JP2003248000A - Analyzing method using carrier for immobilized biomacromolecule and carrier for immobilized biomacromolecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analyzing method capable of inexpensively measuring biomacromolecules immobilized on carriers of various shapes such as a substrate, a bead, and especially, fiber, and a carrier for immobilized biomacromolecules applicable to mass production. <P>SOLUTION: In this analyzing method, biomacromolecules are immobilized on a surface of a carrier and molecules having an affinity to the biomacromolecules are detected and identified. This analyzing method uses a carrier for immobilized biomacromolecules characterized in that the surface of the carrier has a polymer having at least one structural unit expressed by the following general expression and that a linker having an affinity to the polymer is given to the biomacromolecules. (R<SP>1</SP>to R<SP>3</SP>express alkyl groups, aryl groups, or hydrogen atoms, and they can be the same or different from each other). <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a nucleic acid on the surface of a carrier,
An analytical method for immobilizing a protein or sugar chain (hereinafter referred to as a biopolymer) and detecting / identifying a molecule having an affinity for the biopolymer, which is a method for stably binding the biopolymer to a carrier. And an analysis method using the biopolymer-immobilized carrier.

[0002]

2. Description of the Related Art Studies on the analysis of genetic information of various organisms have been conducted, and many genes including human genes and their nucleotide sequences, proteins encoded by the gene sequences, and proteins produced by these proteins are secondarily produced. Information on the sugar chains that are produced is rapidly being revealed. The functions of biopolymers such as genes, proteins, and sugar chains whose sequences have been clarified can be investigated by various methods. The main ones are Northern hybridization or Southern hybridization for nucleic acids,
The relationship between various genes and their biological function expression can be investigated by utilizing various nucleic acids / complementarity between nucleic acids. Regarding proteins, the function and expression of proteins can be examined by utilizing the reaction between proteins, as represented by Western hybridization.

In recent years, a DNA microarray method (DNA chip method) has been used as a method for analyzing the expression of many genes at once.
A new analytical method, or so-called method, 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 / quantification methods based on nucleic acid / nucleic acid hybridization reactions, and are protein / protein or sugar chain / sugar chain or sugar. It can also be applied to the detection and quantification of proteins and sugar chains based on chain / protein hybridization. These techniques use a large number of D's on a flat substrate piece called a microarray or chip.
A major feature is that NA fragments, proteins, and those in which sugar chains are aligned and immobilized at high density are used. As a specific method of using the microarray method, for example, a sample obtained by labeling an expression gene of a cell to be studied with a fluorescent dye or the like is hybridized on a flat substrate piece to bind mutually complementary nucleic acids (DNA or RNA). Examples of the method include a method of labeling the area with a fluorescent dye or the like and then reading it at high speed with a high-resolution analyzer, and a method of detecting a response such as a current value based on an electrochemical reaction. In this way, the amount of each gene in the sample can be estimated quickly.

As a technique for immobilizing nucleic acids on a substrate, similar to the Northern method, a method of immobilizing the nucleic acid on a nylon sheet or the like with a high density is used. A method for coating polylysine and the like to immobilize it has been developed.

There are various methods for immobilizing preliminarily prepared DNA on the surface of a solid phase carrier depending on the type of DNA and the type of solid phase carrier. (1) The DNA to be immobilized is cDNA (complementary DNA synthesized using mRNA as a template) or PCR product (cDN
In the case of DNA fragments obtained by amplifying A by the PCR method), these are spotted on the surface of the solid-phase carrier surface-treated with a polycation using a DNA chip manufacturing apparatus, and
It is common to use the A 2 charge to electrostatically bond to the solid support and then block excess cations.

(2) When the DNA to be immobilized is a synthetic oligonucleotide, a functional group-introduced oligonucleotide is synthesized, and the oligonucleotide is spotted and covalently bonded to the surface of a solid-phase carrier which has been surface-treated (Lamture).
, J. B et al. , Nucl. Acids Re
s. , 22, 2121-2125, 1994 and Guo, Z. , Et al. , Nucl. Acid
s Res. , 22, 5456-5465, 1994.
). The oligonucleotide is generally covalently bonded to the surface-treated solid phase carrier via a spacer or a crosslinker. A method is known in which minute pieces of polyacrylamide gel are aligned on a glass surface and synthetic oligonucleotides are covalently bonded thereto (Yershov, G.
． , Et al. , Proc. Natl. Acad
． Sci. USA, 94, 4913 (1996)
)). Although it is a special method, an array of microelectrodes is prepared on a silica DNA chip, and a permeation layer of agarose containing streptavidin is provided on the electrodes to serve as a reaction site. There is a method of immobilizing biotinylated DNA by positively charging this site and controlling the charge of the site to enable high-speed and precise hybridization (Sonowowski, RG. Et al. P.
roc. Natl. Acad. Sci. USA94
, 1119-1123 (1997)).

In order to make a DNA chip by spotting pre-prepared DNA on the surface of a solid phase carrier, the DNA must be stably immobilized on the surface of the carrier. The method of immobilizing DNA is typically the method of utilizing the charge of DNA or the method of utilizing covalent bond. As a modification of the method utilizing the charge of DNA, PCR modified with an amino group
The product was suspended in SSC (standard salt-citrate buffer) and spotted on the surface of the silylated glass slide,
After the incubation, a method of performing treatment with sodium borohydride and heat treatment in order has been reported (Schena, M. et al., Proc. N.
atl. Acad. Sci. USA 93, 106
14-10619 (1996)), but there is a problem that it is difficult to obtain sufficient stability by this fixing method. Further, in DNA chip technology, the detection limit is important. Therefore, a sufficient amount of DN can be stably added to the surface of the solid-phase carrier.
It is considered that the immobilization of A 3 directly leads to the improvement of the detection limit of the hybridization between the DNA and the labeled target nucleic acid. Therefore, there is a need to develop an excellent method for immobilizing DNA.

Microarrays using proteins and sugar chains have the same problems as those using these nucleic acids.

[0009] On the other hand, in Japanese Patent Publication No. 2001-527539, an active surface containing a carboxylic acid-derived component and a hydrogen bond are provided for supplying and removing a physiologically and / or pharmacologically active substance in a liquid. Interaction systems have been proposed that take advantage of the interaction of linkers with components that can give rise to

[0010]

Under such circumstances, biopolymers are immobilized at a predetermined concentration on a substrate, beads, in particular carriers of various shapes such as fibers, and are arrayed in a measurable form with high density and reproducibility. The establishment of new systematic methodologies that can be applied to mass production at low cost is highly demanded for biopolymer analysis that is expected to increase in importance in the future.
This is a problem to be solved by the present invention.

Specifically, it provides a method of immobilizing a biopolymer such as a nucleic acid, a protein or a sugar chain, which has been prepared in advance, on the surface of the carrier, and an analytical method using the biopolymer-immobilized carrier. With the goal.

[0012]

[Means for Solving the Problems] That is, the present invention provides an analytical method for immobilizing a biopolymer on the surface of a carrier and detecting and identifying a molecule having an affinity for the biopolymer. Is a polymer having at least one structural unit represented by the following general formula (1), and a biopolymer is provided with a linker having an affinity for the polymer, This is an analysis method using a molecule-immobilized carrier.

[0013]

[Chemical 2]

(R ^{1 to} R ³ represent an alkyl group, an aryl group or a hydrogen atom, and may be the same or different.) Further, the structural unit represented by the general formula (1) is provided on the surface of the carrier. A biopolymer having a polymer having at least one of the following: a biopolymer is provided with a linker having an affinity for the polymer, and the biopolymer is immobilized via the carrier surface and the linker. It is a polymer-immobilized carrier.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The details of the analytical method using the biopolymer-immobilized carrier of the present invention will be described below. In the present invention, as the biopolymer to be immobilized on the carrier, any biopolymer such as nucleic acid, protein and sugar chain can be used. The nucleic acid may be either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and examples include nucleic acid extracted from living cells or purified nucleic acid. Furthermore, the DNA is not limited to biologically derived DNA, and synthetically obtained oligo DNA or the like may be used. As the protein, any of antibody, enzyme, receptor, cytokine and the like can be used. The protein is not limited to a biologically derived protein, and may be a synthetic oligopeptide or the like. Furthermore, a part of the chemical structure may be modified for the purpose of introducing a linker, which will be described later.

In the present invention, the carrier for immobilizing biomolecules is a solid in any shape having a polymer having at least one structural unit represented by the following general formula (1) on the surface of the carrier.

[0017]

[Chemical 3]

(R ^{1 to} R ³ represent an alkyl group, an aryl group or a hydrogen atom and may be the same or different.) As the polymer, a homopolymer or a copolymer is used. The polymer uses at least one type of monomer as a raw material, and the monomer is in the form of a double bond capable of participating in polymerization and a functional group capable of participating in polycondensation, and a form of a ketone or a carboxylic acid or a derivative thereof. And a carbonyl group that does not participate in the polymerization reaction. Further, it is more preferable that the polymer has the structure of the general formula (1). The preferable molecular weight range of the polymer is 10,000 to 1,000,000. Particularly preferably, it is 20,000 or more and 500,000 or less.

In the general formula (1), R ¹ and R ² represent an alkyl group, an aryl group or a hydrogen atom and may be the same or different. The alkyl groups may be linear or branched and preferably have 1 to 20 carbon atoms. The aryl group is
It preferably has 6 to 18, more preferably 6 to 12 carbon atoms. The functional group X is O, NR ³ , or CH ₂
Arbitrarily selected from among. R ³ is a functional group defined in the same manner as R ¹ and R ² described above. However, the alkyl group represented by R ³ is particularly preferably linear or branched, and optionally has a substituent and has 1 to 8 carbon atoms, for example, a methyl group, an ethyl group or a propyl group. Group etc. The optionally present substituents include, for example, one or more halogen atoms such as fluorine, chlorine, bromine or iodine, or a hydroxyl group, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, Examples thereof include alkylthiol groups having 1 to 6 carbon atoms. R
The aryl group of ³ is particularly preferably a monocyclic or bicyclic aryl group, which optionally contains a substituent.
Substituents optionally included include one or more heteroatoms and the like. Examples of the particularly preferable aryl group include a phenyl group, a 1- or 2-naphthyl group, an indenyl group and an isoindenyl group. Examples of the aryl group containing a hetero atom include O
, S 2, and N 3 are heteroaryl groups having 3 to 9 carbon atoms and containing a hetero atom. Examples of the monocyclic heteroaryl group include a pyrrolyl group, a furyl group, a thienyl group, an imidazolyl group, an N-methylimidazolyl group, an N-ethylimidazolyl group, a benzothiazolyl group,
Examples thereof include quinazolinyl group, naphthylpyridinyl group, quinolinyl group, isoquinolinyl group and tetrazolyl group.

Among the above-mentioned polymers containing functional groups, preferred are, for example, polymethylmethacrylate (PMMA) and polyethylmethacrylate (PE).
MA) or polyalkylmethacrylate (PAMA) such as polypropylmethacrylate. Of these, polymethylmethacrylate is particularly preferable. Furthermore, polyvinyl acetate, polycyclohexyl methacrylate, polyphenyl methacrylate, etc. can also be used. Further, a copolymer having a structure in which the constituent elements of the polymer are combined or a constituent element of another kind or plural kinds of polymers is added to the constituent element of the polymer can also be used. Examples of the other polymer include polystyrene, polyacrylonitrile, and polyamide.

The ratio of the constituents in the copolymer is
There is no particular limitation. The abundance ratio of the carbonyl group-containing monomer, for example, alkyl methacrylate, in the copolymer is preferably 20 mol% or more, more preferably 40 mol% or more, and most preferably 60 mol% or more.

The existence form of the polymer on the carrier surface is not particularly limited, but in addition to the carrier itself being the polymer, a different material such as glass is used as a substrate on all or part of the surface thereof. Examples thereof include a polymer layer formed by coating or reaction, and a polymer domain formed on the surface by blending or alloying the polymer with other polymers.

In the present invention, the linker having an affinity with the polymer surface preferably contains a polyalkyl ether having an alkyl chain having 1 to 20 carbon atoms.
Preferably used as the linker is a polyalkylene glycol, particularly preferably polyethylene glycol (PEG). In addition, polyalkyleneimines, polyalkyleneamines or polyalkylene sulfides and polyoxazilines.
nes) etc. are also preferably used. Particularly preferred is the use of polyethylene glycol. The various compounds preferably have a molecular weight of 2,000 to 50,000 (2 kDa to 50 kDa).

In the present invention, the method for attaching the linker to the biopolymer is not particularly limited, but --OH group, --SH group, --NH group already existing in or introduced into the biopolymer. For reactive points such as groups,
Examples thereof include a method of reacting a linker bound to a functional group capable of reacting with the reaction point to form a bond. Examples of such a functional group include succinimidyl succinate,
Succinimidyl propionate
dipropionate), nitrophenyl carbonate (nitrophenyl carbonate)
e), trisylates, epoxides, aldehydes
de), isocyanate (isocyanate), maleimide (maleimide), or the like. Suitable linkers for binding to biopolymers include, for example, Shearwater Polymers, Inc.
Inc. ) (2307 Spring Branch Rd.), Huntsville, Alabama (AL) 358
01 (USA)).

In the present invention, an immobilized carrier can be prepared by contacting the surface of the carrier with a solution of a biopolymer having a linker, much more easily than the conventional method. For example,
In a solution of biopolymer with a given concentration of linker,
It is possible to easily immobilize the biopolymer on the surface of the carrier by immersing the carrier for a predetermined time, then taking it out and washing it. In addition, a solution of a biopolymer having a predetermined concentration of a linker is spotted on an arbitrary portion of a carrier using a commercially available DNA chip manufacturing apparatus or the like, and the biopolymer solution is washed to arrange a large number of biopolymers in a minute region. -It is possible to easily create an immobilized biopolymer array.

The solvent for dissolving the linker-attached biopolymer is not particularly limited, but the physical properties of the biopolymer such as the three-dimensional structure of protein can be retained.
Aqueous solvents such as water, physiological saline, and various buffers are particularly preferable, and are also preferable for storage of the linker-added biopolymer.

Examples of the shape of the carrier include a plate shape, a bead shape, and a fiber shape. From these, it is possible to select a shape according to the detection means of the analysis method using the biopolymer-immobilized carrier of the present invention. Specific shapes of the carrier include a flat plate, an optical fiber and beads. In the case of a flat plate, a large number (10 or more types) of various types of biopolymers can be preferably used in the DNA chip manufacturing apparatus as described above.
In the case of beads, a large number of beads on which different types of biopolymers are immobilized can be preferably used.

The fibrous carrier has the following advantages and is particularly preferable. That is, the end face is immersed in a solution of the biopolymer to immobilize the biopolymer. At this time, it is possible to immobilize biopolymers on the end faces of many fibrous carriers at one time. Then, a bundle of fibrous carriers on which different types of biopolymers thus prepared are bundled, and a bundle of fibrous carriers on which various kinds of biopolymers are substantially and easily produced in a large amount. can do. With such a shape, it becomes possible to react various biopolymers with the specimen by a single operation. In addition, comparing this carrier production method with the method of spotting various biopolymers on a flat surface using a DNA chip production apparatus, etc., when using a fibrous carrier, it can be produced at lower cost and easily. Is. Further, compared to a bead-shaped carrier, it is difficult to distinguish each bead with a bead-shaped carrier, but in the case of a fibrous carrier, a means such as printing an ID such as a barcode is used. As a result, it is possible to easily distinguish each fiber.

Further, it is particularly preferable that the fibrous carrier has a light transmitting property. The reason is that it is difficult to handle R
This is because it is possible to use a fluorescent substance or a luminescent substance that can be easily handled for detection without using I (radioisotope).

Further, in the case of a fibrous carrier or the like in which a linker-attached biopolymer is immobilized on the end face, after the analysis is completed, the end face is dipped in alcohol or the like and washed to remove the linker-attached biopolymer. After that, a new biomolecule can be immobilized again for analysis, which is effective in reducing analysis cost.

The biopolymer-immobilized carrier obtained by the present invention is reacted with a sample using the immobilized nucleic acid, protein, sugar chain or the like as a probe to carry out hybridization, whereby a specific base in the sample is obtained. It can be used to detect a nucleic acid having a sequence, a protein having affinity, and a sugar chain.

For detecting the hybrid, a known means capable of specifically recognizing the hybrid can be used. For example, a labeled substance such as a fluorescent substance, a luminescent substance, or a radioisotope is allowed to act on the nucleic acid, protein, or sugar chain in the sample, and the labeled substance can be detected. There is no limitation on the type of the labeled body, the method of introducing the labeled body, and the like, and various conventionally known means can be used. In particular, it is particularly preferable to use a fluorescent substance or a luminescent substance as a labeling substance when the carrier is fibrous and has a light-transmitting property, the sample labeled at the fiber end is reacted, and the detector is detected at the other end. .

When the carrier has electric conductivity, it is also possible to use a method of detecting a response such as a current value based on an electrochemical reaction. In this case, the sample and the sample immobilized on the carrier that serves as the electrode are accelerated, and the reaction between the sample and the sample is promoted.
By reacting under a material that suppresses the reaction, and the whole or a part of this material is contained in the bound sample and the analyte, and measuring the current value flowing through the electrode, that is, the carrier after the reaction, The presence or absence of binding of the sample and the degree of binding can be detected. Specific examples of such a carrier include:
For example, a material in which the surface of PMMA fibers is coated with a material such as a metal by means of sputtering, vapor deposition, plating, CVD or the like to give conductivity can be mentioned. As the material, a material contained in the second to fourth periods of the groups 3A to 5B of the periodic table or a mixture thereof may be coated on the surface of the fiber by the above-mentioned means. Further, fibers in which the above-mentioned conductive material is dispersed in PMMA can also be preferably used. Also,
When the carrier has electrical conductivity, the hybridization can be speeded up by applying an electric field.

Further, when the carrier is fine particles, by adding a component capable of reacting or hybridizing with the immobilized biopolymer, specific fine particles are cross-linked / aggregated in the dispersion medium, which is visually or microscopically observed. It is also possible to provide an analytical method or an analytical device for detecting a specific chemical structure by detecting by means of.

[0035]

The present invention will be described in more detail by the following examples. However, the technical scope of the present invention is not limited by these examples.

Example 1 Three kinds of deoxyribonucleic acid (DNA) having the following base sequences were synthesized. 5'-GGG GCC ATC CAC AGT CTT CT-3 '(DNA 1) 5'-AGA AGA CTG TGG ATG GCC CC-3' (DNA 2) 5'-CCA TGG GGA AGG TGA AGG TC-3 '(DNA 3) ) In addition, the 5'end of DNA1 is aminated, and the 5'ends of DNA2 and DNA3 are FI which is a fluorophore.
It is labeled with TC (Fluorescein-4-isothiocyanate). These three types of DNA were dissolved in PBS to have a concentration of 100 pmol / l.

Next, Shearwater Corpo
ration mPEG-ALD, MW20,000
(Average molecular weight 21,000) is dissolved in PBS and the concentration is 24
It was set to 0 pmol / l. The chemical formula of this mPEG-ALD is as shown in (a) below, and an aldehyde group is introduced on one side of PEG (polyethylene glycol).

CH ₃ O (CH ₂ CH ₂ O) nCH ₂ CH ₂ —CHO (a) 100 μl of the above DNA1 solution and 42 μl of mPEG-ALD solution were put into one Eppendorf tube and gently stirred, followed by shaking for 20 hours. (Hereinafter referred to as Solution 1). When the reaction was confirmed by thin film chromatography using another solution subjected to the same operation, it was found that the amino group at the 5 ′ end of DNA1 and the aldehyde group of mPEG-ALD reacted to form PEG-DNA. It could be confirmed.

In this solution, PMMA (polymethylmethacrylate) beads Matsumoto Microsphere M
(Matsumoto Yushi-Seiyaku Co., Ltd .: particle size 5 to 20 μm)
15 mg was added and suspended, and the mixture was shaken for 1 hour.
In this way, DNA1 was immobilized on the surface of PMMA beads via PEG. Then, after centrifuging with a centrifuge, the supernatant was discarded, 3 × SSC was further added, and the suspension, centrifugation, and discarding of the supernatant were repeated twice. And 1
After the suspension was added with 00 μl of 3 × SSC, the solution was quickly divided into two Eppendorf tubes.

DNA2 and DNA3 solutions were added to the two divided Eppendorf tubes, respectively, and the mixture was stirred and then incubated at 45 ° C. for 2 hours for hybridization. After incubation, centrifuge,
After discarding the supernatant, suspend with 200 μl of 3 × SSC,
It was washed by centrifugation and discarding the supernatant. Similarly, 1 × S
After washing once with SC and twice with 0.2 × SSC, PMMA particles contained in each Eppendorf tube were placed on a slide glass, and fluorescence from FITC was observed with a fluorescence microscope.

As a result, FITC fluorescence was observed from the PMMA beads contained in the tube containing the DNA2 solution, but no fluorescence was observed from the PMMA beads contained in the tube containing the DNA3 solution.
The nucleotide sequence shows that DNA1 and DNA2 form a double strand (that is, they can hybridize),
DNA1 and DNA3 did not form a double strand (ie, no hybridization was possible). That is, PEG
DNA can be immobilized on PMMA beads via

Example 2 The same reagents as in Example 1 were used. Solution 1 was spotted on a PMMA plate, air-dried, washed with pure water and dried. This PMMA
Two plates were prepared, one of which was filled with the DNA1 solution and the other of which was filled with the DNA2 solution. Then, each was covered with a cover glass and incubated at 45 ° C. for 2 hours under the condition of 100% humidity. After washing, drying, and observation with a fluorescence microscope, fluorescence was observed only from the plate on which DNA2 was suspended. Thus, DNA could be immobilized on the PMMA plate surface via PEG.

Example 3 The same reagents as in Example 1 were used. An optical fiber having a PMMA core was immersed in the solution 1, dried, and washed with pure water. Two of these optical fibers were prepared. This one is D
Put the remaining NA1 solution into the microtube containing the remaining 1
Each plate was placed in a microtube containing the DNA2 solution. And it incubated at 45 degreeC for 2 hours. After washing, drying, and observation with a fluorescence microscope, fluorescence was observed only from the optical fiber immersed in the DNA2 solution. Thus, DNA could be immobilized via PEG to an optical fiber whose core was PMMA.

Example 4 PCR was carried out using 5S ribosomal DNA as a template and the following combinations of primers to obtain a reaction product. Furthermore, after ethanol precipitation, after lightly washing with 70% ethanol,
It was air-dried and purified. (DNA4).

5′-GCC TAC GGC CAT ACC ACG CT-3 ′ (primer 1) 5′-GCT TAC AGC ACC CGG TAT TC-3 ′ (primer 2) The 5 ′ end of primer 2 used at this time was It is aminated.

Next, human placenta c
PCR was performed using the DNA library as a template and the following combination of primers to obtain a reaction product. further,
After ethanol precipitation, it was lightly washed with 70% ethanol and then air-dried for purification. (DNA5).

5′-CCA TGG GGA AGG TGA AGG TC-3 ′ (primer 3) 5′-GGG GCC ATC CAC AGT CTT CT-3 ′ (primer 4) The 5 ′ end of primer 4 used at this time was It is aminated. Dissolve these two types of DNA in PBS,
Each concentration was 100 pmol / l.

Next, Shearwater Corpo
ration mPEG-ALD, MW20,000
(Average molecular weight 21,000) is dissolved in PBS and the concentration is 24
It was set to 0 pmol / l. The chemical formula of this mPEG-ALD is as shown in (b) below, and an aldehyde group is introduced on one side of PEG (polyethylene glycol).

CH ₃ O (CH ₂ CH ₂ O) nCH ₂ CH ₂ —CHO (b) 20 μl of the above DNA4 solution and 8.4 μl mPEG-ALD solution were put into one microtube and gently stirred. Strained for 20 hours (hereinafter referred to as Solution 2). Similarly, 20 μl of DNA5 solution and mPEG
-8.4 μl of ALD solution was put into one microfuge tube, gently stirred, and then agitated for 20 hours (hereinafter referred to as solution 3).

In the solution 2, the end surface of the fiber (200 μmφ) in which gold was sputtered around PMMA was immersed in the solution 2.
It was dried and washed with pure water to obtain a fibrous carrier having DNA4 immobilized on the end face. Similarly, using Solution 3, a fibrous carrier having DNA 5 immobilized on the end face was obtained. In addition, such a fibrous carrier can be easily produced in a large amount by repeating impregnation, drying and washing, or simultaneously impregnating a large number of fibers and drying and washing.

Next, separately, primer 3 used in PCR,
The same DNA 4 and DNA 5 were prepared, except that the 5'end of the primer 4 was not aminated. Then, add pure water and buffer to each, and further add 10U Klenow Fragment and 2.5 μl of dNTP mixture.
(2.5 mM dATP, dTTP, dGTP, and 400 mM dCTP), 2 μl of random primer 9mer (6 mg / ml) was added. Furthermore, D
Cy3-dCTP (made by Amersham Pharmacia) in NA4 solution
Cy5-dCTP (manufactured by Amersham Pharmacia) was separately added to the DNA5 solution, incubated at 37 ° C. for 20 hours, and then purified using ethanol precipitation or the like. In this way, DNA4 in which the Cy3 labeled body was incorporated and DNA5 in which the Cy5 labeled body was incorporated were obtained. Next, DNA4 in which the Cy3 labeled body is incorporated,
DNA5 containing the Cy5-labeled substance was incorporated into
Dissolved in 20 μl of × SSC and 0.1% SDS and mixed. Put this solution in a microtube and put D on the end face.
DNA of a fibrous carrier on which NA5 is immobilized and DNA on a fibrous carrier on which DNA4 is immobilized
Simultaneously attacked the fixed end face. At this time, in order to distinguish the fibrous carriers, the side faces of the fibrous carriers were marked with a marker. In this state, incubation was carried out at 65 ° C. for 12 hours, the fibrous carrier was taken out from the microtube, washed and dried.

Fluorescence detection from Cy5 was performed as follows. A laser (wavelength 635 nm) is used as excitation light for fluorescence.
Was used. This condensed beam was applied to the end face of the fibrous carrier on which DNA was immobilized, and the light emitted from the other end face was condensed by the condenser lens. This is followed by a dichroic mirror (Omega Optical; product number XF203
5) was arranged so as to form an angle of 45 degrees with the optical axis, and unnecessary excitation light was removed. Immediately after the dichroic mirror, a bandpass filter (manufactured by Omega Optical; product number XF3076) was placed to further remove unnecessary excitation light. Immediately after this band pass filter, a photo multimeter (manufactured by Hamamatsu Photonics KK; product number H5784)
-02) was placed 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), the fluorescence from the two types of fibrous carriers after the above hybridization was detected by the same method as above except that the wavelength of the laser to be irradiated was 530 nm, for Cy5 and Cy3, respectively. It was measured. Only the fluorescence of Cy3 was observed and the fluorescence of Cy5 was not detected from the optical fiber prepared by immersing it in the nucleic acid solution of DNA4. From the optical fiber prepared by immersing it in the nucleic acid solution of DNA5, only fluorescence of Cy5 was observed, and fluorescence from Cy3 was not detected.

Comparative Example 1 The same reagents as in Example 1 were used. DNA1 solution (PBS
In which DNA1 was dissolved, concentration 100 pmol / l)
And 100 μl of PBS and 42 μl of PBS were placed in an Eppendorf tube and stirred.

Next, 15 PMMA beads were added to this solution.
After adding mg and suspending, it was punished for 1 hour. Then, after centrifuging with a centrifuge, the supernatant was discarded, 3 × SSC was further added, and the suspension, centrifugation, and discarding of the supernatant were repeated twice. Then, 100 μl of 3 × SSC was added and suspended, and this solution was quickly divided into two Eppendorf tubes.

DNA2 and DNA3 were respectively placed in the two divided Eppendorf tubes in the same manner as in Example 1.
After adding and stirring, the mixture was incubated at 65 ° C. for 20 hours for hybridization. After the incubation, the mixture was centrifuged and the supernatant was discarded, followed by washing with 3 × SSC, suspension, centrifugation, and discarding the supernatant. Similarly, 1
PMMA in each Eppendorf tube after washing once with × SSC and twice with 0.2 × SSC
Place the particles on a slide glass and FIT with a fluorescence microscope.
The fluorescence from C was observed.

As a result, no fluorescence was observed from the beads contained in any of the tubes, and under these conditions (when PEG was not used as a linker), DMA was observed on the PMMA bead surface.
It was impossible to fix NA.

[0058]

As described above, according to the analysis method of the present invention, a carrier on which a biopolymer such as a nucleic acid, a protein or a sugar chain is immobilized can be reproducibly and efficiently obtained. By using the biopolymer-immobilized carrier, the type of biopolymer such as nucleic acid, protein or sugar chain in a sample can be easily investigated.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/53 ZNA G01N 33/543 525E 33/543 525 33/566 33/566 37/00 102 37/00 102 C12N 15/00 A F F term (reference) 4B024 AA11 AA19 AA20 CA01 CA09 CA11 HA12 HA14 HA20 4B029 AA07 AA21 BB20 CC01 CC02 CC03 CC13 FA12 FA15 4B063 QA01 QA13 QQ42 QQ52 QR32 QR35 QR38 QR55 QR10 QR83 QR02 QR83 QR02 QR83 QR02

Claims (6)

[Claims] 1. An analytical method for immobilizing a biopolymer on the surface of a carrier and detecting and identifying a molecule having an affinity for the biopolymer, wherein the surface of the carrier is represented by the following general formula (1). Analyzing method using a biopolymer-immobilized carrier, wherein the biopolymer has a polymer having at least one structural unit and a linker having an affinity for the polymer is attached to the biopolymer. . [Chemical 1] (R ^{1 to} R ³ represent an alkyl group, an aryl group or a hydrogen atom, and may be the same or different.) 2. The analysis method according to claim 1, wherein the linker contains at least a polyalkyl ether as a structural element. 3. A molecule contained in a sample is detected and identified by hybridization between a molecule contained in the sample and a biopolymer immobilized on a carrier. 1. The analysis method according to 1. 4. A carrier surface comprising a polymer having at least one structural unit represented by the general formula (1), and a biopolymer having a linker having an affinity for the polymer, A biopolymer-immobilized carrier, wherein a biopolymer is immobilized via a linker and a linker. 5. The biopolymer-immobilized carrier according to claim 4, wherein the biopolymer-immobilized carrier comprises a fibrous carrier having a biopolymer immobilized on an end face thereof or a bundle of the fibers. 6. The biopolymer-immobilized carrier according to claim 5, wherein the fibrous carrier has a light-transmitting property.