JP4167431B2 - Inspection board for biochemical inspection - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a test substrate for use in biochemical tests containing biochemical substances such as nucleic acids, nucleic acid derivatives, proteins, carbohydrates, cells, cell fragments, etc., and a micro-substrate for immobilizing biochemical substances used therefor About.
[0002]
[Prior art]
In the field of gene manipulation technology, technological development for efficient analysis of all gene functions of various organisms, slight differences in nucleotide polymorphisms, or protein diversity is advancing. Inspection boards are used to fix and operate a very small part of the above. 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. Biomaterial analysis technology using test substrates can be applied to biomolecules other than DNA, such as proteins and chemicals, drug discovery research, development of disease diagnosis and prevention methods, energy and environmental countermeasures It is expected to provide a new means for research and development.
[0003]
The realization of DNA chip technology began with the discovery that the base sequence of DNA can be determined by hybridization with oligonucleotides or DNA fragments. Development techniques for DNA chips and microarrays have been developed, and it has become possible to efficiently examine mutations and polymorphisms in a short time with respect to gene expression. A DNA chip is an element in which a large number of oligonucleotides or DNA fragments (also referred to as probes) corresponding to various genes are aligned and immobilized on a solid surface of the chip, and a large number of immobilized probes are formed in a matrix. Usually, thousands to tens of thousands of oligonucleotide individuals are immobilized on one DNA chip.
[0004]
In the method using a DNA chip, cDNA (complementary DNA) of mRNA (messenger RNA) as a sample is synthesized using a replication chain reaction, and after fragmentation, fluorescent fragments are attached to the fragments to form labeled fragments. . These labeled fragments are brought into contact with a DNA chip and hybridized to the immobilized oligonucleotide or DNA fragment. The labeled fragment is held in an oligonucleotide or DNA fragment having a complementary sequence, and an excessive amount of the labeled fragment is removed by a washing operation. Thereafter, the amount and position of the labeled fragment retained by fluorescence measurement is detected, and the type of the corresponding oligonucleotide or DNA fragment is examined. This method is suitable for monitoring the expression of genes of known sequence. Instead of cDNA, a fragment of genomic DNA can be labeled, and the homology with the probe can be similarly examined.
[0005]
There are roughly the following three types of DNA chip manufacturing methods. In the first method, a plurality of linkers modified with a protecting group that can be removed photochemically are bonded to the surface of a solid phase via an amino group or the like and arranged. The photolithographic technique has been developed as a semiconductor manufacturing technique. By applying this technique, light is irradiated through a mask that can irradiate only a desired linker fixing position, and the protecting group is removed. Next, a monomer having a protecting group that can be removed photochemically is introduced, and the first coupling reaction is carried out only at the deprotected site. As a result, the oligonucleotide is extended by that portion. By repeating such photolithography and monomer introduction many times, a desired oligonucleotide matrix is formed.
[0006]
Apart from this, there is also a method in which mononucleotide derivatives are sprayed one after another to the target site by an ink jet method or the like and polymerized to synthesize different oligonucleotides at each site.
[0007]
In the second method, an oligonucleotide to be immobilized is prepared in advance, a small amount of the oligonucleotide or DNA fragment is dropped on the surface of a substrate such as glass or a polymer film, and immobilized at that position by covalent bonding or the like. For example, if an isothiocyanate group is introduced on the substrate surface and the end of the oligonucleotide is an amino group, the oligonucleotide can be easily immobilized by covalent bonding at the isothiocyanate group fixing position.
[0008]
In the third method, an oligonucleotide or DNA fragment to be immobilized is prepared in advance, and a small amount thereof is dropped on the surface of a substrate such as glass or a polymer film, and is immobilized at the dropping position using an adsorption action.
[0009]
[Problems to be solved by the invention]
However, in the first method described above, every time the oligonucleotide is extended, photolithography and introduction of the monomer must be repeated, or the operation of dropping the nucleotide derivative determined according to the design and polymerizing each time is performed. Must be repeated. It took a long time to produce this type of DNA chip, and the production cost was high due to the complexity of the mask and operation to produce oligonucleotides with different sequences on one substrate.
[0010]
In each of the second method and the third method, a plurality of types of oligonucleotides amplified by chemical synthesis or replication chain reaction are taken into a dedicated spotting device and sequentially arranged on the solid phase surface. Therefore, in order to drop various kinds of oligonucleotides on one substrate, it was necessary to arrange and use the same number of stamp pins or to clean and use the stub pins. In this respect, there was a big problem in the apparatus and work. In particular, when the stabbing pins are washed and used again, unless complete washing is performed, different oligonucleotides are mixed and an accurate inspection 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 production cost increases. In any of the above methods for producing a DNA chip, when a large number of DNA chips having the same oligonucleotide matrix are produced, it is necessary to repeat the same operation for each DNA chip and produce them one by one. There was a problem that the manufacturing cost did not decrease so much.
[0011]
  Also, there are many combinations depending on the symptoms and the purpose of the test, and DNA sequences corresponding to each should be prepared as appropriate. A general-purpose DNA identification substrate on which thousands to tens of thousands of oligonucleotides are immobilized is used.
However, in order to perform complementary binding for such a great variety of DNAs, it is necessary to provide a large number of replication cultures of the DNA to be tested, and to each of which an identification functional group such as a fluorescent substance is added. There is a problem that the amount of complementary binding of the DNA to be tested is reduced, the speed of complementation is slow, and it takes a long time and results in obtaining a result.there were. Furthermore, DNA testing is a matter related to personal privacy, and it has not been possible to answer the request to perform only the testing required by the individual.
[0012]
The present invention reduces the amount of a substance to be inspected by miniaturizing a substrate used for detecting, selecting or obtaining nucleic acids, nucleic acid derivatives, proteins, and biochemical substances such as chemical substances, carbohydrates, and cells. The present invention provides an inspection substrate that can accelerate the reaction speed, reduce the inspection time and cost, and can shorten the inspection substrate.
The present invention relates to a nucleic acid, a nucleic acid derivative, a protein, a chemical substance, a sugar cell that specifically interacts with a substance immobilized on its surface by adsorption, chemical interaction, hybridization, etc. using a test substrate. It is intended to provide an inspection substrate capable of purifying and separating the components.
Furthermore, the present invention is to provide a test substrate capable of shortening the preparation time of the test substrate used for detecting the biochemical substance as much as possible and reducing the substrate manufacturing cost.
[0013]
[Means for Solving the Problems]
  The inspection board of the present invention is an inspection board for performing a biochemical inspection using a biochemical substance,Necessary for the biochemical examinationMultiple micro-substrates, each of which has only one kind of biochemical substance immobilized on the fixed surfaceIs mounted on one mounting means.
[0014]
  Furthermore, the present inventionThe micro-substrate is mounted with the back surface of the fixed surface facing the mounting means side, and the back surface can be read in a state of being mounted on the mounting means, and the one type of biochemical substance can be read. The corresponding identification symbol is given
[0015]
In this specification, the biochemical substance includes a nucleic acid, a nucleic acid derivative, a protein, and a carbohydrate. Biochemical substances include cells, cell fragments, and cell constituent substances.
[0016]
The micro substrate of the present invention includes a micro substrate in which only one kind selected from such biochemical substances is fixed on the surface. A large number of such micro substrates are prepared and prepared in advance. Many different types of biochemical substances are fixed. According to the requirement of biochemical inspection, only a micro substrate on which a necessary kind of biochemical substance is fixed is selected from micro substrates prepared in advance and arranged as an inspection substrate, and the inspection substrate is provided for the inspection. .
[0017]
Here, the biochemical examination includes all examinations, tests, experiments, and other operations using the above-described biochemical substances. Biochemical testing includes the process of detecting, purifying, or fractionating these chemicals using nucleic acids, nucleic acid derivatives, proteins, carbohydrates, cells, cell fragments, or chemicals. Such tests, for example, provide new tools for the simultaneous analysis of gene expression, mutation, polymorphism, etc., and drug discovery research, development of disease diagnosis and prevention methods, and research and development of energy and environmental issues. 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 and cell interactions, adsorption, antigen-antibody reactions, etc. The present invention relates to a substrate used for purifying and separating a substance that interacts with a fixed substance.
[0018]
The inspection board of the present invention comprises a plurality of micro-substrates on which a specific kind of biochemical substance is fixed on the surface, and mounting means for arranging and holding these micro-substrates. Only a set of necessary biochemical substances can be arranged, and if a test substrate is used, only necessary tests can be performed quickly and at a low cost.
[0019]
As such mounting means, a flat base on which a large number of minute substrates can be arranged can be used. As the base, a tray can be used in which a large number of depressions having a shape corresponding to the outer shape of the minute substrate are provided in advance at a position where the minute substrate is to be disposed. Positioning and placing the micro substrate becomes possible by placing in the recess.
The mounting means may be in the form of a container, and a container that can be loaded with a micro-substrate and that can be opened or sealed can be used. For example, there are a glass tube and a resin tube.
[0020]
In the present invention, it is preferable that the micro substrate is provided with a label for identifying the micro substrate or a biochemical substance fixed to the micro substrate, that is, an identification code. The identification code is added to the surface, bottom surface, or side surface of the micro substrate as a code for identifying and specifying the micro substrate, or specifying a biochemical substance to be fixed on the micro substrate. Such identification codes are written in advance on the surface of the substrate, and in the subsequent fixing operation, arrangement operation, and required inspection process of the biochemical substance, reading is performed to prevent erroneous operation under computer control. The processing by the automatic machine can be carried out efficiently.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
Since the micro-substrate of the present invention has only one specific type of biochemical substance fixed on the surface in advance on one micro-substrate, the surface required for fixing the biochemical substance, that is, if a fixing surface is provided, Although the outer shape is not limited, it is preferable that the fixing surface of the biochemical substance is a smooth flat surface.
[0022]
A specific type of biochemical substance is fixed and stored in advance on each micro-substrate, and at the time of inspection, a large number of micro-substrates are arranged and provided for the inspection. These many micro-substrates are arranged on the mounting means and provided as inspection substrates. The other mounting substrate is preferably in the form of a flat plate, 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 micro-substrates can be arranged in rows and columns.
As an example of the inspection substrate, FIG. . . Is a test substrate 1 mounted on the upper surface of a tray 3 having a rectangular shape as a mounting means.
[0023]
Regarding each micro-substrate, one specific type of biochemical substance is fixed to each micro-substrate on the fixed surface of the surface, and the fixed surface is a smooth surface having a surface roughness Ra of 0.5 μm or less. Is preferred. This definition of surface roughness is particularly preferable for an assay substrate in which fluorescence determination is performed using a fluorescent label of a biochemical substance in an inspection process. That is, when the surface roughness is more than Ra 0.5 μm, when the inspection is performed with fluorescence in the inspection process, excitation light for fluorescent color development is scattered on the surface, making it difficult to determine the fluorescence. . Further, the fact that the fixed surface is a flat surface improves the inspection accuracy. In particular, a smooth surface having a surface roughness Ra of 0.2 μm or less is preferable.
[0024]
  Micro substrate, polymer, ceramicTheCan be formed from silicon.
These materials are particularly preferably hydrophobic or poorly hydrophilic.
Examples of the polymer include cellulose acetate-based, polyvinyl-based, and polyester-based polymers.
Examples of the polyvinyl polymer include polystyrene and polymethyl methacrylate.
Examples of polyester polymers include polyethylene terephthalate and bisphenol A polycarbonate.
[0025]
The ceramic includes an oxide sintered body, and for example, alumina, sapphire, silica, zirconia, titania, mullite, silicon carbide, and the like can be used. Ceramics includes glass, and for example, silicate glass and borosilicate glass are used. Glass having a silica layer on the surface is also preferably used.
Examples of the material for the micro substrate include metals, in particular, metals and sub-metals which are inert and have high corrosion resistance, and alloys thereof. For example, silicon or another semiconductor material can be used as this material.
[0026]
In particular, micro substrates of glass, silicon, and ceramics are used, and these materials have an advantage that they can be easily surface-treated as described above and can be easily analyzed by a fluorescence scanning apparatus.
[0027]
The micro-substrate is made of such a material, has a smooth flat surface on the fixed surface, and is not particularly limited in shape. Usually, a thin plate having a rectangular upper surface can be used. For example, the outer dimensions may be those having a length and width in the range of 0.2 to 3.0 mm and a thickness in the range of 0.1 to 2.0 mm. FIG. 3A shows such a small substrate 20 having a rectangular shape.
[0028]
In the manufacture of a micro substrate, a plate material, for example, a flat plate, which is relatively larger than the micro substrate can be formed from the above materials, and can be divided into appropriate micro substrates. For the division, a plate material can be cut and cut out using a cutting blade such as a die saw. For example, it is cut out from a flat plate made of silica glass, sapphire glass, silicon, polymer or the like with a die saw.
[0029]
  As a preferable dividing method, the above plate material is used.OrWhen forming the flat plate, it is possible to adopt a method in which a lattice-shaped dividing groove is provided on the surface in advance, and the dividing groove is bent and divided into a large number of rectangular minute substrates.
The division by the dividing groove can be preferably applied to relatively brittle materials such as silica glass, sapphire glass, silicon and ceramics.
After the plate material or flat plate is given an identification symbol to be described later or necessary surface treatment before dividing, the substrate 20 is divided along the dividing groove to obtain the micro substrate 20.
[0030]
  It is preferable that the micro substrate includes a surface layer formed in advance on the fixing surface of the micro substrate according to the biochemical substance to be fixed. FIG. 3C shows an example in which the surface layer 6 is formed on the entire surface of the rectangular minute substrate 20. The surface layer 6 is used as a bonding layer for securely fixing the biochemical substance on the fixing surface 21 of the micro substrate. Material of micro substrate 20ButIs it hydrophobic,Alternatively, when the surface layer 6 is poor in hydrophilicity, the surface layer 6 is composed of nucleic acid, nucleic acid derivative, protein, carbohydrate, cell, cell fragment.OrIt is bonded to other substances to be fixed, such as chemical substances, by electrostatic bonding or covalent bonding to ensure fixation on the micro substrate.
[0031]
As the surface layer on the fixed surface, a thin film of a polycationic material 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 treatment with a silane coupling agent having a functional group such as an amino group, an aldehyde group, or an epoxy group. The treatment using poly-L-lysine or the like may be combined with the treatment with a silane coupling agent.
[0032]
A surface layer can be further formed on the fixed surface or the thin film formed thereon. Such a surface layer includes a layer containing a charged hydrophilic polymer substance and a layer made of a crosslinking agent. The surface layer of the solid surface can also be formed by containing or impregnating the silane coupling agent or a hydrophilic or crosslinkable polymer in a micro substrate.
[0033]
An identification code 5 is assigned in advance to the minute substrate of this embodiment. The identification code may identify the substrate or display the substance to be fixed, but it identifies the substrate or the fixed substance by detecting the identification code and comparing it with the data. It is.
[0034]
The identification code can be given to an arbitrary position such as the back surface or the side surface in addition to the surface or the fixed surface of the minute substrate. As the identification code, a known one can be used, written on one of the outer surfaces of the minute substrate, letters, numbers, symbols, other codes, code information that can be read optically or magnetically, for example, two-dimensional There are 3D barcodes and dot codes. In particular, since the minute substrate is small, the identification code is preferably a fine form that can be detected optically or magnetically. FIG. 3B shows an example in which the identification code 5 composed of a two-dimensional code is formed on the bottom surface 24 of the minute substrate 2.
[0035]
The identification code can be displayed by printing letters, numbers, symbols, and the above barcode with ink or paint. The ink or paint used in this case is preferably selected from coating materials having chemical resistance and corrosion resistance to a solvent that dissolves or suspends the biological sample in the inspection process. Preferably, an ultraviolet curable resin-containing ink is coated and irradiated with an ultraviolet pattern representing an identification code, or printing can be performed by laser scanning.
[0036]
In addition to printing with ink or the like, the identification code can also be drawn directly on the surface of the minute substrate by laser irradiation as being optically detectable. The example of FIG. 3B shows an example in which a patterned array of dot codes 5 is engraved on the bottom surface 24 of the minute substrate 20 by a laser beam.
Furthermore, as an identification code that can be detected magnetically, a magnetic layer formed on a substrate, magnetized by a magnetic head, converted into a symbol, and read can be used.
[0037]
The identification code displayed by laser irradiation is such that the solid support surface is partially dissolved by the heat of the scanning laser beam on the irradiated surface and engraved with the required mark, so that chemical resistance does not have to be considered. So it is convenient.
On the other hand, in the case of a laser irradiation identification code, a phenomenon in which the edge portion of the engraved code rises is seen. In FIG. 3B, the encircling dot code, that is, the edge portion bulges in an annular shape. This bulge is preferably 3 μm or less, and more preferably 1 μm or less. When the edge portion has a height of 3 μm or more, when storing a large number of minute substrates engraved with the identification code 5, the edge rising portion is rubbed by the contact between the substrates, and missing particles (or “particles”) This is because the particles may be peeled off and the subsequent inspection and purification and other work operations may be difficult.
[0038]
FIG. 3D schematically shows the micro substrate 2 on which the biochemical substance 7 is fixed on the surface layer on the fixing surface 21 of the micro substrate 20. Various methods are used for fixing, for example, a stamp pin method and an ink jet method. In the stamp pin method, for example, a slit method using a stamp pin in which a slit is formed at the tip, a pin method in which a solution is attached to the tip of a needle tip, or a solution is spread in a film in a ring having a diameter of 1 mm or less. A pin-ring system that penetrates the inside with a pin tip can be widely used.
The biochemical substance is processed in advance prior to immobilization on the micro-substrate 20, and a functional group capable of participating in the interaction is formed so as to be electrostatically or covalently bonded to a thin film or a surface layer of the substrate surface at the molecular end. It is preferable to introduce a group. Such functional groups can include thiol groups, amino groups, aldehyde groups, carboxylic acid groups, and biotin. In particular, the functional group is preferably a thiol group or an amino group.
[0039]
As shown in FIG. 3D, only one type of biochemical substance 7 such as a nucleic acid is immobilized on the surface of the immobilization surface 21 of each micro-substrate 2 immobilized and prepared as described above. A large number of micro-substrates 2 in which only one kind of the same or different biochemical substance 7 is fixed to one micro-substrate are prepared so as to cover a large number of biochemical materials. Then, depending on the purpose and specification of inspection, test, experiment, etc., only a large number of substrates on which a specific type and amount of biochemical substances necessary are fixed are selected. The selected micro substrates are mounted on the tray 3 as mounting means, for example, or a large number of the selected micro substrates are incorporated into an appropriate container to form the inspection substrate 1.
[0040]
The mounting means may be formed in a flat plate shape or a dish shape from ceramics, polymer, or metal, although it varies depending on the form of use of the inspection substrate in many inspection processes. The mounting means has a concave portion whose outer shape corresponds to the micro substrate on the upper surface, and the selected set of micro substrates is sequentially placed and placed in each of the concave portions to form an inspection substrate. The Subsequent management and inspection are performed in units of inspection substrates.
[0041]
FIG. 2 shows an example of a flat tray 3 as the mounting means. The upper surface of the tray 3 is provided with a large number of recesses 32 formed so that the lower part of the outer shape of the rectangular micro-substrate 2 is fitted. As described above, a large number of micro-substrates 2a... 2f selected for each biochemical substance are fitted into each of a large number of recesses 32. The inspection substrate 1 is as shown in FIG. The recessed portion 32 can be formed in a shape that penetrates the front and back and has a locking portion (for example, a step) of the micro substrate, and the bottom surface of the micro substrate 2 fitted in the recessed portion 32 is formed on the tray 3. Since it can be visually recognized from the bottom surface 33 side, the identification code on the bottom surface side of the minute substrate can be read from the back side of the tray. Alternatively, the tray 3 may be formed of a glass transparent body, and can be read through the glass with the recessed portion as a bottom.
However, when the minute substrate is identified by the respective identification codes and placed in a predetermined recess, it is unnecessary to read the subsequent identification codes of the minute substrates. As described above, the tray structure considering the reading is not necessarily required.
[0042]
A test substrate using these micro-substrates can be widely used as a DNA substrate on which a nucleic acid is immobilized. It is also used to immobilize biochemical substances other than nucleic acids, such as antibodies, and detect, selectively purify, or sort out antigens, carbohydrates, cells, etc. that interact with the antibodies. In addition, for example, it is possible to fix a chemical substance and detect, purify, or sort out antigens, carbohydrates, cells, etc. that interact with the chemical substance.
[0043]
【Example】
[Example 1]
In this example, a micro substrate was prepared by selecting alumina as the substrate material. The alumina flat plate has an outer shape with dimensions of 65 × 40 mm and a thickness of 0.2 mm, and a large number of orthogonal dividing grooves are formed on the surface at intervals of 0.6 mm in length and 0.35 mm in width. As the alumina flat plate, alumina plates having surface roughness Ra of 0.2 μm, 0.35 μm, 0.5 μm and 0.6 μm were obtained by barrel polishing and sandblasting.
[0044]
The alumina flat plate was irradiated with light having an excitation wavelength of 532 nm and 630 nm and a bandwidth of 1.5 nm using a fluorescence spectrophotometer, and the fluorescence spectrum of each wavelength was measured. For the alumina flat plate having a surface roughness Ra of 0.2 μm, absorption maximum peaks were not observed at 570 and 660 nm, which are detection wavelengths of Cy3 and Cy5, which are often used as fluorescent labels. It was found that it can be used as a substrate to be inspected using these fluorescent labels. When the surface roughness Ra is increased, the bandwidth of the reflected light having the excitation wavelength tends to be increased. When the surface roughness Ra is 0.6 μm, the reflected light bandwidth is extended to the fluorescence detection wavelength region, 570 and 660 nm. It was found that the light of the wavelength overlaps with the reflected light from the substrate, respectively, and the accuracy of the inspection is lowered.
[0045]
An identification code was engraved on the above-mentioned alumina substrate provided with a split groove by laser beam scanning. Thousands of biochemical substances and other types of specimens are considered in the 0.55 x 0.25 mm square area on the surface of the alumina substrate inside the area divided by the split grooves. Then, a YAG laser beam was scanned to dig a two-dimensional barcode. The laser reading of the dug two-dimensional bar code was confirmed, and the reading was possible in 0.5 seconds, which was effective as the identification code 5.
[0046]
The surface of the surface where the identification code was dug was observed using a surface shape microscope, and the surface undulation was observed, and the bulge amount or height of the edge portion of the spot hole formed on the surface was measured by irradiation with a laser beam. The swell of the edge around the beam spot hole was 0.8 μm at a digging depth where the laser irradiation output was reduced and the code could be read by the image sensor.
[0047]
The bulge of the edge part is likely to cause wear-free peeling and drop-off, but in this regard, a test with 60 minutes of horizontal vibration for 1 minute was conducted by laminating substrates with various bulges, and the subsequent bulge part The height of was measured. As a result, at the rise of 5 μm, it was worn to 4.7 μm after the test, and almost no change was seen at 3 μm or less. From this, it was confirmed that as the identification code 5 attached to the substrate by the laser beam, if the bulge of the edge portion is 3 μm or less, there is no abrasion due to the rubbing of the substrate and there is no influence on the inspection caused by the missing particles. .
[0048]
[Example 2]
Based on the knowledge of Example 1, having a flat surface with a surface roughness Ra of 0.35 μm, engraving the identification symbol 5 with a laser on the back surface of the alumina substrate with a split groove, and then dividing according to the split groove to obtain a length. A number of micro substrates 20 having a width of 0.58 mm, a width of 0.30 mm, and a thickness of 0.20 mm were formed, and the following processing was performed using these.
[0049]
In order to clean the micro substrate 20, the divided micro substrate 4 is immersed in a mixed solution of 20% aqueous sodium hydroxide and 95% ethanol, shaken at room temperature for 2 hours, and then with distilled water. Rinse and wash.
[0050]
In order to form the surface layer 6 on the micro substrate 20, it was immersed in a 2% ethanol solution of aminopropyltrimethoxysilane, shaken at room temperature for 1 hour, then taken out and washed with ethanol. Then, it heated and dried at 110 degreeC with the dryer for 15 minutes, and obtained the micro substrate 20 coat | covered with the silano compound thin film.
[0051]
After this micro-substrate 20 is active-esterified, only one type of oligonucleotide having 25 base sequences is spotted on the fixed surface of the micro-substrate, and the micro-substrate 2 on which the 25-base oligonucleotide is immobilized is prepared. Obtained.
[0052]
The microsubstrate 2 is placed on the tray 3, and a fluorescein-binding oligonucleotide complementary to the oligonucleotide immobilized on the microsubstrate 2 is added to the substrate for hybridization. Washed off at After drying, the fluorescence measurement of the fixed surface of the micro substrate was performed. In the minute substrate on which the oligonucleotides were spotted, clear fluorescence with a wavelength of 473 nm was observed, which proved to be sufficiently useful for detection.
[0053]
[Example 3]
In the same procedure as in Example 2, a large number of substrates 2 each having one kind of oligonucleotide having a different number of bases immobilized thereon were prepared, and each of the two substrates was inserted into a transparent glass tube having an inner diameter of 0.5 mm. A test substrate 1 for detecting the base number of the oligonucleotide 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 applied to the bottom surface of the micro substrate 2 using a laser reader.
[0054]
[Example 4]
A poly L-lysine thin film was formed by the following procedure on the surface of the micro-substrate 4 divided from the alumina flat plate shown in Example 2 and similarly washed with alkali. First, the microsubstrate was immersed in a poly L-lysine diluted solution and left for 1 hour, taken out from the solution, and centrifuged at a rotation speed of 500 rpm for 1 minute using a centrifuge to remove excess poly L-lysine solution. The word is put in a suction type incubator, dried at 40 ° C. for 5 minutes, transferred to a storage container and kept at room temperature for 2 weeks to sufficiently fix the poly L-lysine thin film on the micro substrate 20 to form the surface layer 6. did.
[0055]
The micro substrates 20 formed with the poly L-lysine thin film were arranged in a spot-mounted glass tray, and each micro substrate 20 was spotted with a DNA solution having a concentration of about 1 μg / μl prepared in advance. After the spotting, each micro-substrate was treated with a post-treatment solution, washed with enol, and then dried to produce a substrate 2 on which only one kind of DNA 7 was fixed. Similarly, substrates 2 on which only different types of DNA were immobilized were prepared in sequence. Many micro-substrates 2 on which DNA was immobilized were stored in a glass case while being placed on a glass tray and stored at room temperature. Thus, a large number of micro-substrates were prepared in which only different types of DNA were immobilized on each micro-substrate.
[0056]
In the inspection, a necessary combination of DNAs is required corresponding to the inspection of only the necessary items. Accordingly, according to the requirements, the corresponding DNA sets are confirmed by the identification codes from the prepared many DNA substrates. A set of selected micro-substrates was arranged on the tray 3 to form an inspection substrate 1. A concave portion of the substrate size is arranged on the tray, so that the micro substrate can be easily fixed. In this way, according to the inspection request, it is possible to perform the inspection by selecting a micro substrate on which DNA is immobilized from the items necessary for the inspection. Confirmation and selection of the micro substrate to be assembled were made by discriminating the identification code 5 with a laser reading device through a glass case.
[0057]
【The invention's effect】
The microsubstrate of the present invention immobilizes only one kind of biochemical substance, such as a nucleic acid, a nucleic acid derivative, a protein, a carbohydrate, a cell, and a cell fragment, necessary for the inspection. A large number of such small substrates are prepared in advance, and the arrangement of biochemical substances required for inspection and other operations can be selected and arranged from the above-mentioned minute substrates, and therefore the inspection substrate used for detecting the biochemical substances The manufacturing time can be shortened, and the manufacturing cost can be reduced.
[0058]
  Since the micro-substrate fixes a single biochemical substance, it can be miniaturized to an extremely small substrate, and in preparation for inspection.IsSince it is only necessary to select and arrange such a small number of substrates so as to correspond to the inspection purpose, the amount of the substance to be inspected is reduced, and the inspection time and the absence of expression other than the target item are reduced. Costs can be reduced.
[0059]
The inspection substrate is configured by selecting a micro substrate in which only one kind of biochemical substance is fixed to each micro substrate and arranging a large number of selected micro substrates on the mounting means. Easy to operate and manage.
[0060]
In particular, if an identification code is attached to each micro substrate, the micro substrate is prepared and arranged, and in the subsequent inspection process, the identification code is sequentially read to identify the biochemical substance fixed to the micro substrate, The inspection process can be performed while avoiding erroneous operations.
[0061]
By using the test substrate of the present invention, a nucleic acid, a nucleic acid derivative, a protein, a chemical substance, a saccharide, a cell or the like that specifically interacts with a substance immobilized on the surface of a micro substrate is adsorbed, chemically interacted, By operations such as hybridization, purification and fractionation can be easily performed according to the purpose.
[Brief description of the drawings]
FIG. 1 is an external view of an inspection 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.
FIGS. 3A and 3B are external views of a micro substrate, in which FIG. 3A shows the external shape of the micro substrate, FIG. 3B shows the micro substrate with the identification code attached to the bottom, and FIG. 3C shows the micro substrate with the surface layer formed. , (D) respectively show a micro substrate having a biochemical substance fixed on a fixed surface.
[Explanation of symbols]
1 Inspection board
2 Micro substrate
3 trays
5 Identification code
6 Surface layer

Claims (5)

A test substrate for performing a biochemical test using a biochemical substance, wherein a plurality of micro-substrates each having only one type of biochemical substance necessary for the biochemical test are immobilized on a fixed surface, Mounted on one mounting means,
The micro-substrate is mounted with the back surface of the fixed surface facing the mounting means side,
An inspection board, characterized in that an identification symbol corresponding to the one kind of biochemical substance is given to the back surface in a state of being mounted on the mounting means. The mounting means has a plurality of recesses in a flat tray, and the micro substrate is fitted and fixed in the recesses,
The inspection substrate according to claim 1 , wherein the recessed portion has front and back surfaces and has a locking portion for a minute substrate. The mounting means has a plurality of recesses in a flat tray, and the micro substrate is fitted and fixed in the recesses,
The inspection substrate according to claim 1 , wherein the recess has a bottom and the tray is a transparent body. Test board according to any one of claims 1 to 3, characterized in that the surface layer in accordance with the biochemical substance to be fixed is formed on the entire surface of the micro-substrate. 5. The inspection substrate according to claim 4 , wherein the identification code is given by partially dissolving a surface layer formed on a bottom surface of the micro substrate with a laser beam.

Images