labeled by performing amplification in the presence of a nucleoside triphosphate labeled with a fluorescent substance or the like.
When the test substance is an antigen or antibody, the test substance antigen or antibody may be directly labeled by an ordinary method, or the test substance antigen or antibody may be bound to the selective binding substance. After that, the fiber is washed, and a labeled antibody or antigen reacting with the antigen or antibody is reacted with the antigen or antibody to measure the label bound to the fiber.

The step of allowing the test substance to interact with the immobilized substance can be performed in exactly the same manner as in the prior art. The reaction temperature and time are appropriately selected depending on the chain length of the nucleic acid to be hybridized, the type of antigen and / or antibody involved in the immune reaction, and the like. In the case of nucleic acid hybridization,
Usually, it is 1 minute to several hours at about 50 ° C. to 70 ° C., and in the case of an immune reaction, it is usually about 1 minute to several hours at room temperature to about 40 ° C.

According to the above-described method, a test substance such as a nucleic acid, an antibody, or an antigen which selectively binds to the immobilized selective binding substance can be measured. That is, when a nucleic acid is immobilized as a selective binding substance, a nucleic acid having a sequence complementary to a sequence complementary to the nucleic acid or a part thereof can be measured. When an antibody or antigen is immobilized as a selective binding substance, an antigen or antibody immunoreacting with the antibody or antigen can be measured. The “measurement” referred to in the present specification includes both detection and quantification.

By using the present invention, the expression of genes, proteins, and sugar chains in various organisms can be efficiently, rapidly and simply examined. For example, after labeling nucleic acids extracted from normal human liver and liver infected with hepatitis virus, hybridization is performed on each of the fiber arrays in which various known human genes are immobilized on the fibers of the present invention. By comparing the degree of binding of the normal liver nucleic acid and the hepatitis liver nucleic acid to the sequence, changes in gene expression in the hepatitis liver can be examined.

Similarly, a labeled normal brain extract protein and an Alzheimer's brain extract protein are bound to a fiber array to which various monoclonal antibodies, which are proteins, are bound, and the bound proteins are compared with normal to obtain a protein in the Alzheimer's brain. Abnormal expression can be examined.

[0041]

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the following examples.

Example 1 One end of a polymethyl methacrylate optical fiber fiber (250 μm in diameter, 100 mm in length) was immersed in a nucleic acid solution of actin gene (Takara Shuzo Co., Ltd.) (the nucleic acid concentration was 10 μg / ml) and dried in air. UV treatment (using a UV crosslinker manufactured by Stratagene) was performed to obtain a nucleic acid-immobilized fiber. An oligonucleotide complementary to a part of the used nucleic acid sequence was synthesized, and digoxigenin (DIG:
Digoxigenin, Roche Diagnostics, Inc.).

Each of the terminally aminated oligonucleotides was dissolved in 100 mM borate buffer (pH 8.5) to a final concentration of 2 mM. Equivalent amount of digoxigenin-3-O-methylcarbonyl-α-aminocaproic acid-N-hydroxy-succinimide ester (26 mg / ml dimethylformamide solution)
Was added and left at room temperature overnight. Ethanol precipitation was performed using glycogen (Roche Diagnostics Co., Ltd.) as a carrier, and the precipitate was air-dried.
It was dissolved in mM Tris-HCl (pH 7.5) and 1 mM EDTA. The DIG-labeled oligonucleotide thus obtained was used as a model of the sample nucleic acid.

The prepared nucleic acid-immobilized fiber was put into a hybridization bag, and hybridization was carried out by a standard method (performed according to a product manual of Roche Diagnostics Co., Ltd.).

After completion of the hybridization, the nucleic acid-immobilized fiber was washed, and an anti-DIG enzyme-labeled antibody solution was added to allow an antigen-antibody reaction. After the reaction, the nucleic acid-immobilized fiber was washed to remove unbound antibody. DIG detection reagent was added and equilibrated. When the water was removed and the optical signal was detected from the other end of the optical fiber, the signal was detected in accordance with the immobilization of the nucleic acid.

Example 2 Pretreatment of Fiber A glass optical fiber having a diameter of 200 μm (manufactured by Hoyashot Co., Ltd.) was cut with a glass optical fiber cutter, and both end faces were mirror-finished. Next, one end face was cleaned with a mixed solution of pure water, ethanol and NaOH, and then washed with pure water. Further, the cleaned end face was treated with a mixed solution of pure water and poly-L-lysine (composition: 10%
(Poly-L-lysine) to introduce an amino group on one end face of the optical fiber.

Two types of nucleic acid solutions (“λC” manufactured by Takara Shuzo Co., Ltd.)
control Template & Primer S
et-A "; product number TX803 (λ of about 1000 bp)
DNA fragment) and “Human manufactured by Takara Shuzo Co., Ltd.
TFR (1 kb) Template & Prim
er Set "; product number TX806 (about 1000 bp
), The respective nucleic acids were amplified by PCR. PCR
As primers used in the method, those included in each product were used. This was purified to obtain a purified nucleic acid solution. The end face of the optical fiber into which the amino group was introduced was immersed in a purified nucleic acid solution, dried in the air, and then subjected to UV crosslink (120 mJ) to obtain two optical fibers each having one type of nucleic acid fixed to the end face. Next, in order to block unnecessary amino groups on the surface of the optical fiber that has not reacted with the nucleic acid, boric acid, pure water, NaOH for pH adjustment,
A mixed solution of succinic anhydride and 1-methyl-2-pyrrolidone (3 g of succinic anhydride in 187 ml of 1-methyl-2
-Dissolve in pyrrolidone and immediately before use 17 ml 1 M Na
The surface on which the nucleic acid was immobilized was immersed in -borate (pH 8.0 solution added) and shaken. Then, it was washed.

Treatment of RNA RNA solution (Takara Shuzo Co., Ltd., “λpolyA + RNA”
-A "; product number TX802) was prepared. It has a base sequence complementary to one of the above nucleic acids (TX803). This is called reverse transcriptase Superscript II.
(Manufactured by GIBCOBRL; product number 18064-07)
1), 2.5 mM dATP, 2.5 mM dCTP,
2.5 mM dGTP, 1.0 mM dTTP, Cy5
-DUTP (Amersham Pharmacia; product number P
A55022), and incubated at 42 ° C. for 1 hour to perform reverse transcription to obtain a cDNA solution in which Cy5 dye was incorporated.

Similarly, an RNA solution ("H" manufactured by Takara Shuzo Co., Ltd.)
human TFR RNA (1 kb) "; product number TX
805), and Cy5-dUTP is replaced with Cy3-dUT.
P (manufactured by Amersham Pharmacia; product number PA530)
Reverse transcription was carried out under the same conditions as above except that
A cDNA solution incorporating the y3 dye was obtained. This Cy
The cDNA incorporating the three dyes is one of the above nucleic acids (TX
806).

Two types of cDN incorporating the above dyes
A solution was mixed and purified, and further buffer (3.4 x SSC,
(0.1% SDS) to obtain a solution for hybridization.

Hybridization: Two optical fibers having one type of nucleic acid fixed to the end surface and a cDNA solution (a solution for hybridization) incorporating the above-mentioned dye were put in a vinyl bag, and the hybridization solution was added. Was sealed to prevent evaporation. This was left at 65 ° C. for 16 hours. Further, the optical fiber was removed from the plastic bag and washed.

First, detection of fluorescence from Cy5 was performed as follows.
A laser (wavelength: 635 nm) was used as the excitation light for fluorescence. The condensed beam was applied to the end face of the optical fiber in which the DNA solution was immersed, and light coming from the other end face was condensed by a condensing lens. Thereafter, a dichroic mirror (manufactured by Omega Optical; product number XF2035) was arranged at an angle of 45 degrees with respect to the optical axis, and unnecessary excitation light was removed. Immediately after the dichroic mirror, a band pass filter (manufactured by Omega Optical; product number XF3)
076) was arranged, and unnecessary excitation light was further removed.
A photomultimeter (manufactured by Hamamatsu Photonics; product number H5784-02) was placed immediately after this bandpass filter, and the fluorescence from the Cy5 dye was observed.

For the fluorescence from Cy3, a dichroic mirror and a bandpass filter are used for Cy3 (manufactured by Omega Optical; product numbers XF2017 and XF2017, respectively).
F3083), detection was performed in the same manner as described above except that the wavelength of the laser to be irradiated was 530 nm.

In this way, the fluorescence from the two types of optical fibers after the above-mentioned hybridization is Cy.
5, and each of Cy3 was measured. From an optical fiber produced by immersion in a nucleic acid solution of TX803, Cy5
Was observed, and the fluorescence of Cy3 was not detected. From the optical fiber prepared by immersion in the nucleic acid solution of TX806, fluorescence of only Cy3 was observed, and fluorescence of Cy5 was not detected.

Example 3 The same measurement as in Example 2 was carried out except that the optical fibers of Example 2 were bundled, the excitation light beam was scanned, and the fluorescence from each optical fiber was detected. As a result, the same result as in Example 2 was obtained.

Example 4 Example 2 was repeated except that the end face of the optical fiber irradiated with the laser as the excitation light was not immersed in the DNA solution.
The same measurement as described above was performed. That is, the second embodiment is the same as the second embodiment except that the direction of the optical fiber when detecting the fluorescence is reversed. As a result, the same result as in Example 2 was obtained.

Example 5 The treatment of an optical fiber and a nucleic acid was performed in the same manner as in Example 2.
In order to measure the fluorescence from Cy5, the optical system was as follows. First, a laser (wavelength 635 nm) was used as excitation light for fluorescence. First, a bandpass filter (manufactured by Omega Optical; product number X1069) was arranged perpendicular to the optical axis to remove unnecessary light other than the excitation light.
Furthermore, a dichroic mirror (made by Omega Optical; an angle of 45 degrees with the optical axis of the laser beam;
Product No. XF2035) was placed, and this focused beam was irradiated on the end face of the optical fiber opposite to the end face immersed in the DNA solution. Further, the fluorescence returned from the end face immersed in the DNA solution is collected on the end face side on the side to which the excitation light is irradiated, passed through the dichroic mirror (manufactured by Omega Optical; product number XF2035), and further banded. Pass filter (manufactured by Omega Optical; product number XF30)
76), the extra excitation light was cut. The optical system is shown in FIG.

For the fluorescence from Cy3, a dichroic mirror and a band-pass filter are used for Cy3 (manufactured by Omega Optical; product numbers XF1074 and XF1074, respectively).
F2017, XF3083), and detection was performed in the same manner as described above, except that the wavelength of the laser to be irradiated was 532 nm.

In this way, the fluorescence from the two types of optical fibers after the above-described hybridization is Cy
5, and each of Cy3 was measured. From an optical fiber produced by immersion in a nucleic acid solution of TX803, Cy5
Was observed, and the fluorescence of Cy3 was not detected. From the optical fiber prepared by immersing it in the nucleic acid solution of TX806, fluorescence of only Cy3 was observed, and fluorescence from Cy5 was not detected.

Example 6 The optical fiber was an image conduit (manufactured by Edmund Optics Japan; product number CJ53843) in which many thin optical fibers were bundled. This image conduit consists of three 25 μm-thick quartz optical fibers.
012 bundles.

The treatment of the optical fiber and the nucleic acid was performed in the same manner as in Example 5. The detection optical system was the same as in Example 5. At that time, only the fluorescence of Cy5 was observed from the optical fiber prepared by immersing in the nucleic acid solution of TX803,
No fluorescence of Cy3 was detected. From the optical fiber prepared by immersion in the nucleic acid solution of TX806, fluorescence of only Cy3 was observed, and fluorescence of Cy5 was not detected. Further, assuming that the intensity of the fluorescence from Cy5 and Cy3 in Example 5 is 1, in the experiment of this example, the intensity of the fluorescence is 3
And the S / N became higher. It is considered that the reason for this is that, when the image conduit is used, the converged light does not diverge inside the optical fiber, and the excitation light can be efficiently sent to the sample.

Example 7 Treatment of Optical Fiber A 200 μm diameter glass optical fiber (manufactured by Hoyashot) used in Example 1 was coated with a Pt film by sputtering to obtain a conductive optical fiber. Furthermore, it cut | disconnected with the cutter for exclusive use of a glass optical fiber, and the end surface of both sides was made into the mirror surface state. Further, the end face is immersed in a mixed solution of pure water and poly-L-lysine (composition: 10% poly-L-lysine),
An amino group was introduced on one end face of the optical fiber.

The subsequent treatment of the nucleic acid and the like was performed in the same manner as in Example 2, except that one type of nucleic acid was fixed to the end face.
An optical fiber and a solution for hybridization were obtained.

Hybridization Two optical fibers with one type of nucleic acid immobilized on the end face and a cDNA solution (a solution for hybridization) containing Cy5 and Cy3 dyes were placed in a microtube (1.5 ml). Further, a hole was made in the lid of the microtube, an optical fiber was passed therethrough, and the end face of the optical fiber to which DNA was fixed was immersed in the hybridization solution in the microtube. Furthermore, the hole of the lid of the microtube was sealed with a paper bond and sealed so that the solution did not evaporate. The hybridization solution at this time was 50 μl. The solution was prepared by dissolving DNA labeled with a fluorescent substance in pure water. This was left at 65 ° C. for 10 minutes.
At this time, paste the copper foil on the bottom of the micro tube,
A voltage of 100 V was applied using the optical fiber as the anode and the copper foil at the bottom of the microtube as the cathode. Further, the optical fiber was taken out of the microtube and washed. When the light was measured in the same manner as in Example 2, only the fluorescence of Cy5 was observed and the fluorescence of Cy3 was not detected from the optical fiber prepared by immersion in the nucleic acid solution of TX803. T
From the optical fiber prepared by immersion in the nucleic acid solution of X806, fluorescence of only Cy3 was observed, and fluorescence of Cy5 was not detected. The fluorescence intensity at this time was the same as in Example 2. On the other hand, the conditions of the optical fiber were the same as in Example 2 (that is, no voltage was applied), and the hybridization time was 10 minutes. As a result, the intensity of the fluorescence was 1/3 of that of Example 2. Thus, if the optical fiber is made conductive and an electric field is applied, only 10
A minute of hybridization time was found to be sufficient.

[0065]

According to the present invention, there are provided a fiber having a selective binding substance immobilized thereon and a fiber array having a selective binding substance immobilized thereon. Advantageous Effects of Invention According to the present invention, it is possible to provide a fiber on which a selective binding substance is immobilized with good efficiency and reproducibility, and by combining these fibers into a fiber array, the selective binding substance can be arbitrarily dense and accurate. Can be efficiently obtained with good reproducibility. Furthermore, by using an optical fiber, it is possible to easily and efficiently detect a sample that is efficiently bound through the fiber. In addition, by attaching a label to each fiber, it is possible to identify each fiber to which the test substance is bound regardless of the position in the array.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an optical system portion of a measuring device used in Example 5.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Band pass filter 2 Dichroic mirror 3 Band pass filter 4 Condensing lens 5 Optical fiber 6 Sample (DNA) 7 Photomultiplier 8 Laser light source

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) G01N 21/78 G01N 33/543 521 33/543 521 33/552 33/552 33/566 33/566 37/00 102 37 / 00 102 C12N 15/00 F (72) Inventor Koji Watanabe 1-1-1, Sonoyama, Otsu City, Shiga Prefecture Inside Shiga Plant of Toray Industries, Inc. (72) Inventor Hitoshi Shinmasa 1-1-1 Sonoyama, Otsu City, Shiga Prefecture No. F-term in the Shiga Plant of Toray Industries, Inc. (reference) 2G043 AA03 BA16 DA02 DA06 EA01 GA01 GB01 HA01 HA05 HA09 JA03 KA02 KA05 KA09 LA02 2G054 AA06 CA21 CA22 FB04 GA04 GB02 4B024 AA11 AA20 CA04 HA12 4B030 AB08 AB03 AB01 AB08 AB08 AB08 AB08 AB08 AB08 AB08 AB08 AB08 AB08 AB12 QA20 QQ08 QQ43 QR42 QR56 QS25 QS34 QX02

Claims (15)

[Claims] 1. A fiber array comprising a fiber on which a selective binding substance is immobilized or a bundle of the fiber. 2. The fiber array according to claim 1, wherein the selective binding substance is a nucleic acid, a protein, a saccharide, or an antigenic compound. 3. The fiber array according to claim 2, wherein the selective binding substance is a nucleic acid, an antibody or an antigen. 4. A fiber array comprising the fiber according to claim 1, wherein the fiber has a light transmitting property, or a fiber bundle. 5. The fiber according to claim 1, wherein the fiber has electrical conductivity.
A fiber array comprising the fiber according to any one of claims 1 to 3, and a bundle of the fiber. 6. A fiber array comprising the fixed fibers and a bundle of the fibers according to claim 1, wherein the fibers have flexibility. 7. A fiber array comprising a fiber and a bundle of fibers according to claim 1, wherein the selective binding substance is fixed to a fiber end. 8. The fiber array according to claim 1, wherein the type of the selective binding substance is different in all or a part of the fiber array. 9. The fiber array according to claim 8, wherein each fiber on which the selective binding substance is immobilized can be distinguished. 10. The substance which has reacted with the immobilized selective binding substance can be directly detected through a fiber.
The fiber array according to any one of the above. 11. The fiber array according to claim 1, wherein the fiber is an optical fiber. 12. A corresponding selectively binding substance that selectively binds to the fiber array according to any one of claims 1 to 11 with the selective binding substance immobilized on the fibers of the fiber array pair. A method for measuring the result of a selective binding reaction, comprising reacting a test sample containing, and washing, and then measuring the corresponding selective binding substance bound to the fiber. 13. The fiber according to claim 1, wherein the fiber is light-transmissive, and the selective binding substance is fixed to one end of the fiber.
2. The method according to 2. 14. The corresponding selective binding substance is labeled with a fluorescent or luminescent substance, or selectively binds to a measurement substance labeled with a fluorescent or luminescent substance, and after the reaction and washing, 14. The method according to claim 13, wherein a reaction result is measured by measuring fluorescence or luminescence from a fluorescent or luminescent substance using the corresponding selective binding substance or the label of the measurement substance from the other end of the fiber. 15. A test comprising applying a voltage or a current to the fiber array according to claim 5 and including the selective binding substance and the corresponding selective binding substance immobilized on the fibers of the fiber array pair. A method for selectively binding a sample.