JP2006184056A - Method for manufacturing selective-bonding substance immobilization carrier - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enhance yield in manufacturing a carrier with a selective-bonding substance immobilized on its projection parts by using pins for immobilizing a selective-bonding substance. <P>SOLUTION: A method is provided for immobilizing the selective-bonding substance on the plurality of projection-part upper surfaces among uneven parts on the carrier by using the pins for immobilizing the selective-bonding substance. This method for manufacturing the selective-bonding substance immobilization carrier is characterized in that the area of each pin head for immobilizing the selective-bonding substance is larger than the area of each projection-part upper surface. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing 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, with respect to nucleic acids, the relationship between various genes and their expression of biological functions can be examined by utilizing complementarity between various nucleic acids / nucleic acids such as Northern hybridization or Southern hybridization. With respect to proteins, the function and expression of proteins can be examined using protein / protein reactions, as typified by Western hybridization.

  In recent years, an 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. As a specific method of using the microarray method, for example, a sample obtained by labeling a gene expressed in a cell to be studied with a fluorescent dye or the like is hybridized on a flat substrate piece, and complementary nucleic acids (DNA or RNA) are bound to each other. There are a method of reading the part 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, it is called a spotter having a pin for immobilizing nucleic acid by coating poly-L-lysine, aminosilane, etc. on a flat substrate such as a glass slide. A method of immobilizing each nucleic acid using this pin using an attachment device has been developed (see, for example, Patent Document 1).

  In addition, in order to obtain better S / N (signal / noise), a carrier for immobilizing DNA or the like on a convex portion instead of a flat substrate has also been proposed (see, for example, Patent Document 2). Such a substrate suppresses noise around the spot and has high performance. However, when a selective binding substance is immobilized on the upper surface of the convex portion using a pin, a pin head position having a diameter of about 100 μm and a diameter of about 100 μm are used. It is necessary to align the position of the upper surface of the convex part. If this alignment is insufficient, the pin head contacts only a part of the convex part, and a selective binding substance such as DNA is placed only on the contacted part. I can't. In the worst case, there is a problem that the upper surface of the convex portion and the pin head do not come into contact with each other, and DNA or the like cannot be placed on the upper surface of the convex portion.

  In order to solve such a problem, for example, using a spotter equipped with imaging means such as a camera, position information is calculated from image information from the imaging means, and the pin head is calculated based on the position information. It is also conceivable to use a spotter that controls the position of (see Patent Document 3, for example).

However, a spotter having such a photographing means is complicated and expensive. Furthermore, in order to increase productivity at the time of spotting, it is a regular method to spot using a plurality of pins at once, but in this case even if using a spotter equipped with photographing means and means for calculating position information The problem that occurred could not be solved. This problem is as follows. That is, a plurality of pins are generally fixed to a jig called a pin block. The pitch of the pin fixed to the fixing jig (pin block) and the pitch of the convex portion of the carrier are designed to be the same, but a manufacturing error of about several tens of microns of the pin block or the substrate is inevitable. . Therefore, even if the position of one pin head of a plurality of pins and the top surface of the convex portion is accurately aligned, the position of the other pins and the top surface of the convex portion cannot be accurately aligned as shown in FIG. 1 due to this error. There is a problem that a spot where DNA or the like can be immobilized is formed only on a part of the upper surface of the convex portion, which becomes a defective spot. That is, there is a problem that the yield during production is very low.
JP 10-503841 A (Claims) JP 2004-264289 A (Claims) JP 2003-149094 A (Claims)

  The problem to be solved by the present invention is to provide a method capable of reliably immobilizing DNA or the like on the upper surface of a convex portion with a simple apparatus. Furthermore, when DNA is fixed on the upper surface of the convex portion using a plurality of pins as described above, the yield in production is improved due to the positional deviation of the pin head due to the manufacturing error of the substrate or the error of the pin position. It is.

  That is, the present invention is a method for immobilizing a selective binding substance on the upper surfaces of a plurality of convex portions of a concavo-convex portion on a carrier using a pin for immobilizing the selective binding substance, wherein the selective binding substance is immobilized. This is a method for producing a selective binding substance-immobilized carrier, wherein the area of the pin head for use is larger than the area of the upper surface of the convex portion.

  According to the present invention, there is provided an efficient method for producing a carrier on which a selective binding substance having a small amount of non-specific analyte adsorption, a good hybridization efficiency, and a good S / N ratio is immobilized. Can be provided. Specifically, it is possible to provide a manufacturing method capable of reliably immobilizing a selective binding substance typified by DNA on the entire upper surface of a plurality of convex portions of a concavo-convex portion on a substrate, and significantly improving the production yield. it can.

  Hereinafter, a method for producing an immobilization carrier for a selective binding substance of the present invention will be described.

  The carrier on which the selective binding substance of the present invention is immobilized has a concavo-convex part, and the selective compatibility 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.

  When the selective binding substance fixing pin is used for fixing on the upper surface of the convex portion of the carrier, the present invention requires that the area of the pin head of the fixing pin is larger than the area of the upper surface of the convex portion. . The effect will be described with reference to FIGS. FIG. 1 shows a case where a solution of a selective binding substance such as DNA is spotted on the upper surface of a convex portion of a substrate using a plurality of pins, and shows a case where the area of the pin head and the upper surface of the convex portion are the same. It shows a case where the position of the pin 12a and the upper surface of the convex portion 14a directly below it are aligned. Even if the positions of the pin head of 12a and the upper surface of the convex portion of the substrate of 14a are matched, another pin head position (shown by 12b) and the upper surface of the convex portion shown by 14b are caused by the error of the pin block 11 for fixing the substrate 13 and the pin. Is often out of position. Thus, if the position of the pin head and the upper surface of the convex portion is shifted, it is spotted only on a part of the convex portion, resulting in a defective product, resulting in a problem that the yield at the time of manufacture becomes very poor. When the upper surface of the convex portion is a circle, the diameter is approximately 50 to 400 μm. Therefore, if there is an error of several tens of microns in the position of the pin head or the position of the substrate, the above-described problem occurs.

  Such a problem can be solved at once by increasing the area of the pin head, as shown in FIG. Some positional errors can be absorbed by making the area of the pin head larger than the area of the upper surface of the convex portion. The relationship between the area Sp of the pin head and the area Ss of the upper surface of the convex portion is preferably between Sp / Ss ≧ 1.4. In this figure, if the shape of the upper surface of the convex portion and the shape of the pin head are circles, the range of Rp and Rs of each diameter is approximately Rp / Rs ≧ 1.2. Within this range, position errors due to pin blocks and the like can be absorbed. The upper limit of Sp / Ss and Rp / Rs is preferably 25 ≧ Sp / Ss, that is, 5 ≧ Rp / Rs. Particularly preferably, 9 ≧ Sp / Ss, that is, 3 ≧ Rp / Rs. Within this range, spotting is possible without greatly separating the distance between the adjacent convex portion and the upper surface of the adjacent convex portion (that is, without reducing the spot density of the chip).

  As the shape of the pin, it is possible to use a capillary having a capillary formed on the pin itself like a fountain pen and holding a solution of a selective binding substance therein, and such a capillary is formed. No pins can be used. In the case of a pin that does not have a capillary tube, since a certain amount of liquid can be dropped on the upper surface of the convex portion, a pin head having a single-letter or cross-shaped groove can be preferably used.

  When actually spotting, a large number of substrates are arranged on the stage of a spotting device (spotter). Next, the tip of the selective binding substance is spotted by immersing the pin tip in a solution of the selective binding substance (hereinafter referred to as probe solution), moving the pin to the target convex part, and lightly contacting the pin tip and the upper surface of the convex part. Do. In the case of a pin that does not form a capillary tube, every time it is spotted, the operation of “immerse the tip of the pin in the probe solution → spot on the top surface of the convex portion” is repeated to spot the same type of probe solution on all the substrates. After that, the pins are washed and the next probe solution spotted. On the other hand, in the case of a pin formed with a capillary, the pin is immersed in the probe solution and the solution is held in the capillary. However, if the substrate is brought into contact with the substrate as it is, a large excess of the probe solution flows out, so the dummy substrate is tapped several times to discard the excess probe solution (tapping). Next, the probe tip is spotted by moving the pin tip to the desired convex portion and bringing the upper surface of the convex portion into contact with the pin tip lightly. And a spot is continuously performed on the convex part of the following board | substrate. After spotting the same type of probe solution on all substrates, wash the pins, discard the remaining probe solution, and perform the operation of `` holding the next probe solution in the capillary → tapping → spot DNA spot → washing ''. Will repeat.

  The position of the pin head and the convex portion of the substrate is calculated by calculating position information from image information from the image pickup means using a spotter equipped with image pickup means such as a camera, and the position of the pin head is determined based on the position information. A controllable spotter may be used, and the positions of the pin head and the upper surface of the convex portion may be photographed with a micro camera or the like, and fine adjustment may be performed while visually confirming.

  As the composition of the probe solution, a selective binding substance serving as a probe dissolved in a buffer solution such as SSC (standard saline-citrate buffer solution) can be used. The concentration of the selective binding substance is preferably in the range of 1 to 100 μM. Especially preferably, it is the range of 10-50 micromol. In the probe solution, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 4- (4,6-dimethoxy-) is obtained depending on the combination of the functional group of the substrate surface and the functional group of the selective binding substance. A condensing agent such as 1,3,5-triazin-2-yl) -4-methyl-morpholinium chloride (DMT-MM) can also be preferably contained. In addition, when a probe solution such as sodium dodecyl sulfate (SDS) is included in the probe solution, the probe solution “extends” on the upper surface of the convex portion, and a spot without unevenness can be obtained. It can be particularly preferably used.

  As a method for placing the substrate on the stage, a method as shown in FIG. 3 can be preferably used. That is, the reference surface of the substrate is pressed against the abutment 33. The pressing against the abutment can be achieved, for example, by pressing the substrate with a spring as shown by 34. It is preferable to arrange a large number of sets of abutments and springs on the stage and set the substrate on the stage.

  Next, the shape of the carrier will be described. Although the selective binding substance is immobilized on the plurality of convex portions of the concavo-convex portion, the height of the upper surface of the convex portion is preferably substantially the same with respect to this height. 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 substantially same height means that the difference in height is less than 100 μm, and more preferably 50 μm or less.

  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 part, it may happen that accurate fluorescence intensity cannot be measured due to the depth of focus of the scanner, and the difference in height during spotting may cause unexpected variation. It can be a cause.

  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 the microarray is abutted against a jig 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 41 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 42. 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.

  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.

  In the selective binding substance-immobilized carrier of the present invention, the carrier surface is preferably made of a low autofluorescent resin in order to immobilize the selective binding substance. Here, the low autofluorescent resin is a 1 mm thick clean flat plate using GenePix 4000B manufactured by Axon Instruments, under conditions of excitation wavelength of 532 nm, photomultiplier setting gain of 700, and laser power of 33%. When the fluorescence intensity is 1000 or less. Resins that do not satisfy this condition are not preferable because the S / N ratio during detection deteriorates. Examples of such a low autofluorescent resin include a polymer represented by the following general formula (1).

(R1, R2, and R3 in the general formula (1) represent an alkyl group, an aryl group, or a hydrogen atom.)
A homopolymer or a copolymer is used as the polymer. The polymer uses at least one type of monomer as a raw material, and the monomer includes a double bond that can participate in polymerization and a functional group that can participate in polycondensation, and a form of a ketone or a carboxylic acid or a derivative thereof. Exists. The polymer preferably has a structure represented by the general formula (1).

  In addition, when the said polymer is a copolymer, it is preferable to contain the structural unit represented by General formula (1) 10% or more of all the monomer units. When the content of the structural unit represented by the general formula (1) is 10% or more, many carboxyl groups can be generated on the surface and many probe nucleic acids can be immobilized in steps as described later. As a result, the S / N ratio is further improved.

  In the present invention, the polymer means a polymer having a number average degree of polymerization of 50 or more. The preferred range of the number average degree of polymerization of this polymer is 100 to 10,000. Particularly preferably, it is 200 or more and 5000 or less. The number average degree of polymerization can be easily measured by measuring the molecular weight of the polymer by a conventional method using GPC (gel permeation chromatograph).

In the general formula (1), R1 and R2 each represents an alkyl group, an aryl group or a hydrogen atom, and may be the same or different. The alkyl group may be linear or branched and preferably has 1 to 20 carbon atoms. The aryl group preferably has 6 to 18, more preferably 6 to 12 carbon atoms. Functional group X O, NR3, or chosen arbitrarily from among CH _2. R3 is a functional group defined in the same manner as R1 and R2.

  Preferred examples of the polymer containing various functional groups include polymethyl methacrylate (PMMA), polyethyl methacrylate (PEMA), polypropyl methacrylate polyalkyl methacrylate (PMMA), and the like. Among these, polymethyl methacrylate is most preferable because it can be easily molded by injection molding or hot embossing and has a relatively high glass transition temperature. Furthermore, polyvinyl acetate, polycyclohexyl methacrylate, polyphenyl methacrylate, or the like can also be used. Further, a copolymer having a structure in which the constituent elements of the polymer are combined or the constituent elements of the polymer are added with one or more kinds of constituent elements of the polymer can also be used. Examples of the other polymer include polystyrene.

  In the case of a copolymer, the range of the ratio of each component is preferably 10 mol% or more in the proportion of the monomer containing a carbonyl group, for example, alkyl methacrylate. By doing so, a large amount of carboxylic acid can be generated on the surface and a large amount of probe nucleic acid can be immobilized. As a result, the S / N ratio is further improved. Among the structural units of the polymer, the more preferable proportion of the monomer is 50 mol% or more.

  Furthermore, in the present invention, 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), it is preferable to pre-treat this with an alkali or an acid. By doing so, a carboxyl group can be formed on the surface of the carrier. . As a means for generating a carboxyl group on the surface of the carrier, not only a method of treating with an alkali or an acid is used alone, but also a method of exposing the carrier to ultrasonic treatment, oxygen plasma, argon plasma, radiation, etc. in warm. You may combine. Among these methods, it is preferable to generate a carboxyl group on the surface by immersing the carrier in an alkali or acid whose temperature has been increased from the viewpoint that the carrier is less damaged and can be easily carried out. As a specific example, if the carrier is immersed in an aqueous solution of sodium hydroxide or sulfuric acid (preferred concentration is 1N to 20N), preferably at a temperature of 30 ° C. to 80 ° C. and held for 1 to 100 hours. Good.

  It can be confirmed by XPS (X-ray photoelectron spectroscopy) whether or not a carboxyl group is generated on the surface of the carrier by the above method. Specifically, the carboxyl group is labeled with fluorine using a labeling reagent containing fluorine (for example, trifluoroethanol). Then, it is possible to estimate the functional group amount in consideration of the reaction rate with respect to the C1s and F1s peak area intensity in the labeled sample. To further improve the accuracy, the surface of the sample labeled with trifluoroethanol is observed with TOF-SIMS (time-of-flight secondary ion mass spectrometry) to generate carboxyl groups on the surface of the carrier. You can check whether you are doing.

  If the carboxyl group is generated on the surface of the carrier in this way, using this as a foothold, the carrier side is modified with biotin or avidin, the selective binding substance is modified with avidin or biotin, and selective binding is achieved by the interaction between avidin and biotin. It is also conceivable to immobilize the substance on the carrier, or to react the carrier side with a linker such as ethylenediamine, and further react and immobilize this linker with a selective binding substance. However, in these methods, since a two-step reaction is performed, the selective binding substance that can be immobilized on the carrier tends to be less due to the reaction yield. Therefore, it is preferable to immobilize the selective binding substance by directly reacting the carboxyl group of the carrier with the functional group of the selective binding substance. That is, it is preferable to fix the carboxyl group on the surface of the carrier and the amino group or hydroxyl group of the selective binding substance 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. Another promising condensing agent is 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methyl-morpholinium chloride (DMT-MM). 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 above-described method, since a carboxyl group having a negative charge is present except for the spot portion, a non-specific sample (typically DNA) is not present. It is estimated that the selective binding substance can be immobilized firmly and densely by covalent bond, and the degree of spatial freedom of the immobilized selective binding substance is higher than that of glass. For this reason, it is possible to obtain a carrier having a high hybridization efficiency with the specimen. The advantage of the high degree of spatial freedom is that the efficiency of hybridization with a specimen is greatly improved, particularly when the immobilized selective binding substance is DNA having a base length of 10 to 100 bases called oligo DNA. Gives excellent characteristics.

  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. As a support body layer, metals, such as glass and iron, chromium, nickel, titanium, stainless steel, are preferable. In addition, as the support layer, a resin that can withstand relatively high temperatures such as polycarbonate, polyimide, and polyamide may be used. 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, antigenic substances 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 and treatment of DNA immobilization carrier)
A mold for injection molding was produced using a known method LIGA (Lithographie Galvanoformung Abformung), and a substrate made of PMMA having a shape as described later was obtained by the injection molding method. The average molecular weight of PMMA used in this example is 150,000, PMMA contains 1% by weight of carbon black (Mitsubishi Chemical # 3050B), 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 the spectrum when specularly reflected light from a carrier is captured using an apparatus (manufactured by Minolta Camera, CM-2002) equipped with an illumination / light receiving optical system that conforms to 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, a recessed portion having a size of 13 × 10 mm and a depth of 0.2 mm is provided. In this recess, 256 convex portions having a diameter of 0.2 mm and a height of 0.2 mm are provided. The breakdown of the 256 convex portions has 64 (8 × 8) convex portions as one block, and there are four such blocks. 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 height of 256 convex portions) and the flat portion was measured, it was 5 μm or less. In addition, the height of 256 convex top surfaces varies (the difference in height between the highest convex top surface and the lowest convex top surface), and the average value of the convex top surface and the flat portion. The difference in height between the upper surfaces was measured and found to be 5 μm or less. A specific convex layout is shown in FIG. The 8 × 8 portion shaded in the upper left in FIG. 7 corresponds to one block, and the pitch with the adjacent block is 4.5 mm.

The PMMA substrate was immersed in a 10N aqueous sodium hydroxide solution at 65 ° C. for 12 hours. This was washed in the order of pure water, 0.1N HCl aqueous solution, and pure water to generate carboxyl groups on the substrate surface. When a PMMA flat plate containing carbon black used in this example was measured under the conditions of an excitation wavelength of 532 nm, a photomultiplier setting gain of 700, and a laser power of 33% using GenePix 4000B of Axon Instruments. The autofluorescence intensity of this plate was 250.
(Preparation of probe solution)
The DNA of SEQ ID NO: 1 (70 bases, 5 ′ terminal amination) was synthesized. This DNA was dissolved in pure water at a concentration of 0.27 nmol / μl to obtain a stock solution. When spotting on the substrate, PBS (NaCl 8 g, Na ₂ HPO ₄ · 12H ₂ O 2.9 g, KCl 0.2 g, KH ₂ PO ₄ 0.2 g in pure water was diluted to 1 l. In order to condense the carboxyl group on the surface of the carrier and the amino group at the end of the probe DNA, the final concentration of the probe is 0.027 nmol / μl with hydrochloric acid for pH adjustment added to the pH 6.5) 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added to bring the final concentration to 50 mg / ml. Further, sodium dodecyl sulfate (SDS) was added at 0.005% in order to reduce spot unevenness. Then, 30 μl of these mixed solutions were dispensed into 4 locations of a 384 well microtiter plate.
(Spotting)
Four stainless steel pins were set on a pin block of a spotter (Gene Stamp II) manufactured by Nippon Laser Electronics in the form of being set at the four corners of the apex of 4.5 mm. Then, the 20 substrates prepared above were spotted on the spotter stage, and the probe solution was spotted. In addition, although the capillary is not provided in the pin, the 1-letter-shaped groove | channel which is 50 micrometers in width and 30 micrometers in depth is carved in the pin head.

  Then, while observing with the micro camera, fine adjustment was performed on each of the 20 substrates so that the position where the pin descends when spotting the upper surface of a certain convex portion matches the position of the upper surface of the convex portion. Later, four pin tips were dipped at once into the four probe solutions placed in the 384 well microtiter plate described above, and the probe solution was spotted onto the four protrusions at a time. “Dip the tip of the pin in the probe solution” → “spot on the upper surface of the convex portion” was repeated 64 times, and the probe solution was spotted on a total of 256 convex portions. In order to examine the size of the pin head, the pin head size was changed to 220 μm, 240 μm, 300 μm, 400 μm, and 600 μm.

(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: 5 solution 2 μl, SEQ ID NO: 6 solution 2 μl, Template (SEQ ID NO: 7 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 was dissolved in 1% by volume BSA (bovine serum albumin), 5 × SSC (5 × SSC is 43.8 g of NaCl and 22.1 g of trisodium citrate hydrate is dissolved in pure water. Concentrated to 1 liter, 43.8 g of NaCl and 22.1 g of trisodium citrate hydrate dissolved in pure water, quantified to 5 liter, expressed as 1 × SSC, 10 times concentrated The solution is expressed as 10 × SSC, and the 5-fold diluted solution is expressed as 0.2 × SSC.), 0.1 wt% SDS (sodium dodecyl sulfate), 0.01 wt% salmon sperm DNA solution (each concentration is All were dissolved in 400 μl to obtain a solution for hybridization.

(Hybridization)
The sample DNA was hybridized to the substrate on which the probe DNA obtained above was immobilized. Specifically, 50 μl of a hybridization solution was dropped on a carrier on which the probe nucleic acid prepared previously was immobilized, and a cover glass was placed thereon. This was placed in a plastic container and incubated at 65 ° C. and 100% humidity for 10 hours. After incubation, the cover glass was peeled off, washed and dried.

(Measurement)
The carrier after the above treatment was set in a scanner for DNA chip (GenePix 4000B manufactured by Axon Instruments) for measurement. And when there is a chip in the spot, that is, when the pin head is in contact with only a part of the upper surface of the convex portion, it was counted as a defective spot. Table 1 shows the results of observation of 20 × 256 = 5120 spots for one type of pin diameter.
(Comparative Example 1)
The same experiment as in Example 1 was performed except that the diameter of the pin head was set to 200 μm. The results of observing the spot shape in the same manner as in Example 1 are shown in Table 1.

  Thus, when the area of the pin head is made larger than the area of the upper surface of the convex portion, the normal spot rate is clearly improved. It can also be seen that it is particularly effective to increase the area ratio of the pin head to the upper surface of the convex portion by 1.4 times or more.

The figure which shows a mode when the area of a convex part and a pin head is the same The figure which shows a mode when the area of a pin head is larger than the convex part upper surface area Schematic diagram when the carrier is set on the spotter stage Schematic diagram of the carrier used in the present invention Schematic cross-sectional view of the carrier used in the present invention Example of jig for hitting the carrier The figure which shows the layout of the convex part of the support | carrier used in the Example

Explanation of symbols

11 Pin block 12 Immobilization pin 13 Substrate 14 Convex portion 21 Pin block 22 Immobilization pin 23 Substrate 24 Convex portion 31 Flat portion 32 Convex portion 33 Abutment portion 34 Abutment spring 41 Flat portion 42 Concavity and convexity portion 63 DNA chip 64 Objective lens 65 Excitation light 66 Spring 71 for filling DNA chip with jig 71 Convex part

Claims (2)

A method of immobilizing a selective binding substance on the upper surfaces of a plurality of convex portions of a concavo-convex part on a carrier using a pin for immobilizing a selective binding substance, wherein the area of the pin head for immobilizing the selective binding substance is A method for producing a selective binding substance-immobilized carrier, wherein the area is larger than the area of the upper surface of the convex portion. The method for producing a selective binding substance-immobilized carrier according to claim 1, wherein the relationship between the area Sp of the pin head of the immobilizing pin and the area Ss of the upper surface of the convex portion is Sp / Ss≥1.4.

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