JP2006078197A - Immobilized carrier of selectively bindable substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a carrier where a selectively bindable substance having an excellent S/N ratio at detection is immobilized. <P>SOLUTION: This immobilized carrier of the selectively bindable substance wherein the selectively bindable substance is immobilized on the carrier surface has characteristics wherein the carrier surface comprises a saturated annular polyolefin-based resin, and the carrier is provided with an irregularity part, and the selectively bindable substance is immobilized on the upper surfaces of a plurality of projection parts of the irregularity part. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a carrier on which a substance that selectively binds to a test substance (herein, “selective binding substance”) is immobilized.

  Research on genetic information analysis of various organisms has been started, and information on many genes and their base sequences including human genes, proteins encoded by the gene sequences, and sugar chains that are secondarily produced from these proteins Is being revealed rapidly. The functions of macromolecules such as genes, proteins, and sugar chains whose sequences have been clarified can be examined by various methods. Mainly, for nucleic acids, various nucleic acids / nucleic acid complementarity such as Northern blotting or Southern blotting can be used to examine the relationship between various genes and their biological function expression. With respect to proteins, the function and expression of proteins can be examined using protein / protein reactions, as typified by Western blotting.

  In recent years, a new analysis method or methodology called a DNA microarray method (DNA chip method) has been developed and attracted attention as a method for analyzing many gene expressions at once. In principle, these methods are the same as conventional methods in that they are nucleic acid detection and quantification methods based on nucleic acid / nucleic acid hybridization reactions, and are performed between proteins / proteins or between sugar chains / sugar chains and sugars. It can also be applied to the detection and quantification of proteins and sugar chains based on hybridization between chains / proteins. These techniques have a great feature in that a large number of DNA fragments, proteins, and sugar chains are arranged and fixed at high density on a flat glass substrate called a microarray or chip. Specific usage of the DNA microarray method is, for example, by hybridizing a sample labeled with the expression gene of the cell under study with a fluorescent dye on a flat substrate piece and binding nucleic acids (DNA or RNA) complementary to each other. And a method of reading the location 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. Thus, the amount of each gene in the sample can be quickly estimated.

As a technique for immobilizing nucleic acid on a substrate, poly-L-lysine, aminosilane, etc. are coated on a flat substrate such as a glass slide, and each nucleic acid is dispersed using a spotting device called a spotter. A method for immobilization has been developed (see, for example, Patent Document 1). And as materials for substrates other than glass, plastics such as saturated cyclic polyolefins have been proposed (see, for example, Patent Document 2).
JP 10-503841 A (Claims) 2003-161731 (Claims and Examples)

  However, when oligo DNA is immobilized on a flat glass or plastic substrate, there are the following problems. That is, 1) When hybridization is performed, the sample DNA is easily adsorbed nonspecifically to portions other than the spot where the probe DNA is immobilized, and this nonspecific adsorption is performed when fluorescence detection is performed with an apparatus called a scanner. As a result, there is a problem that the S / N ratio is not sufficient.

  The problem to be solved by the present invention is to provide a carrier on which a selective binding substance with high detection sensitivity is immobilized, preventing the deterioration of the S / N ratio as described above.

  That is, the present invention is a selective binding substance-immobilized carrier in which a selective binding substance is immobilized on the surface of the carrier, the carrier surface is made of a saturated cyclic polyolefin resin, and the carrier is provided with uneven portions. The selective binding substance-immobilized carrier, wherein the selective binding substance is immobilized on the upper surfaces of the plurality of convex portions of the concavo-convex portion.

  According to the present invention, it is possible to provide a carrier on which a selective binding substance having a low non-specific sample adsorption, a good hybridization efficiency, and a good S / N ratio is immobilized.

  Hereinafter, the carrier for immobilizing a selective binding substance of the present invention will be described. In the carrier for immobilizing a selective binding substance of the present invention, the surface of the carrier for immobilizing the selective binding substance needs to be a saturated cyclic polyolefin. The saturated cyclic polyolefin resin used in the present invention has a low background (autofluorescence) and excellent heat resistance and solvent resistance when a detection target is measured with a fluorescent label. Specific examples of the molecular structure include a polymer having a cyclic olefin structure or a saturated polymer obtained by hydrogenating a copolymer of a cyclic olefin and an α-olefin, and preferably represented by the general formula (1). And a hydrogenated product of norbornene ring-opening polymer.

(However, formation in the above formula (1), R ₁ and R ₂ may be the same or different each a hydrocarbon residue having 1 to 10 carbon hydrogen or carbon, also, R ₁ and R ₂ to each other ring You may do it.)
The polymer having a structural unit represented by the general formula (1) is norbornene as a monomer and an alkyl or alkylidene substituted product thereof, specifically, 5-methyl-2-norbornene, 5,6-dimethyl-2- There are norbornene, 5-ethylidene-2-norbornene, etc. In addition, dicyclopentadiene, 2,3-dihydrodicyclopentadiene, and alkyl substituents such as methyl and ethyl are used for ring-opening polymerization. There is a saturated polymer produced by hydrogenating a ring-opened polymer.

  In addition, a polymer of a cyclic olefin monomer represented by the general formula (2), or an α-olefin such as ethylene, propylene, isopropylene, 1-butene, 3-methyl-1-butene, 1-pentene, 1-hexene And a saturated polymer produced by hydrogenating a random copolymer of cyclic olefin monomers represented by the general formula (2).

(However, in the above formula (2), R ₁ and R ₈ is selected from the group consisting of hydrogen and halogen atoms and hydrocarbon residues, R ₅ to R ₈ are each may form a ring.)
Furthermore, a polymer of a cyclic olefin monomer represented by the general formula (3), or α- such as ethylene, propylene, isopropylene, 1-butene, 3-methyl-1-butene, 1-pentene, 1-hexene. A saturated polymer produced by hydrogenating a random copolymer of an olefin and a cyclic olefin monomer represented by the general formula (3) may be used.

(However, in the above formula (2), R ₁ and R ₁₂ is selected from the group consisting of hydrogen and halogen atoms and hydrocarbon residues, R ₉ to R ₁₂ are each may form a ring.)
A base material to be used as a support for immobilizing a biochemically active substance is molded using the saturated polyolefin resin described in the general formulas (1) to (3) as a raw material, but the base material molding method and shape are particularly limited. There is no. In view of moldability, methods such as extrusion molding, compression molding, and injection molding are preferable, and among these, injection molding is most preferable.

  As other resin materials other than cyclic polyolefin, polypropylene is also preferably used since it has less autofluorescence.

  In order to immobilize a selective binding substance on a carrier having a polymer having at least one structural unit represented by the general formula (1), pretreatment is performed to form a carboxyl group or a hydroxyl group on the surface of the carrier. It is preferable to make it. Examples of means for generating a carboxyl group on the surface of the carrier include oxygen plasma, argon plasma, and a method in which the carrier is exposed to radiation. However, since the substrate is less damaged and the amount of carboxyl groups on the surface can be increased, oxygen plasma can be used. The treatment of can be particularly preferably used. In particular, the low-temperature oxygen plasma treatment can be most preferably used because it causes little damage to the substrate.

  When the carboxyl group is generated on the surface of the carrier in this way, it becomes possible to immobilize a selective binding substance having an amino group or a hydroxyl group on the surface of the carrier by a covalent bond. In general, various condensing agents such as dicyclohexylcarbodiimide, N-ethyl-5-phenylisoxazolium-3'-sulfonate are used to facilitate the reaction of these bonds. Among these, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) is less toxic and relatively easy to remove from the reaction system. It is one of the most effective condensing agents for the condensation reaction with a carboxyl group. These condensing agents such as EDC may be used by mixing with a solution of a selective binding substance, or a carrier having a carboxyl group formed on the surface is previously immersed in an EDC solution to activate the surface carboxyl group. You may make it.

  When such a condensing agent is used and the carboxyl group on the support surface is reacted with the amino group of the selective binding substance, the support surface and the selective binding substance are immobilized by an amide bond. When the carboxyl group of this is reacted with the hydroxyl group of the selective binding substance, the support surface and the selective binding substance are fixed by an ester bond. The temperature at which the sample containing the selective binding substance is allowed to act on the carrier is preferably 5 ° C to 95 ° C, more preferably 15 ° C to 65 ° C. The treatment time is usually 5 minutes to 24 hours, preferably 1 hour or longer.

  By immobilizing a selective binding substance on the polymer surface by the method described above, nonspecific analyte adsorption can be suppressed, and the selective binding substance can be immobilized firmly and densely by covalent bonding. Furthermore, a carrier having high hybridization efficiency with a specimen can be obtained for the presumed reason that the spatial freedom of the immobilized selective binding substance is higher than that of glass.

  By the way, when producing a carrier with a polymer containing a structural unit represented by the general formula (1), a fine shape can be obtained by using an injection molding method, a hot embossing method, or the like as compared with glass, ceramic, metal, or the like. It is possible to mass-produce the carrier provided with Therefore, the shape of the carrier on which the selective binding substance is immobilized will be described. It is preferable that the carrier on which the selective binding substance of the present invention is immobilized has a concavo-convex part, and the selectivity compatible substance is immobilized on the upper surface of the convex part. By adopting such a structure, at the time of detection, a non-specifically adsorbed specimen is not detected as described later, so that the selective binding substance substance with low noise and consequently better S / N is obtained. Can be provided. And about the height of the some convex part of an uneven | corrugated | grooved part, it is preferable that the height of the upper surface of a convex part is substantially the same. Here, when the height is substantially the same, a selective binding substance is immobilized on the surface of a convex part having a slightly different height, and this is reacted with a fluorescently labeled analyte, and then scanned with a scanner. The height at which the difference in signal level strength does not matter. Specifically, “the heights are substantially the same” means that the difference in height is less than 100 μm. Furthermore, the carrier of the present invention is preferably provided with a flat portion. Specific examples are shown in FIGS. 1 is a flat portion, and a selective binding substance (for example, nucleic acid) is immobilized on the upper surface of the convex portion of the concavo-convex portion indicated by 2. And it is preferable that the upper surface of the convex part of this uneven | corrugated | grooved part is substantially flat. Here, the fact that the upper surface of the convex portion is substantially flat means that there is no unevenness of 50 μm or more. Furthermore, the height of the upper surface of the convex portion of the concavo-convex portion and the height of the flat portion are substantially the same. Here, the heights of the flat portion and the uneven portion are substantially the same height when the signal level is not lowered when scanned by the scanner. Specifically, the difference in height is substantially the same, which means that the difference between the height of the upper surface of the concavo-convex portion convex portion and the height of the flat portion is smaller than 100 μm.

  That is, in general, a microarray generally reacts a fluorescently labeled specimen with a selective binding substance immobilized on a carrier, and reads fluorescence with a device called a scanner. The scanner condenses the laser light by narrowing down the laser light as excitation light with an objective lens. The condensed light is irradiated on the surface of the microarray, and the laser beam is focused on the surface of the microarray. Under this condition, the fluorescence generated from the microarray is read by scanning the objective lens or the microarray itself.

  When such a scanner is used to scan the carrier on which the selective binding substance is immobilized on the upper surface of the convex portion of the present invention, fluorescence (noise) of the sample DNA adsorbed nonspecifically in the concave portion of the concave and convex portion is detected. Demonstrate the effect that you want. This is because the laser beam is focused on the upper surface of the convex portion, so that the laser beam is defocused in the concave portion. In other words, the difference between the height of the upper surface of the highest convex portion and the height of the upper surface of the lowest convex portion among the plurality of convex portions to which the selective binding substance is immobilized is preferably 50 μm or less. This is because if there is more variation in the height of the upper surface of the convex portion, it may happen that accurate fluorescence intensity cannot be measured due to the depth of focus of the scanner.

  The difference between the height of the upper surface of the highest convex portion and the height of the upper surface of the lowest convex portion among the plurality of convex portions to which the selective binding substance is immobilized may be 50 μm or less, but 30 μm or less. It is more preferable that the height is the same. In addition, the same height as used in this application shall also include the error by the dispersion | variation which generate | occur | produces by production etc.

  The plurality of convex portions on which the selective binding substance is immobilized means a portion on which the selective binding substance (for example, nucleic acid) necessary as data is immobilized, and is simply a dummy selective binding substance. The part where is fixed is excluded.

  In general, the method of adjusting the focus of the scanner is as follows. That is, when the scanner focuses the excitation light on the surface of the microarray, the excitation light is focused at the corner of the microarray, or as shown in FIG. Fit to microarray surface. Then, the entire microarray is scanned under the conditions. Therefore, the carrier of the present invention is particularly preferably provided with an uneven portion and a flat portion. Specific examples are shown in FIGS. Reference numeral 11 denotes a flat portion, and a selective binding substance (for example, nucleic acid) is immobilized on the upper surface of the convex portion of the concavo-convex portion indicated by 12. Furthermore, it is preferable that the difference between the height of the upper surface of the convex portion of the concavo-convex portion and the height of the flat portion is 50 μm or less. In this way, when scanning the carrier on which the selective binding substance is immobilized, it is possible to focus the excitation light once on the upper surface of the flat portion or to hit the flat portion against the jig. . That is, the scanner can be easily focused. Thus, since the excitation light is focused on the flat portion, the upper surface of the convex portion on which the selective binding substance is immobilized is flat, and the difference between the height of the upper surface of the convex portion and the height of the flat portion is the same. Is preferably 50 μm or less.

  If the difference between the height of the upper surface of the convex part and the height of the flat part is larger than 50 μm, the following problems may occur. That is, since the focal point of the excitation light is adjusted on the upper surface of the flat part, if the height of the upper surface of the convex part is different, the focal point of the excitation light on the upper surface of the convex part is blurred. It may happen that no fluorescence due to the reaction between the substance and the analyte is detected. The same thing can occur even when a flat portion having the same height as the upper surface of the convex portion is not provided.

  In addition, when the upper surface of the convex portion is not flat, the focal spot size of the excitation light on the upper surface of the convex portion varies, and as a result, the intensity of fluorescence detected in one upper surface of the convex portion varies. . If this happens, later analysis becomes difficult. In the case of the present application, the above problem does not occur, and a good signal (fluorescence) can be obtained.

  The difference between the height of the upper surface of the convex portion and the height of the flat portion may be 50 μm or less, more preferably 30 μm or less, and the height of the upper surface of the convex portion and the height of the flat portion are the same. It is still preferable if it exists. In addition, the same height as used in this application shall also include the error by the dispersion | variation which generate | occur | produces by production etc.

  In the present invention, the selective binding substance is not spotted on the planar carrier, but is fixed only on the upper surface of the convex portion of the concavo-convex portion. Therefore, even if the specimen sample is non-specifically adsorbed on a portion other than the top surface of the convex portion, the focus of the excitation light is blurred on the portion other than the top surface of the convex portion. No fluorescence is detected. For this reason, the noise is reduced, and as a result, the S / N ratio is improved.

  Moreover, it is preferable that the area of the upper surface of a convex part is substantially the same. By doing in this way, the area of the part by which various selective binding substances are immobilized can be made the same, which is advantageous for later analysis. Here, the area of the upper part of the convex part is substantially the same means that the value obtained by dividing the largest upper surface area in the convex part by the smallest upper surface area is 1.2 or less.

The area of the upper surface of the convex portion is not particularly limited, but is preferably 4 mm ² or less and 10 μm ² or more from the viewpoint that the amount of the selective binding substance can be reduced and handling is easy.

  The height of the convex portion in the concavo-convex portion is preferably 0.01 mm or more and 1 mm or less. If the height of the convex portion is lower than this, a non-specifically adsorbed specimen sample other than the spot may be detected, resulting in a poor S / N. Further, when the height of the convex portion is 1 mm or more, there may be a problem that the convex portion is easily broken and damaged.

  Moreover, it is preferable that the conductive material is provided at least on the side surface of the convex portion. In this case, for example, in the case of nucleic acid, hybridization can be accelerated by providing a counter electrode and applying a current and voltage between the counter electrode and the conductive material. Preferred regions to be coated with the conductive material are all the concave portions and all the side surfaces of the convex portions. An example is shown in FIG.

  As a range of the voltage to be applied, a range of 0.01 V or more and 2 V or less is preferable when a current flows. A particularly preferable range is 0.1 V or more and 1.5 V or less. From this, when a large voltage is applied, water causes electrolysis, which may adversely affect the surface selective binding substance. The material of the conductive material is not particularly limited, but carbon, magnesium, aluminum, silicon, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, tin, zirconium, niobium, molybdenum, palladium, silver, hafnium, Examples include tantalum, tungsten, platinum, gold, stainless steel, mixtures thereof, and conductive polymers. Among these, platinum, gold, and titanium are particularly preferably used. Examples of a method for forming these conductive material films include vapor deposition, sputtering, CVD, and plating.

When the conductive material is coated on the convex portion as described above, it is preferable to further provide a layer of an insulating material other than the upper surface of the convex portion. When there is a layer of insulating material, it is possible to draw the subject only on the upper surface of the convex portion when a current is passed. Examples of the insulating material include metal oxides (for example, Al—O, SiO ₂ , TiO ₂ , VO, SnO, Cr—O, Zn—O, GeO ₂ , Ta ₂ O ₅ , ZrO ₂ , Nb—O). , Y etc. ₂ O _3), nitride _{(Al-N, Si 3 N} 4, TiN, Ta-N, Ge-N, Zr-N, NbN , etc.), sulfides (ZnS, PbS, SnS, CuS), An insulating polymer may be mentioned.

  The selective binding substance-immobilized carrier obtained by the above-described method can be appropriately treated after fixing the selective binding substance. For example, the fixed selective binding substance can be denatured by performing heat treatment, alkali treatment, surfactant treatment, or the like.

  In addition, the selective binding substance-immobilized carrier is generally subjected to a hybridization reaction between a fluorescently labeled specimen and the selective binding substance immobilized on the carrier, and the fluorescence is read by a device called a scanner. The scanner condenses the laser light by narrowing down the laser light as excitation light with an objective lens. However, when autofluorescence is generated from the surface of the carrier, the emitted light becomes noise and may lead to a decrease in detection accuracy. In order to prevent this, the polymer having the structural unit of the general formula (1) exhibits a black color and contains a substance that does not emit light by laser irradiation to make the surface black, thereby reducing autofluorescence from the carrier itself. This is preferable. By using such a carrier, autofluorescence from the carrier can be reduced at the time of detection, so that a carrier with a selectively binding substance substance immobilized with a smaller noise and a better S / N ratio is provided. can do.

  Here, the black carrier means that the spectral reflectance of the black part of the carrier does not have a specific spectral pattern (such as a specific peak) in the visible light (wavelength range of 400 nm to 800 nm) and is a uniformly low value. In addition, the spectral transmittance of the black portion of the carrier also has a specific spectral pattern and is uniformly low.

  As the values of the spectral reflectance and the spectral transmittance, the spectral reflectance in the range of visible light (wavelength is 400 nm to 800 nm) is 7% or less, and the spectral transmittance in the same wavelength range is 2% or less. Is preferred. Note that the spectral reflectance here refers to the spectral reflectance when regular reflected light from a carrier is captured in an illumination / light-receiving optical system conforming to JIS Z 8722 Condition C.

  As a means for blackening, it can be achieved by adding a black substance to the carrier. Preferred examples of the black substance include carbon black, graphite, titanium black, aniline black, Ru, Mn, Ni, Cr, Black materials such as Fe, Co and Cu oxides, carbides of Si, Ti, Ta, Zr and Cr can be used.

  These black materials can be contained alone or in combination of two or more. Among these black materials, carbon black, graphite, and titanium black can be preferably contained, and carbon black can be particularly preferably used because it can be uniformly dispersed in the polymer.

  In addition, the selective binding substance immobilization comprising a polymer having at least one structural unit represented by the general formula (1) on a support layer composed of a material that is difficult to be thermally deformed such as glass or metal as the shape of the carrier. It is preferable to provide a chemical layer because it prevents the shape of the carrier from changing due to heat or external force. This conceptual diagram is shown in FIG. As a support body layer, metals, such as glass and iron, chromium, nickel, titanium, stainless steel, are preferable. In order to improve the adhesion between the support layer and the selective binding substance fixing layer, the surface of the support layer is subjected to a plasma treatment with argon, oxygen or nitrogen gas or a treatment with a silane coupling agent. It is preferable. Examples of such silane coupling agents include 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane, 3- (2-aminoethylaminopropyl) trimethoxysilane, 3- (2-aminoethylaminopropyl) dimethoxymethylsilane, 3-mercaptopropyltrimethoxysilane, dimethoxy-3-mercaptopropylmethylsilane and the like. As a means for providing the selective binding substance-immobilized layer on the support layer, known means such as spin coating and dipping can be used by dissolving the polymer in an organic solvent. More simply, it can be attached to the support layer with an adhesive.

  Here, the “selective binding substance” means a substance that can selectively bind to a test substance directly or indirectly, and representative examples thereof include nucleic acids, proteins, saccharides and other antigenic properties. A compound can be mentioned. The nucleic acid may be DNA, RNA or PNA. 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. Falls under the category of Examples of the protein include an antibody and an antigen-binding fragment of an antibody such as a Fab fragment or F (ab ′) 2 fragment, and various antigens. Since an antibody or an antigen-binding fragment thereof selectively binds with a corresponding antigen, and the antigen selectively binds with a corresponding antibody, it corresponds to a “selective binding substance”. As the saccharide, a polysaccharide is preferable, and various antigens can be exemplified. In addition, substances having antigenicity other than proteins and saccharides can be immobilized. The selective binding substance used in the present invention may be a commercially available substance or may be obtained from a living cell or the like. Particularly preferred as the “selective binding substance” is a nucleic acid. Among these nucleic acids, nucleic acids called oligonucleic acids having a length of 10 to 100 bases can be easily artificially synthesized with a synthesizer, and the amino group at the end of the nucleic acid can be easily modified. It is preferable because it can be easily fixed on the surface of the carrier. Furthermore, if it is less than 20 bases, 20 to 100 bases are more preferable from the viewpoint that the stability of hybridization is low. In order to maintain the stability of hybridization, the range of 40 to 100 bases is particularly preferable.

  Examples of the test substance used in the measurement method using the carrier of the present invention include nucleic acids to be measured, for example, genes such as pathogenic bacteria and viruses, causative genes of genetic diseases, and a part thereof, and various living organisms having antigenicity. Examples include components, antibodies against pathogenic bacteria, viruses and the like, but are not limited thereto. Examples of specimens containing these test substances include body fluids such as blood, serum, plasma, urine, feces, spinal fluid, saliva, various tissue fluids, various foods and beverages, and dilutions thereof. It is not limited to these. Moreover, the nucleic acid to be the test substance may be labeled with a nucleic acid extracted from blood or cells by a conventional method, or may be 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 nucleotide 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 a conventional method, or the test substance antigen or antibody may be bound to a selective binding substance. Thereafter, the carrier is washed, and a labeled antibody or antigen that reacts with the antigen or antibody with an antigen-antibody is reacted, and the label bound to the carrier can be measured.

  The step of causing the immobilized substance and the test substance to interact can be performed in the same manner as in the past. The reaction temperature and time are appropriately selected according to the chain length of the nucleic acid to be hybridized, the type of antigen and / or antibody involved in the immune reaction, and in the case of nucleic acid hybridization, usually 50 ° C. to 70 ° C. In the case of an immune reaction, it is usually from room temperature to about 40 ° C. for about 1 minute to several hours.

  The invention is illustrated in more detail by the following examples. However, the present invention is not limited to the following examples.

Example 1
(Preparation of DNA immobilization carrier)
A mold for injection molding was prepared using a known method LIGA (Lithographie Galvanoformung Abformung), and a substrate having a shape as described later was injection molded by the injection molding method. As a substrate material, a hydrogenated random copolymer of ethylene and dicyclopentadiene was used. Carbon black (Mitsubishi Chemical # 3050B) is contained in the substrate at a ratio of 1% by weight, and the substrate is black. When the spectral reflectance and the spectral transmittance of this black substrate were measured, the spectral reflectance was 5% or less at any wavelength in the visible light region (wavelength 400 nm to 800 nm), and in the same range of wavelengths, The transmittance was 0.5% or less. Neither spectral reflectance nor spectral transmittance had a specific spectral pattern (such as a peak) in the visible light region, and the spectrum was uniformly flat. Note that the spectral reflectance is a spectrum obtained when specularly reflected light from a substrate is captured using an apparatus (manufactured by Minolta Camera, CM-2002) equipped with an illumination / light-receiving optical system conforming to the condition C of JIS Z 8722. The reflectance was measured.

  The shape of the substrate was 76 mm in length, 26 mm in width, and 1 mm in thickness, and the surface was flat except for the central portion of the substrate. In the center of the substrate, there is a recessed portion having a diameter of 10 mm and a depth of 0.2 mm. In this recess, 64 (8 × 8) convex portions having a diameter of 0.2 mm and a height of 0.2 mm are provided. Provided. When the height difference between the height of the upper surface of the convex portion of the concavo-convex portion (the average value of the heights of the 64 convex portions) and the flat portion was measured, it was 3 μm or less. Further, the height variation of the 64 convex upper surfaces (the difference in height between the highest convex upper surface and the lowest convex upper surface), and further, the average height of the convex upper surface and the flat portion The difference in height of the upper surface was measured and found to be 3 μm or less. Furthermore, the pitch (the distance from the central portion of the convex portion to the central portion of the adjacent convex portion) of the concave and convex portion was 0.6 mm.

And this board | substrate was set to the vacuum chamber of the reactive ion etching apparatus (Samco Co., Ltd. product: RIE-10NR). Then, after high vacuum evacuation to 1 × 10 ⁻³ Pa, the substrate was exposed to oxygen plasma for 30 seconds under the conditions of an oxygen flow rate of 50 sccm, a pressure of 10 Pa, and an input power of 100 W. In this way, carboxyl groups were generated on the substrate surface.

(Immobilization of probe DNA)
And DNA of sequence number 1 (60 bases, 5 'terminal amination) was synthesize | combined. This DNA is aminated at the 5 ′ end. This DNA was dissolved in pure water at a concentration of 0.27 nmol / μl to obtain a stock solution. When spotted on the substrate, PBS _{(8 g} of NaCl, dissolved Na ₂ HPO 4 · 12H ₂ O and 2.9 g, the KCl 0.2g, a _KH 2 PO ₄ in 0.2g deionized water, for pH adjustment Hydrochloric acid added to make up to 1 liter, pH 5.5) to a final probe concentration of 0.027 nmol / μl, and condensation of the carboxyl group on the carrier surface with the amino group at the end of the probe DNA Therefore, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added to make the final concentration 50 mg / ml. Then, these mixed solutions were taken in a glass capillary, and this probe DNA was spotted at four locations on the convex portion of the substrate under a microscope. Next, the substrate was placed in a sealed plastic container, incubated at 37 ° C. and 100% humidity for about 20 hours, and then washed with pure water.

(Adjustment of sample DNA)
As the sample DNA, DNA of SEQ ID NO: 2 (968 bases) having a base sequence capable of hybridizing with the DNA-immobilized substrate was used. The adjustment method is shown below.

  The DNAs of SEQ ID NO: 3 and SEQ ID NO: 4 were synthesized. This was dissolved in pure water to a concentration of 100 μM. Next, pKF3 plasmid DNA (Takara Bio Inc. product number: 3100) (SEQ ID NO: 5: 2264 bases) was prepared, and this was used as a template. Amplification was performed by Polymerase Chain Reaction.

  The conditions for PCR are as follows. That is, ExTaq 2 μl, 10 × ExBuffer 40 μl, dNTP Mix 32 μl (the above is attached to product number RR001A manufactured by Takara Bio Inc.), SEQ ID NO: 3 solution 2 μl, SEQ ID NO 4 solution 2 μl, Template (SEQ ID NO 5 0.2 μl was added, and the volume was made up to 400 μl with pure water. These mixed solutions were divided into four microtubes, and PCR reaction was performed using a thermal cycler. This was purified by ethanol precipitation and dissolved in 40 μl of pure water. When a part of the solution after the PCR reaction was taken and confirmed by electrophoresis, it was confirmed that the base length of the amplified DNA was about 960 bases and that SEQ ID NO: 2 (968 bases) was amplified.

  Next, a 9-base random primer (manufactured by Takara Bio Inc .; product number 3802) was dissolved at a concentration of 6 mg / ml, and 2 μl was added to the DNA solution purified after the PCR reaction. This solution was heated to 100 ° C. and then rapidly cooled on ice. 5 μl of buffer attached to Klenow Fragment (Takara Bio Inc .; product number 2140AK) and 2.5 μl of dNTP mixture (dATP, dTTP, and dGTP concentrations of 2.5 mM and dCTP concentration of 400 μM, respectively) were added thereto. . Furthermore, 2 μl of Cy3-dCTP (manufactured by Amersham Pharmacia Biotech; product number PA53021) was added. To this solution, 10 U of Klenow Fragment was added and incubated at 37 ° C. for 20 hours to obtain sample DNA labeled with Cy3. Since the random primer is used for labeling, the length of the sample DNA varies. The longest sample DNA is SEQ ID NO: 2 (968 bases). When the sample DNA solution was taken out and confirmed by electrophoresis, the strongest band appeared in the vicinity corresponding to 960 bases, and the area corresponding to a shorter base length was thinly smeared. This was purified by ethanol precipitation and dried.

  This labeled sample DNA is dissolved in 1% by weight BSA (bovine serum albumin), 5 × SSC (5 × SSC is 43.8 g of NaCl and trisodium citrate hydrate is dissolved in 22.1 g of pure water. 200 ml of NaCl, 43.8 g of NaCl and 22.1 g of trisodium citrate hydrate dissolved in pure water, and 1 ml of the mess up to 1 l. The solution is expressed as 10 × SSC, and the 5-fold diluted solution is expressed as 0.2 × SSC.), 0.1% (w / v) SDS (sodium dodecyl sulfate), 0.01% (w / v) salmon sperm DNA Solution (each concentration is a final concentration) was dissolved in 400 μl to obtain a hybridization solution.

(Hybridization)
10 μl of the DNA solution prepared in the above (Preparation of sample DNA) is taken out, and 30 μl of 1% (w / v) BSA, 5 × SSC, 0.1% (w / v) SDS, 0.01% (W / v) Salmon sperm DNA solution was added to make a total of 40 μl. And this was dripped at the uneven | corrugated | grooved part of the support | carrier with which DNA mentioned above was fix | immobilized, and the cover glass was carefully covered. Then, the periphery of the cover glass was sealed with paper bond to prevent the hybridization solution from drying. That is, the molecular weight of the sample DNA was the same as in Example 1 and Comparative Example 1. This was put in a plastic container and incubated at a humidity of 100% and a temperature of 45 ° C. for 10 hours. After incubation, the cover glass was peeled off, washed and dried.

(Measurement)
The carrier after the above treatment was set on a DNA chip scanner (Axon Instruments GenePix 4000B), and measurement was performed with the laser output set to 33% and the photomultiplier voltage set to 500. The results are shown in Table 1. Here, the fluorescence intensity is the average value of the fluorescence intensity in the spot, and the noise is the average value of the fluorescence intensity around the spot (portion where DNA is not spotted). The results are shown in Table 1.

Comparative Example 1
A substrate made of a hydrogenated product of a random copolymer of flat ethylene and dicyclopentadiene was injection molded. This substrate is the same as that of Example 1 except that it is flat. In the same manner as in Example 1, carboxyl group generation, probe DNA immobilization, specimen preparation, hybridization, and measurement were performed. The results are shown in Table 1. As can be seen from this, the signal intensity hardly changes between the two substrates, but it can be seen that the noise is reduced and the S / N can be improved as a result of the uneven shape.

Example 2
Next, an experiment was performed for the case where the height of the convex portion varied. The convex part of the injection molded product used in Example 1 was shaved with wrapping paper, and a difference was provided in the height of the upper surface of the convex part. That is, there are carriers (carriers) having protrusions (4 places) lower by 30 μm than the upper surfaces of other protrusions (reference protrusions), and protrusions (4 places) lower by 50 μm than the upper surfaces of the other protrusions. A certain carrier (carrier a) was produced. The difference between the height of the upper surface of the convex portion (reference convex portion) other than the low portion of the carrier and the height of the flat portion was 3 μm or less. In the same manner as in Example 1, the probe DNA to be spotted was adjusted. Next, in the same manner as in Example 1, the probe DNA solution was spotted at four locations on the upper surface of the reference convex portion and at four locations on the lower convex surface. Furthermore, DNA preparation for hybridization and hybridization operation were performed in the same manner as in Example 1, and measurement was performed in the same manner as in Example 1. Table 2 shows the average value of the fluorescence intensity on the upper surface of the convex portion serving as a reference and the average value of noise around it, the average value of the fluorescence intensity on the upper surface of the convex portion having a low height, and the average value of noise around it.

Thus, it can be seen that a sufficiently large S / N is obtained compared to the comparative example even if the height of the convex portion varies (50 μm or less).
Example 3
Furthermore, the case where there was a difference between the upper surface of the convex part and the flat part was examined. The flat part of the injection-molded product used in Example 1 was scraped with wrapping paper, and two types of carriers having a difference in height between the upper surface of the flat part and the upper surface of the convex part of 30 μm (carrier c) and 50 μm (carrier d) were produced. . That is, the height of the convex portion of the carrier c is 30 μm higher than the height of the flat portion. In the same manner as in Example 1, adjustment of the spotted probe DNA, spotting of the probe DNA solution on the upper surface of the convex portion, adjustment of the hybridization DNA, and hybridization were performed, and measurement was performed in the same manner as in Example 1. It was. In addition, the convex part which spotted the DNA solution on the upper surface is four places about each board | substrate. And the fluorescence intensity of the spot (4 places) on which DNA was spotted and the average value of the surrounding noise (4 places) were determined. The results are shown in Table 3.

  Thus, it can be seen that even when there is a difference in height between the upper surface of the flat part and the upper surface of the convex part (50 μm or less), a sufficiently large S / N is obtained as compared with the comparative example.

Schematic diagram of the carrier of the present invention Schematic cross-sectional view of the carrier of the present invention Example of jig for microarray abutment Sectional view of carrier irregularities when electrodes are formed Conceptual diagram of a carrier having a support layer and a selective binding substance immobilization layer

Explanation of symbols

3 Support layer (glass)
4 selective binding substance immobilization layer 11 flat portion 12 uneven portion 13 microarray 14 objective lens 15 excitation light 16 spring 21 for abutting the microarray to the jig convex portion upper surface 22 conductive film 23 insulating film

Claims (7)

A selective binding substance-immobilized support in which a selective binding substance is immobilized on the surface of the support, wherein the support surface is made of a saturated cyclic polyolefin resin, and the support is provided with uneven portions, and the selective binding substance Is immobilized on the upper surfaces of a plurality of convex portions of the concavo-convex portion. 2. The selective binding substance immobilization according to claim 1, wherein the upper surface of the convex part of the uneven part is substantially flat, and the height of the upper surface of the convex part to which the selective binding substance is immobilized is substantially the same. Carrier. The selective binding substance-immobilized carrier according to claim 1 or 2, wherein the carrier is provided with a flat portion. 4. The difference between the height of the highest convex portion and the height of the lowest convex portion among the plurality of convex portions to which the selective binding substance is immobilized is 50 μm or less. The selective binding substance-immobilized carrier according to claim 1. The selective binding substance-immobilized carrier according to any one of claims 1 to 4, wherein the difference between the height of the upper surface of the convex portion of the concave and convex portion and the height of the flat portion is 50 µm or less. 6. The selective binding substance-immobilized carrier according to claim 1, wherein at least the surface of the carrier is black. The selective binding substance-immobilized carrier according to claim 6, comprising carbon black.

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