JP3893585B2 - Biomolecule microarray substrate, method for producing the same, and biomolecule microarray - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a biomolecule microarray substrate capable of increasing sensitivity and digital analysis, a method for producing the same, and a biomolecule microarray.
[0002]
[Prior art]
A DNA microarray capable of comprehensively analyzing gene expression profiles analyzes thousands to tens of thousands of genes per sheet. A conventional DNA microarray uses a DNA stamping device (DNA arrayer), sucks DNA with a broken quill pin, and simultaneously strikes about 4 to 48 quill pins on a slide glass to produce a DNA spot. Since the DNA spot produced in this way has a non-constant shape and area, it adversely affects reproducibility and quantification. In addition, the DNA spot position is different for each quill pin and the quill pin is rotated at the time of stamping. As a result, the DNA spot is distorted without accurately aligning the DNA spots. . The DNA microarray thus prepared is subjected to a hybridization reaction with a fluorescently labeled nucleic acid sample, and then a fluorescence image of the entire DNA microarray is obtained by a fluorescence scanner based on a confocal fluorescence microscope.
[0003]
Since the fluorescence image of the DNA microarray cannot directly measure the fluorescence intensity for each spot, an operation called gridding is performed by analysis software. Gridding is an operation in which the number of spots on an array, the interval between spots, the size of the spot diameter are input, and the spots are circled. Since this gridding cannot be accurately enclosed when the spot position is deviated, it has a function of automatically correcting the position by software. However, not all operations are automatic, and it is necessary to manually check and correct the grid of all spots by setting the start point of the spot or by visual observation. Therefore, this operation is very complicated, and when the number of DNA spots is several thousand or more, it becomes a very time-consuming work, which causes the analysis speed to be delayed.
[0004]
Therefore, the present invention makes it possible to automatically perform gridding, and can automatically collect fluorescence data from a biomolecule microarray, for example, a DNA microarray, without bothering humans, thereby enabling digital analysis. An object is to provide a substrate.
[0005]
The present invention for achieving the above object is as follows.
[Claim 1] A biomolecule microarray substrate having a plurality of biomolecule probe immobilization spots on a substrate surface, wherein the biomolecule probe immobilization spot is a light reflecting layer spot between the substrate surface The substrate.
[2] The substrate according to [1], wherein the light reflecting layer spot has substantially the same planar shape as the biomolecular probe immobilization spot.
[Claim 3] The biomolecular probe immobilization spot includes biotin, avidin, streptavidin and poly-L-lysine, and an amino group, aldehyde group, thiol group, carboxyl group, succinimide group, maleimide group, epoxide group, and the like. The substrate according to claim 1 or 2, comprising at least one biomolecular probe immobilizing compound selected from the group consisting of compounds having an isothiocyanate group.
[4] The spot for immobilizing a biomolecule probe is circular and has a diameter in the range of 10 to 500 μm or a rectangle and has a side length in the range of 10 to 500 μm. 4. The substrate according to any one of 3 above.
[5] The substrate according to any one of [1] to [4], wherein the light reflecting layer is a metal layer.
[6] The substrate according to any one of [1] to [5], wherein the substrate is a glass substrate, a silicon substrate, or a quartz glass substrate.
[7] The substrate according to any one of [1] to [6], wherein a water repellent layer is provided on the surface of the substrate that is not covered with the spot for immobilizing the biomolecule probe.
[Claim 8] A step of providing a light reflecting layer on the substrate surface, a step of providing a biomolecular probe immobilization layer on the light reflecting layer, a step of providing a photoresist layer on the biomolecule probe immobilizing layer, a photoresist layer The step of exposing through the spot forming mask, the step of developing the photoresist layer, the portion of the light reflecting layer that is not protected by the photoresist layer is etched to form spots on the biomolecule probe immobilization layer and the light reflecting layer. The method for producing a substrate for a biomolecule microarray according to any one of claims 1 to 6, comprising a step of forming and a step of removing the photoresist layer.
[9] The method according to [8], further comprising a step of water-repellent treatment of the substrate surface between the etching step and the photoresist layer removing step.
[10] The method according to [8] or [9], wherein the biomolecule probe immobilization layer is formed by immobilizing a biomolecule probe immobilization compound after the light reflecting layer surface is treated with aminosilane. .
[11] The method according to [10], wherein the light reflecting layer is an aluminum layer, and the surface of the aluminum layer is oxidized with ethanol to form an aminosilane-treated light reflecting layer.
[Claim 12] The manufacturing method according to any one of claims 8 to 11, wherein the light reflecting layer is formed by vapor deposition of metal.
[13] A plurality of biomolecule probe fixing spots in which biomolecule probes are immobilized on the biomolecule probe immobilization spots of the biomolecule microarray substrate according to any one of [1] to [7]. Biomolecular microarray.
[14] The biomolecule microarray according to [13], wherein the immobilized biomolecule probe is oligo DNA, cDNA, RNA, or PNA.
[15] The biomolecule microarray according to [14], wherein the immobilized biomolecule probes have the same or different base sequences between the biomolecule probe fixation spots.
[16] The biomolecule microarray according to [13], wherein the immobilized biomolecule probe is a protein or a peptide.
[17] The biomolecule microarray according to [16], wherein the immobilized biomolecule probes have the same or different residue sequences between the biomolecule probe fixing spots.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
A first aspect of the present invention is a biomolecule microarray substrate having a plurality of spots for immobilizing biomolecule probes.
The substrate of the biomolecule microarray substrate can be, for example, a glass substrate, a silicon substrate, or a quartz glass substrate.
The biomolecular probe immobilization spot has biotin, avidin, streptavidin and poly-L-lysine, and an amino group, aldehyde group, thiol group, carboxyl group, succinimide group, maleimide group, epoxide group and isothiocyanate group. It is formed by immobilizing a predetermined amount of at least one biomolecular probe immobilizing compound selected from the group consisting of compounds.
Examples of the biomolecular probe immobilization compound include the following compounds. However, these compounds are merely examples and are not intended to be limited thereto.
[0007]
<Compound having an amino group>
3-Aminopropyltrimethoxysilane N- (2-aminoethyl) -3-aminopropyltrimethoxysilane (EDA)
Trimethoxysilylpropyldiethylenetriamine (DETA)
3- (2-aminoethylaminopropyl) trimethoxysilane <compound having an aldehyde group>
Glutaraldehyde <Compound with thiol group>
4-mercaptopropyltrimethoxysilane (MPTS)
<Compound having an epoxide group>
3-glycidoxypropyltrimethoxysilane <compound having isothiocyanate group>
4-Phenylenediisothiocyanate (PDITC)
<Compounds having succinimide and maleimide groups>
Disuccinimidyl carbonate (DSC)
Succinimidyl 4- (maleimidophenyl) butyrate (SMPB)
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)
m-maleimidopropionic acid-N-hydroxysuccinimide ester (MPS)
[0008]
The amount of the biomolecular probe immobilization compound immobilized on the biomolecular probe immobilization spot can be appropriately determined according to the type of the compound.
The shape of the biomolecular probe immobilization spot is not particularly limited, but may be, for example, a circular shape, and the diameter thereof may be, for example, in the range of 10 to 500 μm. The shape of the biomolecular probe immobilization spot may be a rectangle or a polygon, and the length of one side of the rectangle or the polygon may be, for example, in the range of 10 to 500 μm.
[0009]
The substrate of the present invention has a light reflection layer between the biomolecular probe immobilization spot and the substrate surface. Furthermore, it is preferable that this light reflecting layer has substantially the same shape as the spot for immobilizing a biomolecular probe provided thereon. Accordingly, there is an advantage that the position and area of the biomolecule spot can be automatically recognized by detecting the spot of the light reflecting layer.
[0010]
The substance constituting the light reflecting layer is not particularly limited as long as it is a substance having light reflectivity. The light reflecting layer can be, for example, a metal layer, and examples of the metal layer include aluminum, gold, silver, platinum, nickel, and the like. Alternatively, the light reflection layer can be a single-layer or multilayer reflection layer made of an oxide or the like.
[0011]
The biomolecule microarray substrate of the present invention can have a water-repellent layer on the surface of the substrate not covered with the biomolecule probe immobilization spot. Since the substrate surface that is not covered with the biomolecule probe immobilization spot is treated with water repellency, the biomolecule probe can be more accurately immobilized on the biomolecule probe immobilization spot. The water repellent layer can be formed using an existing water repellent. As the water repellent, for example, a silicon coating agent, a fluorine coating agent, a nylon coating agent, a Teflon coating agent (Teflon is a registered trademark), or the like can be used.
[0012]
The biomolecule probe immobilized on the biomolecule microarray substrate of the present invention can be, for example, oligo DNA or cDNA, and the biomolecule microarray substrate of the present invention includes a DNA microarray substrate.
[0013]
The biomolecule microarray substrate of the present invention can be manufactured by the manufacturing method of the present invention including the following steps.
Providing a light reflecting layer on the substrate surface;
Providing a biomolecular probe immobilization layer on the light reflection layer;
Providing a photoresist layer on the biomolecular probe immobilization layer;
Exposing the photoresist layer through a spot forming mask;
Developing a photoresist layer;
Etching the light reflecting layer portion not protected by the photoresist layer;
It can be manufactured by the manufacturing method of the present invention including the step of removing the photoresist layer.
[0014]
The step of providing the light reflecting layer on the substrate surface can be performed, for example, by evaporating metal on the substrate surface. As the metal deposition method and conditions, known methods and conditions can be used as they are. In addition, it is preferable to wash the substrate surface before depositing the metal on the substrate surface.
Next, a biomolecule probe immobilization layer is formed on the light reflection layer. When the light reflection layer is an aluminum layer, the surface of the aluminum layer is oxidized with ethanol before the biomolecule probe immobilization layer is formed. An aminosilane compound can be used for aminosilane treatment. Since the light reflecting layer is treated with aminosilane, biomolecules such as DNA can be chemically immobilized instead of physically adsorbed, so that the loss of biomolecules such as DNA in subsequent operations can be reduced. .
[0015]
The step of providing the biomolecule probe immobilization layer on the light reflection layer can be performed by immobilizing the biomolecule probe immobilization compound on the light reflection layer. The compound for immobilizing a biomolecular probe can be appropriately selected from the substances listed in the substrate of the present invention. When the biomolecular probe immobilization compound is biotin, the immobilization can be performed, for example, by reacting biotin succinimide ester with the surface of the light reflection layer treated with aminosilane. When the compound for immobilizing a biomolecular probe is glutaraldehyde, which is a compound having an aldehyde group, the immobilization can be carried out by reacting glutaraldehyde with the surface of the light reflecting layer treated with aminosilane.
[0016]
The step of providing a photoresist layer on the biomolecular probe immobilization layer can be performed, for example, by coating an existing resist material by a conventional method, for example, a spin coating method, and then thermally curing the resist material. There are no particular restrictions on the type of resist material, coating method, and curing conditions. For example, a positive resist material is coated to a thickness of about 1 μm with a spin coater and heated in an oven to thermally cure the resist. Can do.
[0017]
The step of exposing the photoresist layer through the spot forming mask is such that spots for immobilizing biomolecule probes are formed with desired dimensions, shapes and intervals depending on whether the resist material is a positive type or a negative type. A mask is prepared and the process is performed through this mask. The exposure conditions are appropriately determined according to the type of photoresist material.
[0018]
The step of developing the photoresist layer is performed by treating the exposed photoresist layer with a developer and washing as necessary. The developer can be appropriately selected according to the resist material to be used.
[0019]
After the development, the light reflection layer portion not protected by the photoresist layer is etched. This step is performed by appropriately selecting an etchant that can etch the light reflecting layer. For example, when the light reflection layer is a metal layer, the etching solution can be an acid aqueous solution, and the type and concentration of the acid contained in the acid aqueous solution are appropriately determined in consideration of the type of metal to be etched and the thickness of the layer. Can be determined.
[0020]
After the etching, the photoresist layer is removed. This step can be performed using an organic solvent that can dissolve the photoresist, for example, acetone. After removing the photoresist layer, the substrate is washed as necessary to obtain the biomolecule microarray substrate of the present invention.
[0021]
The production method of the present invention may include a step of subjecting the substrate surface to a water repellent treatment between the etching step and the photoresist layer removing step. By performing water-repellent treatment at this stage, a water-repellent layer can be formed on the surface of the substrate that is not covered with the biomolecular probe immobilization spot. The water repellent treatment can be appropriately performed according to the type of the water repellent, and for example, can be performed by immersing the substrate in the water repellent.
[0022]
The present invention includes a biomolecule microarray, and the biomolecule microarray of the present invention is obtained by immobilizing a biomolecule probe on a biomolecule probe immobilization spot on the biomolecule microarray substrate of the present invention.
A plurality of biomolecular probe fixing spots are provided on the substrate. The number of biomolecular probe fixing spots is not particularly limited, and is determined appropriately in consideration of the size of the substrate, the size of the spots, the interval between spots, and the like.
[0023]
The immobilized biomolecular probe can be, for example, oligo DNA or cDNA. That is, the biomolecule microarray of the present invention includes a DNA microarray. In this case, the immobilized biomolecule probe can have the same or different base sequence between the biomolecule probe fixing spots. Furthermore, the immobilized biomolecular probe can be, for example, a protein or a peptide. In this case, the immobilized biomolecule probe can have the same or different residue sequence between the biomolecule probe fixing spots.
[0024]
The biomolecule probe is immobilized on the biomolecule probe immobilization spot by preliminarily immobilizing the biomolecule probe immobilizing substance or functional group having a binding property with the biomolecule probe immobilization compound forming the biomolecule probe immobilization spot. For example, this can be performed by dropping a solution containing the biomolecule probe onto the biomolecule probe immobilization spot on the biomolecule microarray substrate of the present invention.
Examples of the substance having the binding property include avidin, streptavidin, and biotin. Examples of the functional group having the binding property include a carboxyl group, an amino group, an aldehyde group, a thiol group, and a succinimide group. , And maleimide groups.
[0025]
【The invention's effect】
The present invention makes it possible to increase the sensitivity by making the shape, area, and position of the biomolecule spot of the biomolecule microarray constant, and providing a light reflecting layer under the biomolecule spot, and substantially the same plane as the biomolecule spot. By detecting the light reflecting layer having a shape, the position and area of the biomolecule spot can be automatically recognized. Thereby, the fluorescence intensity of the biomolecule spot can be analyzed digitally, and the analysis man-hour can be greatly reduced and the analysis time can be shortened. Since the position and area of the biomolecule spot are recognized, only the fluorescence intensity of the biomolecule spot is measured, and the detection time is not measured without measuring the fluorescence intensity of unnecessary parts (regions other than the biomolecule spot on the microarray). It can be shortened more.
[0026]
EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
Substrate creation method
1． 20 glass slides with no scratches were selected, placed in a slide rack, ultrasonically washed in 200 ml of acetone for 30 minutes, and then rinsed with ultrapure water (500 ml x 2,200 ml x 1). Hydrogen peroxide and sulfuric acid (1: 1, 200 ml) are mixed in an ice bath, immersed in a glass slide, ultrasonically cleaned for 30 minutes, rinsed with ultrapure water (500 ml x 3), and then in 200 ml of ultrapure water. Ultrasonic cleaning was performed for 30 minutes. The slide rack was thoroughly rinsed with ultrapure water (running water) and then immersed in 500 ml of ultrapure water for cleaning. The washed glass slide was covered with aluminum foil and dried in an oven at 200 ° C. for 15 minutes, wrapped in aluminum foil, cooled to room temperature in a desiccator, and stored as it was until use.
[0027]
2． Aluminum tungsten vapor wiped with acetone is set in a vacuum vapor deposition device, and then wiped with acetone and 1cm long aluminum wire (Φ1.Omm, 99.999%) is placed on it. The pressure was reduced to 10 ⁻³ Pa, and aluminum was deposited at a speed of 10 nm / sec so as to obtain a film thickness of 300 to 500 nm.
[0028]
3． 70% ethanol prepared by mixing ultra pure water and ethanol for 5 minutes with ultrasonication and adding 70ml ethanol to a co-plinger containing 5 aluminum-deposited glass slides and sonicating for 30 minutes . The glass slides treated with ethanol were rinsed one by one with ultrapure water (500ml x 3), then placed in a slide rack and rinsed with ultrapure water (500ml) together with the rack.
[0029]
Four. Aminosilane treatment
Add 4 ml of 3- (2-aminoethylaminopropyl) trimethoxysilane to 196 ml of ultrapure water and stir for 30 minutes in the dark room, soak the ethanol-treated slide glass together with the slide rack, and react for 10 minutes in the dark room. It was. After rinsing gently with ultrapure water (200 ml × 2), the slide rack was placed in an oven at 110 ° C., covered with aluminum foil, heated for 45 minutes, wrapped in aluminum foil, and cooled to room temperature in a desiccator.
[0030]
Five. Biotinylation
Biotin (Long Arm) NHS-Water Soluble (Vector Laboratories) was dissolved in 50 mM Na ₂ C0 ₃ -NaHCO ₃ (pH 8.7) buffer to a concentration of 1 mg / ml to prepare a biotinylation buffer. Five glass slides treated with aminosilane were arranged in a square petri dish, 25 ml of biotinylated buffer was added thereto, the lid was covered, and the mixture was gently stirred overnight with a mild mixer. It was washed with ultrapure water (500 ml × 3) and dried in an oven at 60 ° C. for 45 minutes.
[0031]
6． Apply a positive resist (Shipley Far East Co., Ltd., MP S1813) to a thickness of 1 μm with a spin coater on the patterned biotinylated slide (300 rpm, 3 seconds; slope, 5 seconds; 3000 rpm, 10 seconds), Heated in an oven at 60 ° C. for 45 minutes. Next, the slide glass coated with resist was exposed to ultraviolet rays through a mask on which a pattern of spots for DNA probe immobilization was formed. For the exposure operation, a prototype device capable of scanning exposure of the third harmonic of a YAG laser (wavelength: 355 nm, laser power: 80 mW) was used. The scanning time was determined as 120 seconds (30 × 80 mm area) by finding an appropriate time experimentally. Develop for 45 seconds with vigorous shaking with developer (Shipley Far East Co., Ltd., MF CD-26), rinse with ultrapure water (500ml x 2), then etch solution (phosphoric acid: acetic acid: nitric acid: super (Pure water = 16: 2: 1: 1, 200 ml) was sonicated for 3 minutes. The etched slide glass was thoroughly rinsed with ultrapure water (500 ml × 5). By this operation, only the aluminum layer and the spot for immobilizing the DNA probe as the resist lower layer remained on the slide glass.
[0032]
7． Silicon coating (water repellent treatment)
5 ml of a silicon coating reagent (Iwaki Glass Co., Ltd., SIL-COAT 5) was added to 195 ml of ultrapure water heated to 70 ° C., and stirred for 10 minutes. After immersing the etched slide glass for 10 seconds, thoroughly wash it with ultrapure water (500ml × 3), cover the slide glass with aluminum foil and bake at 110 ° C for 30 minutes, wrap in aluminum foil and room temperature in a desiccator The solution was cooled to 0 and stored as it was until use.
[0033]
8． Resist dissolution etching and silicon-coated slide glass were washed one by one with acetone (70 ml × 3) to dissolve the resist remaining on the spot. After thoroughly rinsing with ultrapure water (500ml × 2), it was washed in 70ml ultrapure water for 30 minutes while sonicating, and then rinsed with 70ml ultrapure water.
[0034]
Example 2
Preparation of biomolecular microarray 0.05 mg / ml streptavidin solution (Vector, dissolution buffer: 1XPBS; 137 mM NaCl, 8.10 mM Na ₂ HPO ₄ , 2.68 mM KCl, 1.47 mM KH ₂ PO _{4 on the} entire surface of the substrate produced in Example 1 , PH 7.4) and allowed to stand at room temperature for 30 minutes. Thereafter, the plate was washed 3 times with 1 × PBS for 10 minutes to remove unreacted streptavidin. The substrate to which streptavidin was bound was rinsed with ultrapure water, and then water was removed by a centrifuge and dried. Streptavidin is bound to the DNA immobilization spot on the substrate.
Next, in order to immobilize the biotinylated oligonucleotide DNA on the substrate, the biotinylated oligonucleotide DNA solution was accurately stamped on the DNA probe immobilization spot on the substrate by a spot device. After stamping, 1XSSPE buffer (150 mM NaCl, 8.65 mM NaH ₂ PO ₄ , 1.25 mM) was used to remove unreacted DNA.
Washed with EDTA; pH 7.4) to prepare a DNA microarray.
[0035]
Example 3
Detection sensitivity comparison Example of biomolecule microarray substrate (DNA microarray substrate) obtained in Example 1 and a commercially available glass substrate (PLL slide glass) coated with poly-L-lysine for DNA microarray Spot the Cy3-oligo DNA dilution series (0, 0.06, 0.13, 0.25, 0.50, 1.0, 2.0, 4.0, 8.0 pmol / μl) in the same manner as in Step 2, and measure the fluorescence intensity of each spot without washing as follows. Measured with The obtained results are shown in FIG. From the results shown in FIG. 1, in the DNA microarray substrate obtained in Example 1, a clear fluorescent signal was measured from a spot of 0.13 pmol / μl, whereas a commercially available DNA without a light reflecting layer was measured. In the microarray substrate, a clear fluorescence signal was measured from 0.50 pmol / μl. That is, it can be seen that the DNA microarray substrate of the present invention can be about 4 times more sensitive than a DNA microarray substrate having no light reflecting layer.
[0036]
The fluorescent spot was measured by the following method.
Fluorescence images of both substrates were obtained using a fluorescence microarray scanner (Nippon Laser Electronics Co., Ltd.). At that time, the voltage applied to the detector (photomultiplier tube) of the scanner was measured under the same conditions in the measurement of both substrates. Graphing of the intensity of the fluorescent spot was performed using image analysis software (NIH image).
[0037]
Example 4
Identification of biomolecular probe immobilization spot The biomolecular probe immobilization spot of the DNA microarray obtained in Example 1 was identified by the following method.
Remove the optical cut filter installed on the incident light side of the detector (photomultiplier tube) of the fluorescent microarray scanner (Nihon Laser Electronics Co., Ltd.), and the laser light reflected from the surface of the DNA microarray is directly photomultiplier tube. It was made to enter. At that time, the applied voltage of the photomultiplier tube was usually set lower than that in the case of measuring fluorescence. FIG. 2 shows the light reflected from the DNA microarray surface obtained in Example 1 by this method. The higher the intensity of light incident on the detector, the more white it is displayed. According to this result, it is understood that the spot for immobilizing the biomolecule probe having the light reflection layer spot has higher intensity of the reflected light of the laser beam than the substrate surface not covered with the spot. By using this characteristic, it is possible to specify a spot for immobilizing a biomolecular probe.
[Brief description of the drawings]
1 shows the results obtained in Example 3. FIG. The result obtained in Test Example 1 is shown.
FIG. 2 shows the results obtained in Example 4.

Claims (17)

A biomolecule microarray substrate having a plurality of biomolecule probe immobilization spots on a substrate surface, wherein the biomolecule probe immobilization spot has a light reflection layer spot between the substrate surface and the biomolecule probe immobilization spot. The substrate according to claim 1, wherein the light reflecting layer spot has substantially the same planar shape as the spot for immobilizing a biomolecule probe. The biomolecular probe immobilization spot has biotin, avidin, streptavidin and poly-L-lysine, and an amino group, aldehyde group, thiol group, carboxyl group, succinimide group, maleimide group, epoxide group and isothiocyanate group. The substrate according to claim 1 or 2, comprising at least one biomolecular probe immobilizing compound selected from the group consisting of compounds. The spot for immobilizing a biomolecule probe is circular and has a diameter in the range of 10 to 500 μm, or a rectangle or a polygon, and one side has a length in the range of 10 to 500 μm. The substrate according to any one of the above. The substrate according to claim 1, wherein the light reflection layer is a metal layer. The substrate according to claim 1, wherein the substrate is a glass substrate, a silicon substrate, or a quartz glass substrate. The board | substrate of any one of Claims 1-6 which have a water-repellent layer on the said board | substrate surface which is not coat | covered with the said spot for biomolecule probe fixation. A step of providing a light reflecting layer on the substrate surface, a step of providing a biomolecule probe immobilization layer on the light reflection layer, a step of providing a photoresist layer on the biomolecule probe immobilization layer, and a mask for spot formation of the photoresist layer A step of exposing via a photolithographic layer, a step of developing a photoresist layer, a step of etching a light reflection layer portion not protected by the photoresist layer to form spots on the biomolecule probe immobilization layer and the light reflection layer, photo The manufacturing method of the board | substrate for biomolecule microarrays of any one of Claims 1-6 including the process of removing a resist layer. The manufacturing method according to claim 8, further comprising a step of water-repellent treatment of the substrate surface between the etching step and the photoresist layer removing step. The manufacturing method according to claim 8 or 9, wherein the biomolecular probe immobilization layer is formed by immobilizing a biomolecular probe immobilization compound after the light reflecting layer surface is treated with aminosilane. The manufacturing method according to claim 10, wherein the light reflection layer is an aluminum layer, and the surface of the aluminum layer is oxidized with ethanol to form an aminosilane-treated light reflection layer. The manufacturing method according to claim 8, wherein the light reflecting layer is formed by metal deposition. The biomolecule microarray which has two or more biomolecule probe fixed spots by which the biomolecule probe was fix | immobilized in the spot for biomolecule probe fixation which the board | substrate for biomolecule microarrays of any one of Claims 1-7 has. The biomolecule microarray according to claim 13, wherein the immobilized biomolecule probe is oligo DNA, cDNA, RNA, or PNA. The biomolecule microarray according to claim 14, wherein the immobilized biomolecule probe has the same or different base sequence between the biomolecule probe fixing spots. The biomolecule microarray according to claim 13, wherein the immobilized biomolecule probe is a protein or a peptide. The biomolecule microarray according to claim 16, wherein the immobilized biomolecule probes have the same or different residue sequences between the biomolecule probe fixing spots.

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