JP5660468B2 - Method for producing gel microarray for detecting bio-related substances - Google Patents

Description

  The present invention relates to a method of manufacturing a microarray for detecting a biological substance.

  In recent years, an analysis method called a DNA microarray method (DNA chip method) has been developed that enables collective expression analysis of a large number of genes. As an example of such a DNA microarray, a microarray for detecting a biological substance has a plurality of compartments, each compartment is formed by a hole or a hollow fiber, and a gel-like material containing a capture probe is held in each compartment Has been proposed (see Patent Document 1 and Patent Document 2).

  Such a microarray is manufactured by, for example, a method of sequentially performing the following (1) to (3). (1) A step of producing a hollow fiber bundle in which hollow fibers made of a plurality of organic materials are three-dimensionally arranged so that the fiber axes of the hollow fibers are in the same direction, and the arrangement is fixed with a resin; (2 ) A step of holding the gel-like material containing the capture probe in the hollow part of the hollow fiber by introducing a gel precursor solution containing the capture probe into the hollow part of each hollow fiber and reacting the solution; (3) A process of cutting in a direction crossing the longitudinal direction of the fiber to make a thin piece.

  Compared with the photolithography method and spotting method used when fixing the probe to the flat substrate, such a manufacturing method does not require expensive manufacturing equipment and can produce a large amount of stable quality products. Have

  In addition, according to the microarray manufactured by the manufacturing method, since the gel-like material containing the capture probe is held in the hollow portion of the hollow fiber, compared with the microarray that simply holds the capture probe on the flat substrate, Since a large amount of capture probe can be retained, high detection sensitivity can be achieved. Furthermore, since the hybridization reaction can be performed by electrophoresis, the inspection time can be shortened.

International Publication No. 00/40942 Pamphlet International Publication No. 01/98781 Pamphlet JP-A-11-108928 JP 2001-133453 A JP 2005-37303 A Japanese Patent No. 3834519

  However, as the microarray is arranged on the outer side (periphery), the capture probe is immobilized via a gel-like material (hereinafter referred to as “gel spot”) held in the hollow portions of the arranged hollow fibers. In some cases, the sample capturing ability, that is, the reactivity between the oligo DNA immobilized on the gel (capture probe) and the sample is low. That is, a uniform gel spot may not be formed in the entire microarray.

  Accordingly, a main object of the present invention is to provide a method of manufacturing a microarray having a uniform gel spot in the entire microarray.

As a result of intensive studies to solve the above problems, the present inventors have found that the above problems can be achieved by polymerizing the gel precursor solution filled in the hollow fibers in the aqueous phase. The present invention has been completed.

That is, the present invention relates to a method for producing a microarray for detecting a biological substance including the following steps.
(1) A step of manufacturing a hollow fiber bundle by arranging a plurality of hollow fibers in a three-dimensional manner such that the fiber axes of the hollow fibers are in the same direction, and fixing the arrangement with a resin (2) required According to the step of introducing a liquid into the hollow portion of the hollow fiber and holding it for a certain time (3) introducing the gel precursor solution containing the capture probe into the hollow portion of the hollow fiber (4) in the aqueous phase, The step of polymerizing the gel precursor solution filled in the hollow portion of the hollow fiber (5) The step of cutting the hollow fiber bundle in the direction crossing the longitudinal direction of the fiber to make it into a thin piece.

  According to the present invention, there is no difference depending on the position where the sample capturing ability (reactivity with the sample) of the probe in the gel spot held in the hollow portion of the hollow fiber is arranged (a gel that is homogeneous in the entire microarray) A microarray (with spots) can be produced.

It is a conceptual diagram of an arrangement | sequence fixing device. 2 is a photomicrograph showing a part of the gel spot of the microarray obtained in Example 1. 2 is a photomicrograph showing a part of a gel spot of a microarray obtained in Comparative Example 1. It is a conceptual diagram which shows the spot arrangement | sequence of the produced microarray.

  Each step in the method for producing a bio-related substance detection microarray (hereinafter sometimes referred to as “microarray”) according to the present invention will be described in detail below.

(1) A step of producing a hollow fiber bundle by arranging a plurality of hollow fibers in a three-dimensional manner so that the fiber axes of the hollow fibers are in the same direction, and fixing the arrangement with a resin (first step) )
(1-1) The component of hollow fiber "hollow fiber" is not limited, The hollow fiber which has an organic material as a main component is preferable. Examples of hollow fibers mainly composed of organic materials include polyamide hollow fibers such as nylon 6, nylon 66 and aromatic polyamide, and polyester hollow fibers such as polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid and polycarbonate. Fiber, acrylic hollow fiber such as polyacrylonitrile, polyolefin hollow fiber such as polyethylene and polypropylene, polymethacrylate hollow fiber such as polymethyl methacrylate, polyvinyl alcohol hollow fiber, polyvinylidene chloride hollow fiber, polyvinyl chloride Examples include hollow fibers, polyurethane-based hollow fibers, phenol-based hollow fibers, fluorine-based hollow fibers made of polyvinylidene fluoride and polytetrafluoroethylene, polyalkylene paraoxybenzoate-based hollow fibers, etc. It is.

  The manufacturing method of the said hollow fiber is not limited, It can manufacture by the well-known method as described in patent document 3. FIG. For example, a melt spinning method is preferable, and a horseshoe type, C type nozzle, double pipe nozzle, or the like can be used as the nozzle. In the present invention, it is preferable to use a double tube nozzle in that a continuous and uniform hollow portion can be formed.

  The hollow fiber used in the present invention may be porous, and can be obtained by combining a known spinning technique such as a drawing method, a microphase separation method, and an extraction method with a melt spinning method or a solution spinning method. The porosity is not particularly limited, but from the viewpoint of increasing the density of the biopolymer immobilized around the fiber material unit length, the porosity is desirably high so that the specific surface area is increased.

  The inner diameter of the hollow fiber is not limited, and can be appropriately selected according to the experimental purpose or experiment using the microarray obtained by the present invention. The inner diameter of the hollow fiber is preferably 10 to 2000 μm, and more preferably 150 to 1000 μm.

  If necessary, hollow fibers containing a suitable amount of black pigment such as carbon black can also be used. By containing a black pigment, optical noise derived from foreign substances such as dust can be reduced during detection, and the strength of the resin can be increased. The content of the pigment is not limited and can be appropriately selected according to the size of the hollow fiber, the purpose of use of the microarray, and the like. Content of a pigment can be 0.1-10 mass%, for example, 0.5-5 mass% is preferable and 1-3 mass% is more preferable.

(1-2) Hollow fiber bundle A plurality of hollow fibers are arranged in three dimensions so that the fiber axes of the hollow fibers are in the same direction, and fixed with a resin such as an adhesive so that the arrangement is not disturbed. Thus, a hollow fiber bundle can be obtained.

  As a method for producing a hollow fiber bundle, for example, a method in which a plurality of hollow fibers are arranged in parallel at a predetermined interval on a sheet-like material such as an adhesive sheet and the sheet is spirally wound. (Patent Document 3).

  Further, two porous plates each having a plurality of holes provided at a predetermined interval are overlapped so that the hole portions coincide with each other, the hollow fibers are passed through the hole portions, and the interval between the two porous plates is opened. A method (Patent Document 4) in which the periphery of the hollow fiber between two porous plates is filled with a curable resin raw material and cured.

  As a curable resin raw material, what consists of organic materials, such as a polyurethane resin and an epoxy resin, is preferable. In more detail, what is formed from one or more types of materials comprised from the organic polymer etc. which are illustrated below is preferable.

  Organic polymers include rubber materials such as polyurethane, silicone resin, epoxy resin, polyamide resins such as nylon 6, nylon 66, aromatic polyamide, polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid, polycarbonate, etc. Polyester resins, acrylic resins such as polyacrylonitrile, polyolefin resins such as polyethylene and polypropylene, polymethacrylate resins such as polymethyl methacrylate, polyvinyl alcohol resins, polyvinylidene chloride resins, polyvinyl chloride resins, Examples thereof include phenol resins, fluorine resins made of polyvinylidene fluoride and polytetrafluoroethylene, polyalkylene paraoxybenzoate resins, and the like.

  An appropriate amount of a black pigment such as carbon black can be contained in the organic polymer. By adding a black pigment, optical noise derived from impurities such as dust can be reduced during detection, and the strength of the resin can be increased.

In addition, an organic block can be used, and a plurality of through holes can be formed in the organic block with a laser or the like to form a microarray. The through holes are preferably arranged in parallel to each other in the organic block and formed with a predetermined interval. The shape of the through hole is a square, a rectangle, a circle, or the like. In the case of a circle, the diameter is about 10 to 2000 μm. The arrangement of the through holes is concentric, spiral or the like. The through holes can be formed in the block at a density of 10 to 10,000 holes / cm ² .

  Furthermore, a hollow fiber bundle can also be produced by fusing the outer surfaces of adjacent hollow fibers to each other.

  The arrangement is fixed to the entire length or a predetermined portion of the arranged hollow fibers. A portion having an appropriate length from at least one end portion of the arranged hollow fibers may be not fixed.

  The number of hollow fibers arranged in the present invention, that is, the number of spots is not limited, and can be appropriately selected according to the intended experiment. Therefore, the distance between the hollow fibers can be appropriately selected according to the area of the microarray and the number of hollow fibers arranged.

(2) A step of introducing a liquid into the hollow part of the hollow fiber and holding it for a certain time as necessary (second step)
The second step is optional and may or may not be performed. By performing the second step, it is preferable because a microarray having a more uniform gel spot can be obtained in the entire microarray.

  The second step is a step of introducing a liquid into the hollow portion of each hollow fiber of the hollow fiber bundle obtained in the first step. This step may be performed in a gas phase or in an aqueous phase.

  As a method for introducing the liquid, for example, a method described in Patent Document 5 can be mentioned. More specifically, the hollow portion of the hollow fiber with one end sealed is once evacuated, the tip of the other open end of the hollow fiber is immersed in a liquid, and then the pressure is changed from vacuum to normal pressure. For example, a method of introducing a liquid into the hollow part using a pressure difference between the inside and the outside of the hollow part can be used. In addition to this method, the both ends of the hollow fiber may be opened, one end may be immersed in the liquid, and the liquid may be sucked up from the other and introduced into the hollow part.

  The liquid introduced into the hollow part of the hollow fiber is discharged after being held for a certain time. The certain time is a time required for a part of the introduced liquid to permeate into the hollow fiber from the hollow inner wall surface of the hollow fiber. The time is not limited as long as the liquid filled in the hollow portion of the hollow fiber can sufficiently penetrate the hollow fiber from the inner wall surface of the hollow portion. For example, when the liquid is allowed to permeate into the hollow fiber by natural diffusion, it is preferably held for at least 3 hours, more preferably 10 hours, and even more preferably 15 hours or more. In addition, after the liquid is introduced into the hollow portion of the hollow fiber, the outside of the hollow fiber bundle is evacuated, and a part of the liquid introduced into the hollow portion is forcibly utilized using the pressure difference between the inside and the outside of the hollow portion. It can also be infiltrated.

  The type of liquid used here is not limited as long as it does not prevent the gel from being sufficiently held in the hollow portion of the hollow fiber in the subsequent step, and can be appropriately selected according to the type of gel. it can. For example, water, a buffer solution, and the like can be used, and water is preferable because water is suitably used as a solvent for the gel precursor solution.

  After holding the liquid for a certain time, excess liquid introduced into the hollow portion of the hollow fiber is discharged. The method of discharging can be performed by the method described in Patent Document 5, for example. More specifically, there is a method in which the hollow portion of the hollow fiber into which the liquid is introduced is evacuated again and the introduced liquid is discharged. In addition, it can also be sucked from one end of the hollow fiber, or gas can be blown into the hollow part from one end and the liquid can be pushed out from the other end of the hollow fiber end. Further, instead of blowing gas, it is possible to extrude the liquid by introducing the gel precursor solution described below.

(3) Step of introducing the gel precursor solution containing the capture probe into the hollow portion of the hollow fiber (third step)
In the third step, a gel precursor solution containing a capture probe is introduced. This step may be performed in a gas phase or in an aqueous phase.

  “Capture probes” include nucleic acids (DNA, RNA, etc.), amino acids, peptides, sugars, lipids, antibodies, and organic and inorganic compounds that can detect biologically related substances through interactions such as chemical bonds and physical bonds. Say. They are prepared by extraction from living cells, chemical synthesis and the like.

  For example, DNA can be prepared from living cells by the method of Blin et al. (Nucleic Acids Res. 3.2303 (1976)). RNA can be prepared by the method of Favaroro et al. (Methods. Enzymol. 65.718 (1980)).

  The “biologically related substance” refers to a macromolecular organic compound existing in a living body, and examples thereof include nucleic acids (DNA, RNA, etc.), amino acids, peptides, sugars, lipids, and antibodies.

  The “gel precursor solution” refers to a solution containing a chemical substance that forms a crosslinked structure and causes gelation. For example, it is a solution containing a monomer, a polyfunctional monomer, a polymerization initiator and water. Alternatively, it may be a solution containing a polysaccharide such as alginic acid and a divalent ion and water, or a solution containing a protein such as gelatin, a polyfunctional monomer capable of binding to an amino acid residue of protein, and water. .

  As monomers, acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, N-vinylpyrrolidone, hydroxyethyl methacrylate, (meth) Examples include acrylic acid and allyl dextrin. Examples of the polyfunctional monomer include methylene bis (meth) acrylamide and polyethylene glycol di (meth) acrylate.

  The concentration of the monomer component is not particularly limited, and can be appropriately selected depending on the type of monomer, the type of target gel, and the like. The density | concentration of a monomer component can be 0.5-20 mass%, for example, 1-10 mass% is preferable and 3-5 mass% is more preferable. For example, in the case of an acrylamide monomer, it is about 2.5-10 mass%, Preferably it is 3-7 mass%, More preferably, it can be 3-5 mass%.

  The gel precursor solution may be introduced by, for example, sucking the solution into a syringe having a fine needle and inserting the needle into the hollow portion of each hollow fiber. When one end of the hollow fiber bundle is not fixed, the gel precursor solution can be introduced into the hollow fiber by the following method. That is, first, a hollow fiber bundle and a gel precursor solution are placed in a desiccator. Next, the end of the hollow fiber bundle where the hollow fibers are not fixed may be immersed in this solution, the inside of the desiccator is decompressed, and this solution may be introduced into the hollow portion of the hollow fiber.

(4) Step of polymerizing the gel precursor solution filled in the hollow portion of the hollow fiber in the aqueous phase (fourth step)
In the fourth step, the gel precursor solution filled in the hollow fiber bundle in the previous (third) step is polymerized in the aqueous phase. By polymerizing the gel precursor solution in the aqueous phase, a homogeneous gel spot can be obtained at all spots in the microarray. Therefore, by using the microarray obtained in the present invention, a more accurate measurement result can be obtained.

  In the present specification, the term “in the aqueous phase” includes not only water but also an environment having a high water vapor concentration. The high water vapor concentration means, for example, 50% or more, preferably 70% or more, more preferably 90% or more at 55 ° C.

The method of placing the hollow fiber bundle filled with the gel precursor solution in the aqueous phase is not limited at all. For example, the hollow fiber bundle may be directly immersed in water. At that time, the hydrostatic pressure applied to the hollow fiber bundle is not limited, and it is sufficient that the polymerization is performed efficiently. The hydrostatic pressure is preferably 50 N / m ² or more, and more preferably 100 N / m ² or more. By setting it to 50 N / m ² or more, polymerization is sufficiently performed, and a microarray having a uniform capture probe can be obtained. Alternatively, a bundle of hollow fibers may be placed in a humidified airtight container and humidified by blowing saturated water vapor.

  Next, the gel precursor solution introduced into the hollow part of the hollow fiber is polymerized to hold the gel-like material containing the capture probe in the hollow part of the hollow fiber. The polymerization conditions are not particularly limited, and can be appropriately selected according to the type of gel precursor to be used. For example, when an acrylamide monomer is used as the gel precursor, it can be polymerized using a radical polymerization initiator, and among the radical polymerization initiators, a thermal polymerization reaction using an azo initiator. Polymerization by is more preferred.

  The necessary types (number) of capture probes can be prepared according to the purpose. For example, the same number of capture probes as the number of hollow fibers can be prepared, and different types of capture probes can be introduced into each hollow fiber. Also, the same capture probe can be introduced into a plurality of hollow fibers.

(5) A step of cutting the hollow fiber bundle in a direction intersecting with the longitudinal direction of the fiber to make a thin piece (fifth step)
In the fifth step, the hollow fiber bundle obtained through the previous step (fourth) is cut in a direction crossing the longitudinal direction of the fiber, preferably in a direction orthogonal thereto, and thinning is performed.

  The cutting method is not limited as long as it can be sliced. For example, it can be performed by a microtome, a laser, or the like. The thickness of the obtained flake is not limited and can be appropriately selected depending on the purpose of the experiment. The thickness of the flakes can be, for example, 5 mm or less, and preferably 0.1 to 1 mm.

Bio-related substance detection microarray The bio-related substance detection microarray thus obtained has sufficient capture probe capture capability (reactivity between capture probe and sample) even at the outermost spot. That is, a microarray having a homogeneous gel spot throughout the microarray.

  It can be confirmed by microscopic observation that the microarray obtained by the present invention has a homogeneous gel spot. In addition, the ability to capture a homogeneous sample can be confirmed by an experiment using a model sample labeled with fluorescence or the like.

  When used in an experiment using a model sample labeled with fluorescence, the microarray obtained in the present invention is represented by the average value of the fluorescence signal intensity of the outermost spot with respect to the average value of the fluorescence signal intensity of the inner spot. Fluorescence intensity ratio (= average value of fluorescence signal intensity of outermost spot / average value of fluorescence signal intensity of inner spot) is 0.7 to 1.2, preferably 0.8 to 1.15, more preferably 0 .9 to 1.10. This is because the microarray can be said to be homogeneous throughout the range.

  Here, the “outermost spot” is a spot located on the outermost side in the microarray as illustrated in FIG. 4. For example, in a 16 × 16 microarray, there are 60 spots. The “inner spot” means a spot other than the outermost spot. For example, there are 196 spots in a 16 × 16 microarray.

  The method for measuring the fluorescence signal intensity is not limited, and can be measured by a known method described in Patent Document 6, for example. More specifically, after the hybridization reaction between the microarray and the specimen at 65 ° C. for 16 hours, the unbound specimen is removed using a washing buffer, and the microarray is irradiated with light having the excitation wavelength of the fluorescent substance (if necessary) The fluorescence emitted can be measured with a detector for a plurality of times. The hybridization reaction and the washing method are not particularly limited, and a known method described in a textbook DNA microarray battle manual (Yodosha) or the like can be used.

  The kind of the fluorescent material is not limited, and the fluorescent materials exemplified in Table 1 below can be used. As a method of labeling the specimen using these fluorescent dyes, for example, in the stage of specimen preparation, UTP previously modified with biotin was introduced into the specimen base sequence when the specimen was amplified, and the fluorescent dye was modified. A method of indirectly fluorescently labeling with streptavidin, specifically, a method described in the Instruction Manual of Ambion's MessageAmp (registered trademark) II-Biotin Enhanced Kit, or A Highly Reproducible, Linear, and Automated Sample Preparation Method For example, the method described in DNA Microarrays, David R. Dorris, Ramesh Ramakrishnan, Dionisios Trakas, et al., Genome Res. 2002 12: 976-984 can be used. In addition, a method of directly labeling a specimen by modifying a fluorescent dye on a DNA intercalator such as ethidium bromide can be used.

Hereinafter, specific embodiments will be described in Examples.

<Example 1>
(1) Preparation of capture probe A nucleic acid in which the 5 'end of SEQ ID NO: 1 (gctgcgaaat cactctatcc ttcccatgga cttacctgcc ggtatagcgt) was aminated was synthesized with a DNA synthesizer (consigned synthesis by Invitrogen). This nucleic acid was dissolved in sterilized water to a concentration of 100 pmol / μL to prepare an aminated nucleic acid solution.

  To 5 μL of this solution, 2.5 μL of 25 mM sodium hydrogen carbonate-25 mM sodium dihydrogen carbonate aqueous solution and 2.5 μL of 10 mM methacrylic anhydride solution (dissolved in DMSO) were added and incubated at 25 ° C. for 30 minutes.

  The reaction was stopped by adding 40 μL of sterilized water to obtain a nucleic acid solution modified with a vinyl group (hereinafter, a capture probe solution).

(2) Production of Hollow Fiber Bundle A hollow fiber bundle was produced using the array fixture shown in FIG. In the figure, x, y, and z are orthogonal three-dimensional axes, and the x-axis coincides with the longitudinal direction of the fiber.

  First, two perforated plates with a thickness of 0.1 mm were prepared in which a total of 256 holes each having a diameter of 0.32 mm were provided in a total of 256 rows in 16 rows each with a distance between the centers of the holes being 0.12 mm. These perforated plates were overlapped, and one polycarbonate hollow fiber having an outer diameter of 280 μm, an inner diameter of 180 μm, and a length of 150 mm was passed through all of the holes.

  The position of the two perforated plates was moved in a state in which a tension of 0.1 N was applied to each fiber in the X-axis direction, and was fixed at two positions of 20 mm and 100 mm from one end of the hollow fiber. . That is, the interval between the two perforated plates was 80 mm. Next, the three surrounding surfaces of the space between the perforated plates were surrounded by a plate-like object. In this way, a container having an open top only was obtained.

  Next, the resin raw material was poured into the container from the upper part of the container. As resin, what added 2.5 mass% carbon black was used with respect to the gross mass of a polyurethane resin adhesive (Nippon Polyurethane Industry Co., Ltd., Nipponran 4276, Coronate 4403). The resin was cured by standing at 25 ° C. for 1 week. Next, the porous plate and the plate-like material were removed to obtain a hollow fiber bundle. The obtained hollow fiber bundle was put in a desiccator and the inside was purged with nitrogen, and then allowed to stand for 16 hours.

(3) A container containing treated water for saturating the hollow portion of the hollow fiber bundle with water was prepared and installed in the above-described desigator. After the inside of the desiccator was decompressed, the end of one hollow fiber (hereinafter referred to as end) where the fiber bundle of the hollow fiber bundle was not fixed was immersed in water. Nitrogen gas was sealed in the desiccator, and water was introduced into the hollow portion of the hollow fiber.

  It was left for 16 hours with water introduced into the hollow part of the hollow fiber. After 16 hours, the water in the hollow part was discharged by vacuum decompression. Water Absorption Rate of Hollow Fiber Bundle (Water Absorption Rate (%) = [(Hollow Fiber Mass after Introducing Water into Hollow Part and Holding for 16 Hours and then Dissipating Water in Hollow Part) − (Hollow before Water is Introduced) (Mass of fiber bundle)] / mass of hollow fiber bundle before introducing water × 100) was 1.4%.

(4) Introduction of Gel Precursor Solution into Hollow Fiber Hollow Part Aqueous solution A shown in Table 2 was prepared and placed in a desiccator. In the same manner as described above, the aqueous solution A was sucked from the end portion and introduced into the hollow portion of the hollow fiber.

(5) Step of placing the hollow fiber bundle in the aqueous phase After introducing the aqueous solution A, the hollow fiber bundle is once taken out from the desiccator and put into a container containing 500 mL of pure water that has been previously degassed and nitrogen substituted. The hollow fiber bundle was immersed so that the hollow fiber bonded portion was completely immersed so that the tip of the unsealed hollow fiber was on the upper side and the tip was not in contact with pure water. At this time, when the water pressure was measured at the uppermost part of the bonded portion of the hollow fiber bundle in the container in which the hollow fiber bundle was immersed, it was 50 N / m ² .

(6) Polymerization reaction Next, a container containing pure water in which the hollow fiber bundle was immersed was placed in the desiccator, and a polymerization reaction was carried out at 55 ° C for 3 hours.

(7) After completion of the flaky polymerization reaction, the hollow fiber bundle was cut into a thickness of 250 μm in the direction perpendicular to the longitudinal direction of the hollow fiber using a microtome. In this way, 100 microarrays having a thickness of 250 μm on which gel spots including 256 capture probes were mounted were obtained.

(8) Evaluation of microarray Gel spots were observed using a microscope (fluorescence microscope Eclipse E600 objective lens × 10 NA = 0.3 manufactured by Nikon).

  The microarray was immersed in a light-permeable petri dish filled with water, the petri dish was placed on the stage of a microscope, and white light was irradiated from the bottom of the petri dish. While adjusting the focus of the lens, the gel spots arranged on the outermost periphery and the gel spots arranged on the inner side of the 100 microarrays obtained above were observed with a microscope.

  As a result, in all the microarrays, no difference was found in the in-focus position between the gel spot arranged on the outermost periphery and the gel spot inside thereof, and it was confirmed that a uniform gel spot was formed. A part of the observation result is shown in FIG.

<Comparative Example 1>
The operation was performed in the same manner as in Example 1 except that the process (5) was not performed. As a result, in all the microarrays, a difference was found in the in-focus position between the outermost circumference and the gel spot inside. A part of the observation result is shown in FIG.

<Example 2>
Next, in order to evaluate the detection signal intensity of each spot of the microarray produced by the production method of the present invention, a hybridization reaction was performed using an oligo DNA serving as a model sample.

(1) Production of microarray Except that the sequence of the capture probe of Example 1 was changed to SEQ ID NO: 2 (tctcgaaagg cttcctactc ggctgtgagt ctttacggca accctgtccg gtattgggaa atctt) (contracted synthesis by Invitrogen), A microarray was produced in the same manner as in 7).

(2) Fluorescent labeling of model sample First, a 1 pmol / μL ultrapure aqueous solution of SEQ ID NO: 3 (aagatttccc aataccggac agggttgccg taaagactca cagccgagta ggaagccttt cgaga) (commissioned synthesis by Invitrogen) was prepared. .

  Next, a label solution of the model specimen was prepared as follows using a fluorescent labeling reagent PlatinumBright (registered trademark) manufactured by Cleatech. It is a component of PlatinumBright 647 infrared (fluorescent labeling agent with excitation light of 647 nm and fluorescence of 665 nm), which is a component of PlatinumBright (registered trademark), and the same reagent. A solution blended as shown in Table 3 of 10x Labeling Solution was prepared.

  A fluorescent labeling solution was prepared by reacting the fluorescent labeling agent and the oligo DNA with the composition shown in Table 3 according to the following procedure.

  First, 20 μL of the solution formulated in Table 3 was incubated at 85 ° C. for 30 minutes to be reacted. Next, after the contents of PlatinumBright (registered trademark) KREAPure purification columns are vortexed, the contents are thoroughly stirred, the tip of the column is folded, the lid is opened 1/4 and placed in a 2 mL tube. And centrifuged at 14,000 rpm for 1 minute. The resulting eluate is discarded and D.P. W. 300 μL was added and centrifuged again at 14,000 rpm for 1 minute. Thereafter, the column was transferred to a 1.5 mL tube, and the previously reacted labeling solution was loaded for resin and further centrifuged at 14,000 rpm for 1 minute.

(3) 1 μL was measured from the labeling solution prepared in hybridization (2), and a hybridization solution was prepared with the composition described in Table 4.

  Next, the previously prepared microarray was completely immersed in the hybridization solution in 200 μL of the hybridization solution, and incubated in a hybridization oven at a set temperature of 65 ° C. for 16 hours.

(4) Washing After incubation at 65 ° C. for 16 hours, the microarray was taken out of the hybrid chamber and washed according to the following procedure.

  First, the hybridized microarray was removed from the chamber using tweezers and washed in advance at 65 ° C. Washing buffer A (0.12 M Tris / HCl / 0.12 M NaCl / 0.05% Tween-20 solution: 10 ml) The microarray was immersed in a tube containing, and allowed to stand at 65 ° C. for 20 minutes.

  Next, the tube was replaced with another tube containing the washing buffer A (0.12M-NaCl / 0.05% -Tween 20 solution: 10 ml) that had been kept at 65 ° C., and further allowed to stand at 65 ° C. for 20 minutes.

  Thereafter, the tube was replaced with a tube containing the washing buffer B (0.12 M Tris · HCl / 0.12 M NaCl solution 10 ml) that had been kept at 65 ° C., and left at 65 ° C. for 10 minutes.

(5) Detection of fluorescence signal intensity Five microarrays obtained by the method of the present invention were prepared, each placed in a detection chamber, and the microarray was filled with washing buffer B. Next, the chamber on which the microarray was placed was placed on a sample stage of a cooled CCD camera type fluorescence microscope, and the microarray was irradiated with excitation light at a wavelength of 650 nm for 4 seconds, and the intensity of the emitted fluorescence was measured.

<Comparative example 2>
A microarray was prepared in the same manner as in Comparative Example 1 except that the capture probe sequence of SEQ ID NO: 2 was used, and the same model sample as in Example 2 was used. A hybridization reaction was performed to obtain fluorescence signal intensity data of each spot.

Comparison of fluorescence signal intensity In order to examine the effect of the method of the present invention, the variation in the detection signal intensity of the microarray was compared between Example 2 and Comparative Example 2 as follows.

(A) Comparison of CV of fluorescence signal intensity First, the variation coefficient CV (coefficient of variation) of all the measured microarray spots (256 spots), that is, the standard deviation of the fluorescence signal intensity values of all spots is averaged. The value divided by the value was calculated for each of the five microarrays, and the average value was obtained. When compared between Example 2 and Comparative Example 2, the CV of Comparative Example 2 was about 26%, whereas the CV of Example 2 was less than 10% (Table 5).

(B) Comparison of fluorescence signal intensity at the spot position Next, 5 values were obtained by dividing the average value of fluorescence signal intensity of the outermost spot (60 spots) by the average value of fluorescence signal intensity of the inner spot (196 spots). The calculation was performed on one microarray, and the average value of the five values was compared between Example 2 and Comparative Example 2. The positions of the outermost peripheral spot and the inner spot are shown in FIG.

  As a result, the numerical value obtained in Example 2 was 0.94, and it was confirmed that a microarray having a homogeneous gel spot was obtained. On the other hand, it was 0.44 in the comparative example 2 (Table 5).

  From the above results, it was confirmed that the fluorescence signal intensity of each spot became more uniform by the method of the present invention.

DESCRIPTION OF SYMBOLS 1 Array fixing tool 11 Hole 21 Perforated plate 31 Hollow fiber 41 Plate-shaped object

Claims (2)

A method for producing a microarray for detecting a biological substance, comprising the following steps.
(1) Step of manufacturing a hollow fiber bundle by arranging a plurality of hollow fibers in a three-dimensional manner so that the fiber axes of the hollow fibers are in the same direction, and fixing the arrangement with a resin (2) Capture Step of introducing a gel precursor solution containing a probe into the hollow portion of the hollow fiber (3) Step of polymerizing the gel precursor solution filled in the hollow portion of the hollow fiber in water having a hydrostatic pressure of 50 N / m ² or more (4) A step of cutting the hollow fiber bundle in a direction crossing the longitudinal direction of the fiber into thin pieces. The method of Claim 1 including the process of introduce | transducing a liquid into the hollow part of a hollow fiber, and hold | maintaining for a fixed time after a process (1) and before a process (2) .