JP2003247990A - Test substrate for biochemical test and minute substrate used therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a test substrate used for biochemical test covering biochemical substances such as nucleic acids, nucleic acid derivatives, proteins, glucides, cells, and cell fragments, and minute substrates used thereon for fixing biochemical substances. <P>SOLUTION: The minute substrates each have only one kind of biochemical substance fixed on its fixation surface and the plurality of minute substrates each having only one biochemical substance of a different kind fixed thereon are arranged in line on a mounting means, which serves as the test substrate used for biochemical test. Each of the fixation surfaces of the minute substrates is a plane having surface roughness of Ra 0.5 μm or less, and an identification code is given to some surface of each minute substrate in order to identify the minute substrate or the biochemical substance fixed thereon. A plurality of minute substrates with biochemical substances of different kinds fixed thereon are prepared in great numbers. Minute substrates with biochemical substances determined according to the prerequisites of test and fixed thereon are selected, and arranged on a tray, which serves as a test substrate and is submitted to test. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a test substrate for use in a biochemical test including a biochemical substance such as a nucleic acid, a nucleic acid derivative, a protein, a carbohydrate, a cell and a cell fragment, and a raw substrate used for the test substrate. The present invention relates to a micro substrate for fixing chemical substances.

[0002]

2. Description of the Related Art In the field of gene manipulation technology, technological development for efficiently analyzing all gene functions of various organisms, slight differences in nucleotide polymorphisms, protein diversity, etc. has been advanced. In the field of, a test substrate for fixing and manipulating a very small part of a gene is used. As an example of a test substrate, a DNA chip is a microarray in which a large number of oligonucleotides or DNA molecules are aligned on a carrier such as a slide glass. The biomaterial analysis technology using a test substrate can be applied to biomolecules other than DNA, for example, proteins and chemical substances, and can be used in drug discovery research, development of diagnostic methods for diseases and their preventive methods, measures for energy and environmental problems. It is expected to provide a new means for research and development such as.

The DNA chip technology has been embodied in
It started with the discovery that the base sequence of DNA can be determined by hybridization with an oligonucleotide or a DNA fragment. A technology for producing DNA chips and microarrays has been developed, and it has become possible to efficiently investigate mutations and polymorphisms of (many) gene expressions in a short time. In a DNA chip, a large number of oligonucleotides or DNA fragments (also referred to as probes) corresponding to various genes are aligned and immobilized on the solid surface of the chip,
This is an element in which a large number of fixed probes are formed in a matrix. Usually, thousands to tens of thousands of oligonucleotides are immobilized on one DNA chip.

In the method using a DNA chip, a replication chain reaction is used to synthesize cDNA (complementary DNA) of mRNA (messenger RNA) as a sample, which is fragmented and then labeled with a fluorescent label. Cut into pieces.
These labeled fragments are brought into contact with a DNA chip and hybridized with the immobilized oligonucleotide or DNA fragment. The labeled fragment is retained on the oligonucleotide or DNA fragment whose sequence is complementary, and the excess amount of the labeled fragment is removed by a washing operation. Then, detect the amount and position of the labeled fragment retained by fluorescence measurement,
Examine the type of corresponding oligonucleotide or DNA fragment. This method is suitable for monitoring the expression of genes of known sequence. Genomic DNA instead of cDNA
It is also possible to label the fragment of Escherichia coli and similarly examine the homology with the probe.

The DNA chip manufacturing methods are roughly classified into the following three types. In the first method, a plurality of linkers modified with protective groups that can be removed photochemically are bound to the surface of the solid phase via amino groups and arranged. The photolithography technology, which was developed in the semiconductor manufacturing technology, is applied to light-irradiate through a mask capable of irradiating only a desired linker-fixing position to remove the protective group. Next, a monomer having a protective group that can be removed photochemically is introduced to carry out the first coupling reaction only at the deprotected site. As a result, the oligonucleotide is extended only in that portion. By repeating such photolithography and the introduction of the monomer a number of times, a desired oligonucleotide matrix is formed.

Separately from this, there is also a method in which mononucleotide derivatives are successively sprayed onto a target site by an ink jet method or the like to be polymerized to synthesize a different oligonucleotide or the like at each site.

In the second method, an oligonucleotide to be immobilized is prepared in advance, and the oligonucleotide or DN is immobilized.
A small amount of the fragment A is dropped on the surface of a substrate such as glass or polymer film, and immobilized at that position by covalent bonding or the like. For example, introducing an isothiocyanate group to the surface of the substrate,
If the terminal of the oligonucleotide is an amino group, the oligonucleotide can be easily immobilized on the isothiocyanate group-immobilized position by a covalent bond.

In the third method, an oligonucleotide or DNA fragment to be immobilized is prepared in advance, a small amount of it is dropped on the surface of a substrate such as glass or polymer film, and the adsorbing action is used to immobilize it at the dropping position. To do.

[0009]

However, in the above-mentioned first method, photolithography and introduction of a monomer must be repeated each time the oligonucleotide is extended, or a nucleotide determined according to the design every time. The operation of dropping and polymerizing the derivative must be repeated. It takes a long time to produce this kind of DNA chip, and in order to produce oligonucleotides having different sequences on one substrate, the manufacturing cost becomes high due to the complexity of the mask and the operation.

In any of the second method and the third method described above, a plurality of kinds of oligonucleotides amplified by chemical synthesis or replication chain reaction are incorporated into a dedicated spotting device, and the oligonucleotide is immobilized on the solid phase surface. Since the droplets are sequentially arranged and dropped, in order to drop various kinds of oligonucleotides on one substrate, it was necessary to align and use the same number of stamp pins or to wash and use the stub pins. In this respect, there was a big problem in the equipment and work. In particular, when the stub pins are washed and used again, if they are not completely washed, different oligonucleotides will be mixed and an accurate test cannot be performed. Due to the complexity of such operations, it takes a long time to produce a DNA chip, and these methods also have a problem that the manufacturing cost is high. Further, in any of the above-described DNA chip manufacturing methods, when a large number of DNA chips having the same oligonucleotide matrix are manufactured, it is necessary to repeat the same operation for each DNA chip to manufacture one by one. However, there was a problem that the manufacturing cost did not decrease so much.

[0011] In the test, there are many combinations depending on the symptom and the purpose of the test, and it is necessary to prepare a DNA sequence corresponding to each of them, but this requires a large manufacturing cost. In addition, a general-purpose substrate for DNA identification on which thousands to tens of thousands of oligonucleotides are immobilized is used. However, due to the complementary binding of such a great variety of DNAs,
It is necessary to give a large number of duplicate cultures of the test DNA and to provide each with a discriminating functional group such as a fluorophore, and as a result, the amount of complementary binding of the test DNA is reduced and the complementary speed is slowed to obtain a result. There was a problem that it took a long time and was expensive. Further, the DNA test is a matter related to the privacy of an individual, and it has not been possible to meet the request to perform only the test required by the individual.

According to the present invention, a substrate used for detecting, selecting or obtaining nucleic acids, nucleic acid derivatives, proteins, and biochemical substances such as chemical substances, sugars and cells is made as small as possible and the amount of the substance to be inspected is reduced. (EN) Provided is an inspection substrate which can reduce the reaction time, accelerate the reaction rate, and reduce the inspection time and cost. The present invention provides a nucleic acid, a nucleic acid derivative, a protein, a chemical substance, a carbohydrate cell that specifically interacts with a substance immobilized on the surface of the test substrate by adsorption, chemical interaction, hybridization, etc. The present invention provides an inspection board capable of purifying and separating. Furthermore, the present invention is to provide a test substrate which can reduce the substrate manufacturing cost by shortening the preparation time of the test substrate used for detecting the biochemical substance as much as possible.

[0013]

The test board of the present invention is a test board for performing a biochemical test using a biochemical substance. The feature is that each test board has one type of biochemistry. It is composed of a plurality of micro substrates on which only a substance is immobilized on a fixed surface, and a mounting means for arranging and mounting the plurality of micro substrates.

The present invention further includes a micro substrate constituting such an inspection substrate. The micro substrate of the present invention is a micro substrate on which only one type of biochemical substance should be immobilized, and a plurality of micro substrates on which only different types of biochemical substances are immobilized are arrayed for biochemical examination. It is a micro board for inspection to be used.

In this specification, biochemical substances include nucleic acids, nucleic acid derivatives, proteins and sugars. The biochemical substance includes cells, cell fragments, and cell constituents.

The micro-substrate of the present invention includes a micro-substrate having only one kind selected from such biochemical substances immobilized on its surface. A large number of such micro-substrates are prepared in advance and prepared. A large number of different types of biochemical substances are immobilized on a micro substrate. According to the request of biochemical inspection, only the micro substrate on which the necessary type of biochemical substance is fixed is selected from the prepared micro substrates and arranged as the inspection substrate, and the inspection substrate is provided for the inspection. .

Here, the biochemical test includes all tests, tests, experiments, and other operations using the above-mentioned biochemical substances. For biochemical tests, nucleic acids, nucleic acid derivatives,
It includes the steps of using proteins, carbohydrates, cells, cell fragments or chemicals to detect, purify or fractionate these chemicals. For example, such tests will provide new tools for simultaneous analysis of gene expression, mutation, polymorphism, etc., and drug discovery research, development of disease diagnosis and prevention methods, research and development of energy and environmental problem countermeasures, etc. Substrates for detecting and quantifying nucleic acids, nucleic acid derivatives, proteins, carbohydrates, cells and chemicals that are highly expected to be provided, or through easy detection of protein-cell interactions, adsorption, antigen-antibody reactions, etc. The present invention relates to a substrate used for purifying and fractionating a substance that interacts with the fixing substance.

The test substrate of the present invention comprises a plurality of micro substrates on the surface of which a specific type of biochemical substance is fixed, and mounting means for arranging and holding these micro substrates.
Only a set of biochemical substances necessary for the inspection can be arranged in advance on the inspection board, and if the inspection board is used, only the necessary inspection can be performed quickly and at a low cost.

For such mounting means, a flat plate-like base on which a large number of minute substrates can be arranged can be used. As the base, a tray in which a large number of depressions having a shape corresponding to the outer shape of the micro substrate are provided in advance at the place where the micro substrate is to be arranged can be used. By placing the micro substrate in the recess, it is possible to position and place the micro substrate. Further, the mounting means may be in the form of a container, and as the container, a container into which a micro substrate can be loaded and which can be left open or sealed can be used. Examples include glass tubes and resin tubes.

In the present invention, the micro-substrate is preferably a label for identifying the micro-substrate or a biochemical substance immobilized on the micro-substrate, that is, a mark provided for identification. The identification code is added to the surface, bottom surface or side surface of the micro substrate as a code for identifying and identifying the micro substrate, or identifying the biochemical substance to be fixed or fixed on the micro substrate. Such an identification code is written in advance on the surface of the substrate, and is read out in the subsequent fixing operation of the biochemical substance, the arraying operation, and the required inspection process, while preventing misoperation under computer control. As a result, the processing by the automatic machine can be efficiently performed.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION In the micro substrate of the present invention, since only one specific type of biochemical substance is immobilized on the surface of one micro substrate in advance, the surface required for immobilizing the biochemical substance, that is, immobilization Although the outer shape is not particularly limited as long as the surface is provided, the fixing surface of the biochemical substance is preferably a smooth flat surface.

One specific type of biochemical substance is fixed and stored in advance on each micro substrate, and upon inspection, a large number of micro substrates are arranged and provided for inspection. A large number of these minute substrates are arranged on a mounting means and provided as an inspection substrate. The other mounting substrate is preferably flat, and a large number of the above-mentioned minute substrates are arranged on the upper surface thereof.
In order to arrange a large number, the minute substrates can be arranged in a matrix in the vertical and horizontal directions. As an example of the inspection board, FIG.
A large number of minute substrates having a rectangular outer shape 2. ． ． As a mounting means, the inspection board 1 mounted on the upper surface of the tray 3 having a rectangular outer shape is shown.

Regarding each of the micro substrates, one specific type of biochemical substance is fixed on the surface of each micro substrate, and the fixed surface is a smooth surface having a surface roughness Ra of 0.5 μm or less. Is preferred. The definition of this surface roughness is
It is particularly preferable for the assay substrate in which the fluorescence determination is performed by utilizing the fluorescent label of the biochemical substance in the inspection process. That is, when the surface roughness Ra exceeds 0.5 μm, when the inspection is performed by fluorescence in the inspection process, the excitation light for fluorescence emission is scattered on the surface, which makes it difficult to determine the fluorescence. . Further, the flatness of the fixing surface improves the inspection accuracy. In particular, a smooth surface having a surface roughness Ra of 0.2 μm or less is preferable.

The micro substrate can be formed of polymer, ceramic or silicon. It is particularly preferred that these materials are hydrophobic or poorly hydrophilic. As examples of polymers, cellulose acetate, polyvinyl, polyester polymers and the like can be used. Polyvinyl-based polymers include, for example, polystyrene and polymethylmethacrylate. Polyester-based polymers include, for example, polyethylene terephthalate and bisphenol A polycarbonate.

The ceramic includes an oxide sintered body, and for example, alumina, sapphire, silica, zirconia, titania, mullite, silicon carbide, etc. can be used. Ceramics includes glass, for example, silicate glass,
Borosilicate glass is used. Glass having a silica layer on its surface is also preferably used. For the material of the micro substrate,
Metals, especially inert or highly corrosion-resistant metals or submetals,
Including these alloys. For example, the material may be silicon or other semiconductor material.

Particularly, minute substrates of glass, silicon and ceramics are used, and these materials have an advantage that surface treatment for smoothing them as described above is easy and analysis by a fluorescence scanning device is easy.

The fine substrate has a smooth flat surface on the fixing surface made of such a material, and its shape is not particularly limited.
A thin plate having a rectangular upper surface can be used. As for the external dimensions, for example, those having a length and width of 0.2 to 3.0 mm and a thickness of 0.1 to 2.0 mm can be used. FIG. 3A shows a micro substrate 20 having such a rectangular outer shape.

In the manufacture of a micro substrate, a plate material relatively larger than the micro substrate, for example, a flat plate, is used from the above materials.
Can be formed and appropriately divided into minute substrates. For the division, a plate blade can be cut and cut using a cutting blade such as a die saw. For example, a flat plate made of silica glass, sapphire glass, silicon, polymer or the like is cut with a die saw.

A preferable dividing method is that when the above-mentioned flat plate without a plate material is formed, lattice-shaped dividing grooves are provided on the surface thereof in advance, and then the dividing grooves are bent to form a large number of rectangular minute substrates. A method of dividing can be adopted. The division by the dividing groove can be preferably applied to relatively brittle materials such as silica glass, sapphire glass, silicon and ceramics. The plate material or the flat plate is subjected to a later-described identification mark and necessary surface treatment before being divided, and then divided along the dividing groove to obtain the micro substrate 20.

The micro-substrate preferably includes a surface layer formed in advance on the immobilization surface of the micro-substrate, depending on the biochemical substance to be immobilized. FIG. 3C shows the rectangular micro substrate 2 described above.
An example in which the surface layer 6 is formed on the entire surface of 0 will be shown. The surface layer 6 is
It is used as a bonding layer for securely fixing the biochemical substance on the fixing surface 21 of the micro substrate. Micro substrate 20
When the material of (1) is hydrophobic or poor in hydrophilicity, the surface layer 6 is formed of nucleic acid, nucleic acid derivative,
It binds to substances to be fixed such as proteins, carbohydrates, cells, and other chemical substances such as cell fragments by electrostatic bonding or covalent bonding to ensure the fixing on the micro-substrate.

For the surface layer on the fixed surface, a thin film of a polycationic substance such as gold, poly-L-lysine or polyethyleneimine can be used. The thin substrate 4 can be surface-treated to form a thin film. The thin film can also be formed by surface-treating it with a silane coupling agent having a functional group such as an amino group, an aldehyde group and an epoxy group. The treatment with poly-L-lysine or the like may be combined with the treatment with a silane coupling agent.

A surface layer can be further formed on the fixed surface or the thin film formed on the fixed surface. Such a surface layer includes a layer containing a hydrophilic polymer substance having an electric charge and a layer containing a crosslinking agent. The surface layer of the above solid surface is
It can also be formed by containing or impregnating the above-mentioned silane coupling agent or hydrophilic or crosslinkable polymer in a micro substrate.

An identification code 5 is given in advance to the minute substrate of this embodiment. The identification code may identify the substrate or indicate the substance to be fixed,
The substrate or the fixed substance is identified by detecting the identification code, collating with the data and decoding.

The identification code can be given to an arbitrary position such as a back surface or a side surface in addition to the front surface or the fixed surface of the micro substrate. As the identification code, a well-known code can be used, and the code can be written on any of the outer surfaces of the micro substrate to provide characters, numbers, symbols, other codes, or optically or magnetically readable code information, such as a two-dimensional code. , Three-dimensional bar code or dot code. In particular, since the micro substrate is small, it is preferable that the identification code be in a fine form and be optically or magnetically detectable. In FIG. 3B, the identification code 5 composed of a two-dimensional code is shown on the micro substrate 2.
The example formed on the bottom surface 24 of FIG.

The identification code can be displayed by printing letters, numbers, symbols or the above bar code with ink or paint. The ink or paint used in this case is preferably selected from coating material having chemical resistance and corrosion resistance to a solvent that dissolves or suspends the biological sample in the inspection step. Preferably, the ink containing the ultraviolet curable resin is coated, and an ultraviolet pattern showing an identification code is irradiated, or laser scanning can be performed for printing.

In addition to printing with ink or the like, it is also possible to use an identification code that is optically detectable and that is drawn directly on the surface of the micro substrate by laser irradiation. Figure 3
In the example of (B), an array of patterned dot codes 5 is engraved on the bottom surface 24 of the micro substrate 20 by a laser beam. Further, as the magnetically detectable identification code, a magnetic layer formed on a substrate and magnetized by a magnetic head to be symbolized and readable can be employed.

The identification code displayed by laser irradiation is one in which the surface of the solid phase carrier is partially melted by the heat of the scanning laser beam on the irradiation surface, and a required mark is engraved and drawn.
This is convenient because it is not necessary to consider chemical resistance. On the other hand, in the case of the identification code of laser irradiation, there is a phenomenon that the edge portion of the engraved code rises. In FIG. 3 (B), around the engraved dot code, that is, the edge portion is raised in an annular shape. This rise is preferably 3 μm or less, and more preferably 1 μm or less. When the height of the edge portion is 3 μm or more, when storing a large number of minute substrates with the identification code 5, the edge protrusions are rubbed by the contact between the substrates and a missing particle (or “particle”) is generated. A), the particles may make subsequent inspection and purification and other work operations difficult.

FIG. 3D shows the fixing surface 21 of the minute substrate 20.
Micro substrate 2 having biochemical substance 7 immobilized on the upper surface layer
Is schematically shown. Various methods are used for fixing, such as a stamp pin method and an inkjet method. In the stamp pin method, for example, a slit method using a stamp pin having a slit formed at the tip, a pin method in which a solution is attached to the tip of a needle point, or a solution is applied in a film form in a ring having a diameter of 1 mm or less, The pin-ring method, which penetrates the inside with a pin tip, can be widely used. The biochemical substance is pre-treated prior to being immobilized on the micro substrate 20, and is capable of participating in interaction such that its molecular end is electrostatically or covalently bonded to a thin film or surface layer on the substrate surface. It is preferable to introduce a group. Such functional groups can include thiol groups, amino groups, aldehyde groups, carboxylic acid groups, biotin. Particularly, the functional group is preferably a thiol group or an amino group.

As shown in FIG. 3 (D), only one kind of the biochemical substance 7 such as nucleic acid is immobilized on the surface of the immobilization surface 21 of each of the micro-substrates 2 immobilized and prepared as described above. It The same or different biochemical substances on one micro substrate 7
A large number of micro-substrates 2 on which only one of these is fixed are prepared so as to cover a large number of biochemical substances. Then, according to the purpose and specifications of inspection, test, experiment, etc., only a large number of substrates on which the required specific type and amount of biochemical substance are fixed are selected.
The selected micro substrates are arranged and mounted as the mounting means, for example, on the tray 3, or a large number of the selected micro substrates are assembled into an appropriate container to form the inspection substrate 1.

The mounting means may be formed in a flat plate shape or a dish shape from ceramics, polymer or metal, though it depends on the form of use of the inspection substrate in many inspection processes. The mounting means is provided with recesses on the upper surface thereof, the outer shape of which corresponds to the minute substrates, and the selected set of the minute substrates is sequentially placed and set in each of the recesses to form an inspection substrate. It Subsequent management and inspection are carried out in units of inspection boards.

FIG. 2 shows an example of a flat tray 3 as the mounting means. On the upper surface of the tray 3, a large number of recesses 32 are formed so that the lower portion of the outer shape of the rectangular micro substrate 2 is fitted. A large number of micro substrates 2a ... 2f selected for each biochemical substance according to the requirements as described above.
Is a large number of recesses 32 ... Formed on the surface of the tray 3.
The test boards 1 as shown in FIG. The recessed portion 32 may have a shape that penetrates the front and back and has an engaging portion (for example, a step) of the micro substrate, and the bottom surface of the micro substrate 2 fitted in the recess 32 is the tray 3. Since it is visible from the bottom surface 33 side, the identification code on the bottom surface side of the micro substrate can be read from the back side of the tray. Alternatively, the tray 3 may be formed of a transparent body of glass, and the recessed portion may have a bottom to read through the glass. However, if the micro-substrates are identified by their respective identification codes and placed in the predetermined recesses, it is not necessary to read the identification code of the subsequent micro-substrates. As described above, the structure of the tray considering the reading is not always necessary.

An inspection board using these micro boards is
It can be widely used as a DNA substrate for immobilizing nucleic acids. Biochemicals other than nucleic acids, such as antibodies,
It is also used for immobilizing a protein, detecting an antigen, a carbohydrate, a cell or the like that interacts with the antibody, selectively purifying, or fractionating. In addition, for example, immobilization of chemical substances, detection of antigens, carbohydrates, cells, etc. that interact with it, purification,
Alternatively, it can be separated.

[0043]

EXAMPLES [Example 1] In this example, a minute substrate was prepared by selecting alumina as a substrate material. Alumina plate is
It has an outer shape with a size of 65 × 40 mm and a thickness of 0.2 mm, and a large number of orthogonal split grooves are formed on the surface at intervals of 0.6 mm in length and 0.35 mm in width. The alumina flat plate was subjected to barrel polishing and sandblasting to obtain an alumina plate having a surface roughness Ra of 0.2 μm, 0.35 μm, 0.5 μm and 0.6 μm.

With respect to this alumina plate, using a fluorescence spectrophotometer at excitation wavelengths of 532 nm and 630 nm,
The fluorescence spectrum of each wavelength was measured by irradiating with light having a band width of 1.5 nm. With respect to an alumina flat plate having a surface roughness Ra of 0.2 μm, no absorption maximum peak was observed at 570 and 660 nm which are detection wavelengths of Cy3 and Cy5 which are often used as fluorescent labels. It has been found that these fluorescent labels can be used as substrates for inspection. As the surface roughness Ra increases, the band width of the reflected light of the excitation wavelength tends to widen, and the surface roughness Ra of 0.
It was found that at 6 μm, the reflected light bandwidth spreads to the fluorescence detection wavelength range, and the light having wavelengths of 570 and 660 nm overlaps with the reflected light from the substrate, which reduces the accuracy of the inspection.

On the above-mentioned alumina substrate having a split groove,
The identification code was engraved by laser beam scanning. On the surface of the alumina substrate, inside the area defined by the split groove, and in each area of 0.55 × 0.25 mm square, consider thousands of types of biochemical substances and other types of test bodies. Then Y
A two-dimensional bar code was dug by scanning the AG laser beam. After confirming the laser reading of the dug out two-dimensional bar code, the reading was possible in 0.5 seconds, and it was effective as the identification code 5.

The surface undulations of the surface in which the identification code was dug were observed using a surface shape microscope, and the amount of swelling or height of the edge portion of the spot hole formed on the surface was measured by irradiating a laser beam. The laser irradiation output is reduced and the code sensor can read the code with a digging depth, and the swelling of the edge around the beam spot hole is 0.8.
was μm.

The swelling of the edge portion is liable to cause abrasion-free peeling and falling off. Regarding this point, substrates having swelling of various heights are superposed and a test is performed in which transverse vibration of 60 cycles is applied for 1 minute, and thereafter, The height of the swelling part was measured. As a result, when the protrusion was 5 μm, it was worn to 4.7 μm after the test, and when the protrusion was 3 μm or less, almost no change was observed. From this, it was confirmed that, as the identification code 5 given to the substrate by the laser beam, if the swelling of the edge portion was 3 μm or less, there was no abrasion due to the rubbing of the substrate and there was no influence on the inspection due to the missing particles. .

Example 2 With the findings of Example 1 as the following,
After engraving the identification symbol 5 with a laser on the back surface of an alumina substrate with a split groove having a surface roughness Ra of 0.35 μm, it is divided according to the split groove to have a length of 0.58 mm, a width of 0.30 mm, and a thickness. A large number of 0.20 mm minute substrates 20 were prepared, and the following processes were performed using these.

To clean the micro substrate 20, the divided micro substrate 4 is immersed in a mixed solution of 20% aqueous sodium hydroxide solution and 95% ethanol and shaken at room temperature for 2 hours, and then, It was washed by washing with distilled water.

In order to form the surface layer 6 on the micro substrate 20, the substrate was immersed in a 2% ethanol solution of aminopropyltrimethoxysilane, shaken at room temperature for 1 hour, then taken out and washed with ethanol. Then, in the dryer 110
It was heated and dried at 15 ° C. for 15 minutes to obtain a micro substrate 20 coated with a silano compound thin film.

After the active esterification of the micro substrate 20, only one kind of oligonucleotide having a base sequence of 25 was spotted on the fixed surface of the micro substrate to immobilize the oligo nucleotide having 25 bases. Substrate 2 was obtained.

This micro-substrate 2 is placed on the tray 3, and a fluorescein-bonding oligo-nucleotide complementary to the oligo-nucleotide immobilized on the micro-substrate 2 is added onto the substrate for hybridization, and then an excess fluorein-bonding is carried out. The oligonucleotide was washed off with ethanol.
After drying, fluorescence measurement of the fixed surface of the micro substrate was performed. On the micro-substrate on which the above oligonucleotide was spotted, clear fluorescence with a wavelength of 473 nm was observed, which proved to be sufficiently useful for detection.

[Example 3] By the same procedure as in Example 2, a large number of substrates 2 each having only one kind of oligonucleotide having a different number of bases immobilized thereon were prepared, and each of the two substrates was made into a transparent substrate having an inner diameter of 0.5 mm. A test substrate 1 which was inserted into a glass tube to detect the number of bases of an oligonucleotide as a sample was prepared. The micro substrate incorporated in the transparent glass tube could be confirmed by reading the identification code 5 of the two-dimensional barcode provided on the bottom surface of the micro substrate 2 using a laser reading device.

[Example 4] A poly-L-lysine thin film was formed on the surface of the fine substrate 4 which was divided from the alumina flat plate shown in Example 2 and similarly washed with alkali, according to the following procedure. First, the micro substrate is immersed in a poly-L-lysine diluting solution, left standing for 1 hour, taken out of the solution, and centrifuged using a centrifuge.
Centrifuge for 1 minute at 0 rpm to remove excess poly L-
The lysine solution was removed. Put the word in the suction incubator 40
The surface layer 6 was formed by drying at 5 ° C. for 5 minutes, transferring to a storage container, and holding at room temperature for 2 weeks to sufficiently fix the poly L-lysine thin film on the micro substrate 20.

Micro substrate 2 on which this poly L-lysine thin film is formed
0 is arranged on a glass tray for spotting, and each micro-substrate 20 has a pre-prepared DNA of a concentration of about 1 μg / μl.
The solution was spotted. After spotting, each micro-substrate was treated with a post-treatment solution, washed with enol, and then dried to prepare a substrate 2 on which only one kind of DNA 7 was immobilized. In the same manner, the substrate 2 on which only different kinds of DNA were immobilized was sequentially prepared. A large number of DNA-immobilized micro-substrates 2 were stored in a glass case while being placed on a glass tray and stored at room temperature. In this way, different types of DN are used for each micro substrate
A large number of micro substrates with only A fixed were prepared

In the inspection, a required combination of DNAs is required in order to inspect only the necessary items. Therefore, the set of corresponding DNAs is identified by the identification code from the prepared DNA substrates according to the requirements. The inspection substrate 1 was prepared by arranging a set of selected micro substrates on the tray 3 while confirming the selection. The recessed portion of the substrate size is arranged on the tray, so that the micro substrate can be easily fixed.
In this way, from the items necessary for inspection, D
It has become possible to select and inspect a micro substrate with a fixed NA. The confirmation and selection of the minute substrate to be incorporated were made by discriminating the identification code 5 by the laser reading device through the glass case.

[0057]

INDUSTRIAL APPLICABILITY The microsubstrate of the present invention has only one biochemical substance, such as nucleic acid, nucleic acid derivative, protein, carbohydrate, cell, cell fragment, etc., immobilized on one microsubstrate, which is necessary for testing. Therefore, such a large number of micro substrates can be prepared in advance, and the array of biochemical substances required for inspection and other operations can be selected from the above micro substrates and arranged, thus detecting the biochemical substances. Therefore, it is possible to reduce the manufacturing time of the inspection board used for that purpose, and it is possible to reduce the manufacturing cost.

Since the micro substrate fixes a single biochemical substance, it can be miniaturized into an extremely small substrate, and such a micro substrate corresponds to the inspection purpose in preparation for inspection. Since it is only necessary to select and arrange the minimum number as described above, it is possible to reduce the amount of the substance to be inspected and to reduce the inspection time and cost because there is no manifestation other than the intended items.

The inspection board is constructed by selecting a micro board on which only one type of biochemical substance is fixed on each micro board and disposing a large number of the selected micro boards on the mounting means. It becomes easy to process and manage it.

In particular, if each micro substrate is provided with an identification code, in the process of preparing and arranging the micro substrates and the subsequent inspection process,
By sequentially reading the identification code and identifying the biochemical substance fixed to the micro substrate, the inspection step can be performed while avoiding an erroneous operation.

By using the inspection board of the present invention,
Nucleic acids, nucleic acid derivatives, proteins, chemical substances, saccharides, cells, etc. that specifically interact with substances immobilized on the surface of a micro-substrate can be adsorbed, chemically interacted, hybridized, etc., depending on the purpose. It becomes possible to easily carry out purification and sorting.

[Brief description of drawings]

FIG. 1 is an external view of a test board according to an embodiment of the present invention.

FIG. 2 is an external view showing a group of micro substrates and a tray according to an embodiment of the present invention.

3A and 3B are external views of a micro substrate, where FIG. 3A shows the outer shape of the micro substrate, FIG. 3B shows the micro substrate with an identification code on the bottom surface, and FIG. 3C shows the micro substrate on which a surface layer is formed. , (D) respectively show the micro substrates having the biochemical substance immobilized on the immobilization surface.

[Explanation of symbols]

1 inspection board 2 Micro substrate 3 trays 5 identification code 6 surface layer

Continued front page    (72) Inventor Kenichi Matsubara             1-34 Suehiro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa             Issue NA Chip Research Institute Co., Ltd.             Within (72) Inventor Saburo Nagano             Kyocera Co., Ltd. 1-1 Yamashita-cho, Kokubun City, Kagoshima Prefecture             Inside the Kagoshima Kokubu Factory (72) Inventor Takashi Tanaka             6 Toba-den, Takeda, Fushimi-ku, Kyoto-shi, Kyoto             Kyocera Corporation (72) Inventor Shunichi Tsuneoka             6 Toba-den, Takeda, Fushimi-ku, Kyoto-shi, Kyoto             Kyocera Corporation (72) Inventor Zenpei Tachibana             6 Toba-den, Takeda, Fushimi-ku, Kyoto-shi, Kyoto             Kyocera Corporation F term (reference) 2G045 CB01 DA12 DA13 DA14 DA30                       DA36 FB02 FB03 FB07 FB12                       FB17 HA10 HA11 HA16

Claims (11)

[Claims] 1. A test substrate for performing a biochemical test using a biochemical substance, the test substrate comprising a plurality of micro substrates each having only one type of biochemical substance immobilized on a fixing surface, An inspection board comprising a mounting means for arranging and mounting a plurality of these minute boards. 2. The fixed surface of the micro substrate has a surface roughness Ra0.
The inspection board according to claim 1, which has a flat surface of 5 μm or less. 3. The inspection substrate according to claim 1, wherein an identification code for identifying the micro substrate or the fixed biochemical substance is attached to either surface of the micro substrate. 4. The inspection board according to claim 3, wherein the minute substrate is made of ceramics, and the identification code is a code engraved on the surface of the ceramics by laser beam irradiation. 5. The inspection board according to claim 4, wherein the swelling of the edge portion of the code engraved on the ceramic surface is 3 μm or less. 6. The mounting means has a flat plate shape and is provided with a recessed portion having a size of a minute substrate arranged on the upper surface, and the minute substrate is placed in the recessed portion. Inspection board described in. 7. The mounting means is a glass tube for accommodating a plurality of micro substrates on each of which only one type of biochemical substance is immobilized on each micro substrate. Inspection board. 8. A micro substrate which is arranged in a large number to form a test substrate in order to perform a biochemical test using a biochemical substance, and which has a fixing surface for fixing only one type of biochemical substance. Micro substrate having. 9. The fixing surface of the micro substrate has a surface roughness Ra0.
The micro substrate according to claim 8, which has a flat surface of 5 μm or less. 10. The micro substrate according to claim 8, wherein an identification code for identifying the micro substrate or the biochemical substance to be fixed is attached to either surface of the micro substrate. 11. The micro substrate according to claim 8, wherein the micro substrate is made of ceramics, and the identification code is a code engraved on the ceramic surface by laser beam irradiation.