JP2002022739A - Living organism related substance immobilization micro- array - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a living organism related substance micro-array having a hollow fiber allowing detection of a low-level specific bond without concealment in background intensity and a living organism related substance probe fixed to the hollow fiber. SOLUTION: A hollow fiber satisfying B/A<; 1 is used as this hollow fiber where A means detected light intensity of the hollow part when fluorescence intensity of a fluorescent molecule Cy3 (concentration 0.01 nmol/ml) existing in the hollow part of the hollow fiber is detected from the fiber cut face by means of a fluorescence microscope while B means detected light intensity of a fiber part in measurement under the same condition as the former without existence of a fluorescent molecule.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biological substance microarray for optically detecting a 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) that enables simultaneous expression analysis of a large number of genes has been developed and attracted attention.

[0003] These methods are basically 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, but they are mounted on a flat substrate called a microarray or chip. There is a great feature that a large number of DNA fragments are arranged and fixed at a high density. As a specific use of the microarray method, for example, a sample obtained by labeling an expression gene or the like of a cell to be studied with a fluorescent dye or the like is hybridized on a flat base piece, and nucleic acids (DN
A or RNA), a label is labeled with a fluorescent dye or the like, and then read at high speed by a high-resolution analyzer. Thus, the amount of each gene in the sample can be quickly estimated. In other words, the essence of this new method is basically the integration of the miniaturization of the reaction sample and the technology to sequence and align the reaction sample in a form that can be analyzed and quantified in a reproducible manner in a large, rapid and systematic manner. It is understood that there is.

As a technique for immobilizing a nucleic acid on a substrate, a method of immobilizing a nucleic acid at a high density on a nylon sheet or the like has been developed as in the Northern method. Further, a method has also been proposed in which nucleic acids and proteins are immobilized on glass capillaries and nylon fibers, the fibers and the like are bundled, immobilized with an adhesive, and cut in the fiber cross-sectional direction to produce a bio-related substance array sheet ( JP-A-11-108928).

[0005]

However, since polymers such as nylon are crystalline polymers, they are inferior in transparency, and when detecting bio-related substances on an array sheet by an optical method, Excitation light and fluorescence are scattered, and the background light intensity becomes very high. Further, glass capillaries are inferior in workability, and are cut into several hundred μm to 1 m.
It is very difficult to work into flakes with a thickness of m.

[0006] When the above-mentioned materials are used for a bio-related substance microarray, there is a serious problem that, when the purpose is to detect low-level specific binding, the detection light is masked by the background intensity.

[0007]

Means for Solving the Problems In view of the above-mentioned problems, as a result of diligent studies, the present inventors have found that a bio-related substance microarray having a hollow fiber and a bio-related substance probe fixed to the fiber has the above-mentioned structure. The present inventors have found that by reducing the intensity of the detection light, it is possible to detect low-level specific binding without being obscured by the background intensity.

[0008] That is, the present invention provides a microarray for immobilizing a bio-related substance probe in which a bio-related substance is immobilized on an inner wall and / or a hollow portion of a hollow fiber, and a cut surface intersecting the fiber axis of the fiber is two-dimensionally arranged. Wherein the hollow fiber satisfies the following formula: B / A <1 [where A is a fluorescent molecule Cy3 (concentration 0.01 nmol / m
l) Detected light intensity of the hollow portion when the fluorescence intensity is detected from the fiber cut surface with a fluorescence microscope, and B is the detected light intensity of the fiber portion when measured under the same conditions as above without the presence of fluorescent molecules. Is shown. ]

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below. A bio-related substance probe-immobilized microarray in which a bio-related substance is fixed to an inner wall and / or a hollow portion of a hollow fiber and a cut surface crossing a fiber axis of the fiber is two-dimensionally arranged in the present invention is a biological The immobilization process of the related substance is performed on the hollow fibers as a one-dimensional structure, and then, after a plurality of hollow fibers on which the bio-related substances are immobilized are formed into a three-dimensional structure arranged in an orderly manner, or After fixing a bio-related substance to individual hollow fibers after forming a three-dimensional structure in which hollow fibers are neatly arranged, fixing the bio-related substance obtained by cutting and thinning the fiber array having the three-dimensional structure Refers to thinned two-dimensionally arrayed fibers.

The hollow fiber used in the present invention has a very small absorption and scattering intensity of light in the ultraviolet to near-infrared light region (wavelength: 300 to 850 nm), and additionally has a fluorescence for incident light in this wavelength region. Refers to small strength.

Specifically, the following equation is satisfied. B / A <1 [where A is the fluorescent molecule Cy3
(Concentration 0.01 nmol / ml), the intensity of the detected light in the hollow part when the fluorescence intensity was detected from the fiber cut surface with a fluorescence microscope, and B
Indicates the detected light intensity of the fiber portion when measured under the same conditions as above without the presence of fluorescent molecules. ]

[0012] Here, Cy3 is one of the fluorescent molecules having a rhodamine skeleton, and is a representative molecule used when quantifying DNA molecules by binding to the end of DNA and measuring the fluorescence intensity from the fluorescent molecules. Fluorescent molecule. Therefore, in the present invention, when the concentration of this representative fluorescent molecule is 0.01 nmol / ml, the hollow fiber having a lower background intensity from the hollow fiber that is the support of the biomaterial probe is used than the fluorescent intensity. It is necessary. Fluorescent molecules for DNA detection include compounds having an allophycocyanin skeleton other than the Cy3, a fluorocene isothiocyanine skeleton,
Although they are called Cy5 and FITC, respectively, and have different excitation and emission wavelengths, the hollow fiber of the present invention can reduce the detection light from the background even when these fluorescent molecules are used.

As shown in FIG. 1, when the detection light intensity of the hollow fiber portion around the sample is high, the interface between the hollow fiber and the hollow portion is illuminated brightly, and the background intensity increases. . Further, in order to increase the fiber density (spot density) per unit area, it is desirable that the inside diameter of the hollow fiber is small. In such a case, the influence of the hollow fiber on the detection light intensity appears more remarkably.

When a hollow fiber having a small detection light intensity in the present invention is used, the background intensity is reduced, and detection with higher sensitivity becomes possible.

The material having a low detection light intensity is, for example, a methacrylate monomer such as methyl methacrylate, ethyl methacrylate or butyl methacrylate, a homopolymer of acrylate monomers such as methyl acrylate or ethyl acrylate, or a copolymer thereof, or Examples include polystyrene, polyethylene terephthalate, and polycarbonate.

The outer diameter of the hollow fiber is 1 mm or less, preferably 0.5 mm or less. Further, the inner diameter is preferably 0.03 mm or more. The fiber used in the present invention may be used as it is in an untreated state, but may be a fiber into which a reactive functional group is introduced, if necessary, or may be a plasma treatment, a γ ray, an electron beam, or the like. Fibers that have been subjected to radiation treatment may be used.

Therefore, the hollow fibers of the present invention are hollow or porous hollow fibers made of these materials.

In the present invention, the biological substance to be immobilized on the hollow fiber includes any one selected from the group consisting of the following substances (1) to (3). Nucleic acids are preferred. (1) Nucleic acid, amino acid, sugar or lipid (2) Polymer consisting of at least one component of the above (1) (3) Substance interacting with the above (1) or (2)

For example, nucleic acids include nucleic acids such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), and oxypeptide nucleic acid (OPNA). The biological substance used in the present invention may be a commercially available substance, or may be a substance obtained from living cells or the like.

For example, when a nucleic acid is used as a biological substance, DNA or RNA is prepared from living cells by a known method. For example, DNA extraction is performed by the method of Blin et al. (Blin et al., Nucleic Acid). Acids Res. 3: 2303 (197
6)), and for extraction of RNA, Favalo
ro et al. (Favaloro et al., Methods Enzymol. 65: 7
18 (1980)). Furthermore, a linear or circular plasmid DNA or chromosomal DNA, a DNA fragment obtained by cleaving them with a restriction enzyme or chemically, DNA synthesized by an enzyme or the like in a test tube, or a chemically synthesized oligonucleotide can also be used. it can.

When a bio-related substance is immobilized on a hollow fiber, various chemical or physical interactions between the fiber and the bio-related substance, that is, the functional groups of the fiber and the bio-related substance Or a chemical or physical interaction between the constituents. In addition, after introducing a liquid containing a bio-related substance into the hollow portion of the fiber constituting the array, the interaction between the functional group present on the inner wall surface of the hollow portion of the fiber and the component forming the bio-related substance is performed. Can be used. For example, when an unmodified nucleic acid is immobilized on a fiber, the nucleic acid and the fiber are allowed to act, and then immobilized by baking or ultraviolet irradiation. When immobilizing a nucleic acid modified with an amino group on a fiber, a functional group of the fiber is immobilized using a cross-linking agent such as glutaraldehyde or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). Can be combined with Further, for example, by performing a heat treatment, an alkali treatment, a surfactant treatment, etc. to modify the immobilized bio-related substance, or when using a bio-related substance obtained from a raw material such as a cell or a bacterial body. Can remove unnecessary cell components and the like. Note that these processes may be performed separately or simultaneously. In addition, it may be appropriately performed before the sample containing the biological substance is fixed to the fiber.

It is also possible to immobilize a biological substance, for example, a nucleic acid, on a gel and introduce the gel into a hollow portion. The type of gel that can be used here is not particularly limited, and examples thereof include acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, and N-methylol. One or more monomers such as acrylamide, N-vinylpyrrolidone, hydroxyethyl methacrylate, (meth) acrylic acid, allyl dextrin, etc. and methylene bis (meth) acrylamide, polyethylene glycol di (meth) acrylate, etc. A gel obtained by copolymerizing a functional monomer is used. In this case, the immobilization of the bio-related substance is performed, for example, by preparing a polymer having a polymerizable functional group introduced into the terminal of the nucleic acid, and passing the solution containing the monomer and the polymerization initiator to the hollow portion or the porous portion of each fiber. It can be carried out by polymerizing and gelling after the introduction into the mass part.

The bio-related substance-immobilized fibers prepared as described above are arranged in a plurality of fibers and embedded in a resin or the like to prepare a bio-related substance-immobilized fiber array. Alternatively, a plurality of fibers may be arranged and embedded in a resin or the like, and then a biomaterial may be immobilized on each fiber. By slicing the array at right angles to the fiber axis, a bio-related substance-immobilized fiber array slice (a bio-related substance-immobilized microarray according to the present invention) is obtained. The array slice can converge an arbitrary number of fibers to form an array, but preferably has a large number of fibers per unit area. Therefore, the outer diameter of the fiber is preferably small, 0.5 mm or less, more preferably 0.3 mm
mm.

Further, using a microarray immobilized with a biological substance using a material having a low detection light intensity, for example, when performing simultaneous expression analysis of a large number of genes, detection and analysis can be performed using a fluorescence microscope or the like. Since the detection light intensity of the hollow fiber serving as the background is low, the biological-related substance-immobilized microarray of the present invention can collectively photograph and detect the entire array including the hollow fiber portion, and the time required for detection There is an advantage that can be shortened.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments. However, the technical scope of the present invention is not limited by these examples. Hereinafter, the fluorescence microscope observation of the examples and comparative examples was performed using a cooled CCD camera C4880 manufactured by Hamamatsu Photonics.
-37 (512 × 512 elements, 24 μm square pixels) and Nikon fluorescence microscope E600 objective lens × 10NA = 0.3. The filter used was a filter block dedicated to a fluorescent reagent manufactured by Nikon [excitation wavelength: 535 ± 25 nm, detection wavelength: 610 ±.
38 nm (Cy3 filter), excitation wavelength: 620 ± 30 nm, detection wavelength: 700 ± 38 nm (Cy5 filter), excitation wavelength: 465
~ 495nm, detection wavelength: 515 ~ 555nm (FITC filter)]
There are three types.

Reference Example 1 (a) Synthesis of Fluorescently Labeled Oligonucleotides Oligonucleotides were synthesized using an automatic synthesizer DNA / RNA synthesizer (model 394) manufactured by PE Biosystems. In the final step of DNA synthesis, An oligonucleotide having a GCAT sequence in which FITC, Cy3 or Cy5 was introduced at the 5 ′ end was synthesized. These were used after deprotection and purification by a general method (see Japanese Patent Application No. 11-59397).

(B) Preparation of fluorescently labeled oligonucleotide having a methacrylate group 50 μl of the oligonucleotide (500 nmol / ml) having FITC, Cy3 or Cy5 at the 5 ′ end obtained in (a), 5 μl of glycidyl methacrylate, dimethylformamide (DM
F) 5 μl was mixed and reacted at 70 ° C. for 2 hours to prepare a fluorescent dye having a methacrylate group.
A fluorescent dye having a methacrylate group of 00 nmol / ml (GMA-modified FITC, GMA-modified Cy3, GMA-modified Cy5) was obtained.

Example 1 (1) A case where no fluorescent molecule exists in the hollow portion. A bundle of 25 polymethyl methacrylate (PMMA) hollow fibers (PMMA manufactured by Mitsubishi Rayon Co., Ltd., melt-spun product) having an outer diameter of 300 μm and an inner diameter of 160 μm was bundled, and about 2 cm at one end of the hollow fiber was opened. It was solidified with urethane resin so as to be in a state, and the other end was free end without being solidified with resin. The hollow fiber bundle was filled from the free end of the hollow fiber into the hollow fiber into the portion solidified with resin by suction from the free end of the hollow fiber in a reaction vessel containing a previously prepared reaction liquid (hereinafter referred to as aqueous solution A). After filling the aqueous solution, polymerization was carried out at 70 ° C. for 3 hours under a nitrogen atmosphere.

<Aqueous solution A>-4.5 parts by mass of acrylamide-0.5 parts by mass of N, N'-methylenebisacrylamide-0.1 parts by mass of 2,2'-azobis (2-methylpropion amidine) dihydrochloride-95 parts by mass of water

After polymerization of the acrylamide gel, the block is sliced in a direction perpendicular to the hollow fiber axis to a thickness of about 1
mm slices were obtained. The cut surface of the slice was observed with a fluorescence microscope by the above-described method, and the detected light intensity of the fiber cross section was quantified.
The detection light intensity of the fiber portion was 3,500 when the Cy3 filter was used.

Similarly, the value was 1000 when the Cy5 filter was used and 1000 when the FITC filter was used.

(2) The case where a GMA-modified fluorescently labeled oligonucleotide is present in the hollow part. Next, an acrylamide aqueous solution containing a predetermined concentration of GMA-modified Cy3, GMA-modified Cy5, and GMA-modified FITC prepared in Reference Example (described below,
The aqueous solutions B) were respectively prepared, and fixed in the hollow fibers of PMMA in the same manner as described above. Further, similarly, a slice having a thickness of about 1 mm was obtained, and the fluorescence intensity in the hollow portion of each slice was measured under the same conditions as those in the case where the fluorescent molecules were not contained (ie, with the same excitation light intensity and detection light detection sensitivity). Measurement was performed and the detected light intensity was digitized.

The detection light intensity of the hollow portion was 4380 when using a Cy3 filter.

Similarly, the value was 1310 when the Cy5 filter was used, and 1070 when the FITC filter was used.

<Aqueous solution B> GMA-modified fluorescently labeled oligonucleotide (GMA-modified Cy3: 0.01 nmol / ml, or GMA-modified Cy5: 0.005 nmol / ml, or GMA-modified FITC: 0.05 nmol / ml) 4.5 parts by mass of acrylamide N, N'-methylenebisacrylamide 0.5 parts by mass-2,2'-azobis (2-methylpropionamidine) dihydrochloride 0.1 parts by mass-water 95 parts by mass Table 1 shows the results. I put together.

Comparative Example 1 Nylon 6 having an outer diameter of 300 μm
After bundling 25 hollow fibers (melt-spun products) and polymerizing the acrylamide gel immobilized with GMA-modified probe A in the same manner as in Example 1, the block was sliced in a direction perpendicular to the hollow fiber axis to obtain a thickness. A slice of about 1 mm was obtained. The thin section was observed with a fluorescence microscope in the same manner as in Example 1, and the detected light intensity at the fiber cross section was quantified. The detected light intensity was 8000 when the Cy3 filter was used, and did not satisfy the condition of (Equation 1) (B / A = 1.8> 1). The number was 1700 when the Cy5 filter was used, and 2250 when the FITC filter was used, indicating that the array was a biological substance probe immobilized with a high detection light background.

Table 1.

[0038]

According to the present invention, a bio-related substance microarray for optically detecting a bio-related substance, by using a transparent material for a hollow fiber or a capillary which is a support thereof, is capable of being covered by a background intensity. Optical detection of the level becomes possible.

[0039]

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of a thin section in which hollow fibers having high detection strength are embedded.

FIG. 2 is a cross-sectional view of a thin section in which hollow fibers having low detection strength are embedded.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) G01N 37/00 102 G01N 37/00 102 (72) Inventor Atsushi Takahashi 20-1 Miyukicho, Otake City, Hiroshima Prefecture Mitsubishi Rayon Inside the Central Research Institute, Inc. (72) Takashi Akita 20-1 Miyukicho, Otake City, Hiroshima Prefecture Mitsubishi Rayon Co., Ltd. Central Research Laboratory F-term (reference) 2G043 AA03 BA16 CA03 DA02 EA01 FA01 FA02 FA06 GA07 GB21 HA05 JA03 KA05 LA03 MA01

Claims (2)

[Claims] 1. A bio-related substance probe-immobilized microarray in which a bio-related substance is immobilized on an inner wall and / or a hollow portion of a hollow fiber, and a cut surface intersecting a fiber axis of the fiber is two-dimensionally arranged. A microarray immobilized with a biological substance, wherein the fibers satisfy the following formula: B / A <1 [where A is the fluorescent molecule Cy3
(Concentration 0.01 nmol / ml), the intensity of the detected light in the hollow part when the fluorescence intensity was detected from the fiber cut surface with a fluorescence microscope, and B
Indicates the detected light intensity of the fiber portion when measured under the same conditions as above without the presence of fluorescent molecules. ] 2. The microarray according to claim 1, wherein the hollow fibers are a homo- or copolymer of a (meth) acrylic monomer, polyethylene, polystyrene, polyethylene terephthalate or polycarbonate.