JP2011072201A - Method for producing flake holding organism-related substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a flake holding an organism-related substance by which collapse of gel by cutting is suppressed. <P>SOLUTION: The method for producing the flake by cutting an embedded product in which a plurality of fibers holding the organism-related substance is embedded through a gel so that each of the fiber axes may be arranged in the same direction, in the direction crossing to the fiber axes of the embedded product by an edged tool includes cutting the embedded product while regulating the relief angle of the edged tool within the range of ≥0° and ≤0.9°. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to a method for producing a biological substance-retaining flake.

In recent years, many analytical methods called DNA microarray methods (DNA chip methods) that enable collective expression analysis of a large number of genes have been developed and attracting attention. These methods are all nucleic acid detection / quantification methods based on hybridization reaction between nucleic acids.
Many types of DNA microarrays are known, and there are DNA microarrays using fibers. As a method for producing a DNA microarray using fibers, a fiber bundle comprising a plurality of arranged hollow fibers is embedded with epoxy resin, polypropylene, polystyrene, polyurethane resin, etc., and then a DNA probe is placed in the hollow portion of each hollow fiber. A gel precursor polymerizable solution containing is introduced, a polymerization reaction is performed, and a DNA probe is held in the hollow portion of each hollow fiber. Thereafter, the embedded material is cut in a direction crossing the fiber axis to produce a DNA-immobilized thin piece, that is, a DNA microarray (Patent Documents 1 and 2). As a technique for cutting, there is known a method (Patent Document 3) in which deformation is suppressed thinly and uniformly by adjusting a blade edge angle of a cutting tool to be used and a cutting speed.
However, in the method disclosed in Patent Document 3, a clearance angle of 1 to 2 ° is adopted, and only the tip portion of the blade is in contact with the embedded material, and the lower surface of the blade is not in contact with the embedded material. Cutting the filling. Then, when cutting, the cutting edge bites into the embedded object and, on the other hand, the cutting edge vibrates due to the force of the cutting edge to return upward due to the elasticity of the cutting tool. In some cases, the gel held in the container breaks down.

JP 2000-270878 A Japanese Patent Laid-Open No. 2002-311028 JP 2002-86391 A

  In the case of a DNA microarray in which DNA or the like is held in a fiber via a gel, if a broken gel adheres to a gel held in another fiber, accurate analysis cannot be performed.

  An object of this invention is to provide the manufacturing method of the biological material holding | maintenance thin piece which suppressed the gel breakage by cutting | disconnection.

In the present invention, an embedded object in which a plurality of fibers holding a biological substance via a gel are embedded so that their fiber axes are substantially the same is crossed with respect to the fiber axis of the embedded object. A method for producing a flakes having a biological material, wherein the flakes are cut so that the clearance angle of the blade is in the range of 0 ° to 0.9 °. is there.

  According to the present invention, it is possible to produce a biological substance-retaining flake that suppresses gel breakage due to cutting.

Schematic diagram of cutting embedded material with a blade Schematic view of the blade from the side Schematic diagram enlarging the measured positional relationship Part of the surface image of a slice obtained by cutting with a clearance angle of 0 ° Part of the surface image of a slice obtained by cutting at a clearance angle of 0.5 ° Part of the surface image of a slice obtained by cutting at a clearance angle of 0.9 ° Part of the surface image of a slice obtained by cutting at a clearance angle of 1 ° Part of the surface image of a slice obtained by cutting at a clearance angle of -1 °

The present invention will be described below including preferred embodiments.
The biological material-retaining flake of the present invention is manufactured by the following steps. In addition, the order of a process (1) and a process (2) is not ask | required.
(1) A fiber bundle composed of a plurality of arranged fibers is embedded with epoxy resin, polypropylene, polystyrene, polyurethane resin or the like.
(2) A gel precursor polymerizable solution containing a DNA probe is introduced into each fiber (a hollow part when the fiber is a hollow fiber, and a porous part when the fiber is a porous fiber), and a polymerization reaction is performed. Hold the DNA probe in the fiber.
(3) Cut the embedded material in a direction intersecting the fiber axis.
In the present invention, the biological substance refers to nucleic acids such as DNA, RNA, peptide nucleic acids, proteins, polysaccharides and the like.
Examples of the fiber that can be used as the fiber holding the biological substance include synthetic fiber, semi-synthetic fiber, regenerated fiber, natural fiber, and the like. Representative examples of synthetic fibers include various fibers such as nylon 6, nylon 66 and aromatic polyamide, various polyester fibers such as polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid and polycarbonate, and acrylic such as polyacrylonitrile. Fibers, polyolefin fibers such as polyethylene and polypropylene, polyvinyl alcohol fibers, polyvinylidene chloride fibers, polyvinyl chloride fibers, polyurethane fibers, phenol fibers, polyvinylidene fluoride And fluorine-based fibers made of polytetrafluoroethylene, polyalkylene paraoxybenzoate-based fibers, and the like. Examples of semi-synthetic fibers include various cellulose-based regenerated fibers (rayon, cupra, polynosic, etc.) made from diacetate, triacetate, chitin, chitosan and the like. Representative examples of natural fibers include plant fibers such as cotton and flax, animal fibers such as wool and silk, and mineral fibers such as asbestos.
As the form of the fiber, a known fiber such as a porous fiber or a hollow fiber can be used. The diameter of the fiber is preferably 1 mm or less and several tens of microns or more.
Holding a biological substance via a gel means that the gel is held using a chemical or physical interaction in the hollow part when the fiber is a hollow fiber or the porous part when the fiber is a porous fiber. And a state in which the biological substance is held in the held gel using chemical or physical interaction.
The gel that can be used in the present invention is not particularly limited. For example, acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, N -At least one monomer selected from vinyl pyrrolidone, hydroxyethyl methacrylate, (meth) acrylic acid, allyl dextrin, etc., and a polyfunctional monomer such as methylene bis (meth) acrylamide, polyethylene glycol di (meth) acrylate, etc. A gel obtained by copolymerizing the body in an aqueous medium can be used.
Next, an embedded object in which a plurality of fibers are embedded will be described.
In order to arrange a plurality of fibers in an orderly manner so that the fiber axes are substantially the same, for example, the fibers are regularly arranged using an arrangement jig described in WO00 / 53736. The number of fibers constituting the array is preferably 10 to 1,000,000 per 1 cm2.
In order to fix the arranged fibers, an embedding agent is uniformly injected between the arranged fibers and cured. Examples of the type of embedding agent include embedding agents composed of crosslinkable prepolymers, embedding agents composed of polymerized oligomers and catalysts, and thermoplastic polymers.
A polyurethane resin is mentioned as an embedding agent which consists of a crosslinkable prepolymer. The polyurethane resin can be prepared by adding a curing agent to the polyisocyanate of the crosslinkable prepolymer which is the main component. Examples of the curing agent include alcohols, ketones, amides, esters, castor oil-based polyols, and the like. Among these, those having a boiling point of 60 ° C. or more are particularly preferable, and specific examples include glycerol, polyethylene glycol, methyl ethyl ketone, acetamide, N, N-dimethylformamide, ethyl acetate and the like. In addition to the polyurethane resin, an epoxy resin, a silicone resin, or the like can be used.
As an embedding agent comprising a polymerizable oligomer and a catalyst, an acrylic type containing a (meth) acrylic ester [A] and a (meth) acrylic polymer or copolymer [B] soluble in the [A] component An example is syrup.
Specific examples of the component [A] include, for example, acrylic esters such as methyl acrylate, ethyl acrylate, n-butyl acrylate, t-butyl acrylate, 2-ethylhexyl acrylate; methyl methacrylate, ethyl methacrylate , N-butyl methacrylate, i-butyl methacrylate, t-butyl methacrylate, lauryl methacrylate, 2-ethylhexyl methacrylate, tridecyl methacrylate, stearyl methacrylate, cyclohexyl methacrylate, benzyl methacrylate, methacrylic acid 2 -Methacrylic acid ester such as hydroxyethyl, 2-hydroxypropyl methacrylate, dimethylaminoethyl methacrylate, diethylaminoethyl methacrylate, glycidyl methacrylate, tetrahydrofurfuryl methacrylate, allyl methacrylate Tell may be mentioned, and these may be used alone or in combination of two or more.
[A] When the glass transition temperature of the resin obtained by curing any one of the above monomers alone (only one type) as the component (temperature changing from a hard state to a soft state) is low, the resin tends to be soft. In addition, when the glass transition temperature is high, the obtained cured product tends to be hard. Therefore, in order to develop the desired embedding hardness, it is preferable to appropriately select and use the [A] component based on the glass transition temperature of the [A] component as a homopolymer.
The (meth) acrylic polymer used as the [B] component needs to be soluble in the [A] component. In the present invention, “soluble” includes a dispersed state. Specific examples of the component (B) include, for example, methyl methacrylate, ethyl methacrylate, n-butyl methacrylate, i-butyl methacrylate, t-butyl methacrylate, lauryl methacrylate, 2-ethylhexyl methacrylate, Examples thereof include homopolymers or copolymers of monomers selected from methyl acrylate, ethyl acrylate, n-butyl acrylate, t-butyl acrylate, 2-ethylhexyl acrylate, and the like. These may be used alone or in combination of two or more. The ratio of the [A] component and the [B] component is such that when the acrylic syrup composed of the [A] component and the [B] component is 100 parts by mass, the [A] component is in the range of 40 to 80 parts by mass, The component is in the range of 20 to 60 parts by mass, preferably the component [A] is in the range of 40 to 70 parts by mass, and the component [B] is in the range of 30 to 60 parts by mass.
Moreover, when performing flaming, it is preferable to add a urethane oligomer, a urethane polymer, or another rubber component to the acrylic syrup composed of the [A] component and the [B] component as appropriate. As the polymerization initiator for acrylic syrup, initiators such as azo, peroxide and redox which can be dissolved in the solvent to be used can be used. Examples include 2,2'-azobisisobutyronitrile, 2,2'-azobis (2-methylbutyronitrile) isobutyronitrile, benzoyl peroxide, or benzoyl peroxide-dimethylaniline. .
Examples of embedding agents that are thermoplastic polymers include polyethylene, polypropylene, polystyrene, ABS resin, methacrylic resin, polyvinyl chloride, polyamide, polycarbonate, polyacetal, PBT resin, polyphenylene sulfide, polyarylate polysulfone, polyethersulfone, polyether Examples include ether ketone, polymethylpentene, and polyetherimide.
When the embedded material embedded with the embedding agent is used as a biological material microarray, low fluorescence intensity may be required for detection of the biological material. In such a case, it is preferable to add carbon black or a quencher to the embedding agent. Moreover, it is preferable to select the embedding agent in consideration of the adhesion between the fiber and the embedding agent according to the type of fiber used. For example, an embedding agent made of acrylate syrup is preferable for polymethyl methacrylate hollow fibers.
It is preferable that the curing temperature of the embedding agent of this invention is below the glass transition temperature of a fiber material. More preferably, it is 100 ° C. or lower. In this way, by embedding an embedded material in which a plurality of fibers are embedded, the hardness is 70 to 95 (measured by JIS K 7215), and the surface of the flakes is smooth, It is possible to obtain a fiber array flake in which a bio-related substance without fibers falling off is retained. In the present invention, the hardness of the embedding agent is measured according to JIS K 7215 and refers to the hardness measured 5 seconds after the pressing surface is in close contact with the embedding material. If the hardness of the embedded material is less than 70 and greater than 95, the surface becomes rough and the fibers fall off, which is not preferable.
Next, thinning of the embedded material in which the fibers are arranged will be described.
When slicing, the embedded material may be cut in a direction intersecting the fiber axis of the embedded material, usually in a direction orthogonal thereto. The blade edge angle of the blade used for cutting is preferably 20 ° or less, and more preferably 15 ° or less. Examples of the material for the blade include carbon steel, SUS, and cemented carbide. Moreover, it is also possible to implement thinning using a commercially available microtome as an apparatus.
The thickness of the flakes can be arbitrarily adjusted, but is usually 1 to 5000 μm, preferably 10 to 2000 μm. The flakes produced in this way can be used as a bio-related substance microarray that can detect the expression state of a large number of genes and genome mutations at once.

  FIG. 1 shows a schematic diagram of cutting an embedded object with a blade. The flakes 3 are manufactured by cutting the embedding 2 in which the fibers 1 are embedded using a blade 5 in a direction 4 intersecting the fiber axis of the embedding 2. The cutting edge angle of the cutter described in this specification corresponds to 6 in FIG. The clearance angle of the blade corresponds to an angle formed by the cut surface and the lower surface of the blade, that is, 7 in FIG. The cutting of the embedded object may be performed by moving the embedded object 2 toward the blade 5 and cutting it, or by moving the blade 5 toward the embedded object 2 and cutting it.

  The clearance angle 7 is preferably 0 ° or more and 0.9 ° or less, and more preferably 0 ° or more and 0.5 ° or less. Further, it is most preferably 0 °. When the relief angle is a negative angle, the lower surface of the blade is always cut while pressing the cutting surface during cutting, and therefore the surface of the cutting surface is damaged by the blade, resulting in an increase in gel breakage. On the other hand, when the clearance angle is 1 ° or more, only the tip portion of the blade comes into contact with the block, and the lower surface of the blade is cut without being in contact with the block. Then, when cutting, the cutting edge bites into the block, and the counterclockwise force causes the cutting edge to move upward due to the elasticity of the cutting edge. The gel is easily broken. Further, if the angle is made larger, the blade may be broken at the time of cutting.

  A method for measuring the clearance angle 7 after being attached to the cutting device will be described. FIG. 2A shows a schematic view of the blade seen from the side. For example, the height of one part of the blade is measured from the lower surface of the blade by the non-contact type laser displacement meter 8. In FIG. 2A, for example, a measurement position A is assumed. Next, the height of the blade at a position moved by an arbitrary distance 9 horizontally from the measurement position A toward the edge of the blade is measured by the displacement meter 8. This is the measurement position B.

FIG. 2B shows a schematic diagram in which the measured positional relationship is enlarged. The vertices A and B of the triangle correspond to the measurement position A and the measurement position B in FIG. 2A, respectively. A vertex C represents a point moved by an arbitrary distance 9 from the vertex A. The height distance 10 can be obtained from the difference in height between the vertex A and the vertex B measured by a displacement meter. From these, the clearance angle 7 is

It is possible to calculate by

  As a method of measuring the amount of broken gel generated on a thin piece by cutting with a blade, it is possible to observe the broken gel and count the broken gel. When the gel is transparent and cannot be recognized, it can be observed, for example, by attaching abrasive grains to the gel surface.

Examples of the present invention will be described below.
Preparation of gel precursor solution N, N-dimethylacrylamide (3.42 parts), N, N′-methylenebisacrylamide (0.38 parts), glycerin (47.5 parts) and 2,2′-azobis (2 -Methylpropionamidine) dihydrochloride (0.1 part) was dissolved in water (48.6 parts) to prepare a gel precursor solution.
Fabrication of embedded DNA-immobilized hollow fiber embedded plate A 0.1 mm-thick perforated plate in which 19 × 12 and 228 holes having a diameter of 0.32 mm are arranged at intervals of 0.42 mm in the center of the plate Using two sheets, a hollow fiber made of polycarbonate colored by carbon black (manufactured by Mitsubishi Rayon Co., Ltd., outer diameter 0.28 mm, inner diameter 0.18 mm, length 500 mm) is passed through all the holes of the perforated plate. A porous plate through which two fibers are passed is separated by 50 mm, and a polyurethane resin colored by carbon black (Nippon Polyurethane Industry Co., Ltd., Nippon Run 4276 and Coronate 4403) is filled between the separated porous plates, and the length is 50 mm. The embedding object which has a part which is not fix | immobilized with resin at the both ends of prismatic shape of 7x12 mm square was obtained.

The hollow portion of the hollow fiber of the obtained embedded material is filled with a gel precursor solution and 40 types of nucleic acid probes of different types while entraining bubbles, the gel precursor solution is polymerized, and the DNA-immobilized hollow fiber An embedding material was prepared.
Method for measuring broken gel adhering to the surface of the flakes The flakes obtained by cutting with a blade were immersed in water mixed with abrasive grains to adhere the abrasive grains to the surface of the gel. Thereafter, the slice is rinsed in clean water, and the abrasive particles on the surface other than the gel surface are washed away, and then placed on a slide glass. Water and a cover glass are placed on the Olympus microscope SZX12 to prevent the gel from drying. It was left on the stage. The zoom was set to 20 times, the focus position of the microscope was adjusted to the surface of the thin piece, and an image was taken with a Nikon CCD camera CS5270B. Using the image processing software Image-Pro, the image of the surface of the imaged slice was extracted from the broken gel region adhering to the outside of the hollow fiber area, and the total number of pixels was taken as the total amount of gel.

A knife with a cutting edge angle of 20 ° (super thin carbide blade manufactured by Sumitomo Electric Hardmetal Co., grade AF1, double-edged type) with a clearance angle of 0 ° and a DNA-immobilized hollow fiber in a direction perpendicular to the fiber axis. The embedded material was cut to a thickness of 0.25 mm at a cutting speed of 200 mm / min. Thereafter, the broken gel on the flakes was measured for the total amount of broken gel according to the method for measuring broken gel.
FIG. 3 is a part of the observed surface image of the flake. The number of broken gel 8 regions was 3, and the total amount was 41 pixels.

  The clearance angle was 0.5 °, and other conditions were the same as in Example 1. FIG. 4 is a part of the surface image of the observed flake. There were 19 broken gel 8 regions, and the total amount was 453 pixels.

The clearance angle was 0.9 °, and other conditions were the same as in Example 1. FIG. 5 is a part of the surface image of the observed flake. There were 31 broken gel 8 regions, and the total amount was 608 pixels.
<Comparative Example 1>

The clearance angle was 1 °, and other conditions were the same as in Example 1. FIG. 6 is a part of the observed surface image of a flake. The number of broken gel 8 regions was 33, and the total amount was 831 pixels.
<Comparative Example 2>

The clearance angle was set to -1 °, and other conditions were the same as in Example 1. FIG. 7 is a part of the surface image of the observed flake. There were 49 broken gel 8 regions, and the total amount was 1181 pixels.

1: Fiber 2: Embedded object 3: Thin piece 4: Direction intersecting the fiber axis of the embedded object 5: Cutting tool 6: Cutting edge angle 7: Escape angle 8: Displacement meter 9: Distance 10 of measurement point: Height Distance 11: Broken gel

Claims (1)

Cuts an embedded object in which a plurality of fibers holding biological substances via a gel are embedded so that each fiber axis is substantially the same with a blade in a direction intersecting the fiber axis of the embedded object. And a method for producing flakes,
A method for producing a biologically relevant substance-carrying flake, wherein the blade is cut so that the clearance angle is in the range of 0 ° to 0.9 °.