JP2003156495A - Substrate for biomolecule microarray, its manufacturing method an biomolecule microarray - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a substrate enabling an automatic gridding to surround spots with a circle, capable of collecting fluorescent data automatically without troubling a person from a biomolecule microarray, for example, a DNA microarray, and enabling digital analysis. SOLUTION: This substrate for the biomolecule microarray has a plurality of spots for immobilizing biomolecule probes on the substrate surface. The spot for immobilizing the biomolecule probe has a light reflection layer spot between itself and the substrate surface. This manufacturing method of the substrate for the biomolecule microarray is also provided. The biomolecule microarray has a plurality of biomolecule probe immobilized spots formed by immobilizing the biomolecule probes on the spots for immobilizing the biomolecule probes held by the substrate for the biomolecule microarray.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biomolecule microarray substrate capable of high sensitivity and digital analysis, a method for producing the same, and a biomolecule microarray.

[0002]

2. Description of the Related Art A DNA microarray capable of comprehensively analyzing a gene expression profile analyzes thousands to tens of thousands of genes per sheet. The conventional DNA microarray is
Using a device for stamping DNA (DNA arrayer), a quill pin with a broken tip absorbs the DNA, and about 4 to 48 quill pins are simultaneously struck on a slide glass to form a DNA spot. The DNA spots produced in this way have a non-uniform shape and area, which adversely affects reproducibility and quantitativeness. Further, the positions of the DNA spots are different depending on the quill pins, and the quill pins are rotated during stamping, which results in the DNA spots being distorted without being aligned accurately. . DNA produced in this way
In a microarray, a nucleic acid sample labeled with fluorescence is subjected to a hybridization reaction, and then a fluorescence image of the entire DNA microarray is acquired by a fluorescence scanner based on a confocal fluorescence microscope.

Since the fluorescence intensity of each spot cannot be measured as it is in the fluorescence image of the DNA microarray, an operation called gridding is performed by the analysis software. The gridding is an operation of inputting the number of vertical and horizontal spots on the array, the interval between the spots, and the size of the diameter of the spot, and enclosing the spot with a circle. Since this gridding cannot be accurately enclosed if 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 grids of all spots by setting the spot start point and visually. Therefore, this operation is
It is very complicated, and when the number of DNA spots is several thousand or more, it takes a very long time, which is a factor of slowing down the analysis speed.

Therefore, the present invention makes it possible to perform gridding automatically and collect fluorescence data from a biomolecule microarray, for example, a DNA microarray.
It is an object of the present invention to provide a substrate which can be automatically analyzed without human labor and enables digital analysis.

The present invention that achieves the above object is as follows. [Claim 1] A biomolecule microarray substrate having a plurality of biomolecule probe immobilization spots on the substrate surface, wherein the biomolecule probe immobilization spots are light reflection layer spots between the biomolecule probe immobilization spots and the substrate surface. The substrate having. [Claim 2] The substrate according to claim 1, wherein the light reflection layer spot has substantially the same planar shape as the biomolecule probe immobilization spot. [Claim 3] The biomolecule probe-immobilized spot comprises biotin, avidin, streptavidin, and poly-L-lysine, and an amino group, an aldehyde group, a thiol group, a carboxyl group, a succinimide group, a maleimide group, an epoxide group, and The substrate according to claim 1 or 2, comprising at least one biomolecule probe-immobilizing compound selected from the group consisting of compounds having an isothiocyanate group. [Claim 4] The biomolecule probe immobilization spot is circular and has a diameter in the range of 10 to 500 µm.
Alternatively, the substrate according to any one of claims 1 to 3, which is a rectangle and has a side length of 10 to 500 µm. [Claim 5] The light reflection layer is a metal layer.
The substrate according to any one of 1. [Claim 6] The substrate according to any one of claims 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], which has a water-repellent layer on the surface of the substrate that is not covered with the biomolecule probe immobilization spot. [Claim 8] A step of providing a light reflecting layer on the surface of a substrate, a step of providing a biomolecule probe immobilization layer on the light reflecting layer, a step of providing a photoresist layer on the biomolecule probe immobilization layer, a photoresist layer Through the mask for spot formation, the step of developing the photoresist layer, the light reflection layer portion not protected by the photoresist layer is etched to form spots on the biomolecule probe immobilization layer and the light reflection layer. The method for producing a biomolecule microarray substrate according to claim 1, comprising a step of forming and a step of removing the photoresist layer. [Claim 9] A step of subjecting the substrate surface to a water repellent treatment between the etching step and the photoresist layer removing step.
The manufacturing method described in. [Claim 10] The method according to claim 8 or 9, wherein the biomolecule probe immobilization layer is formed by immobilizing a biomolecule probe immobilization compound after the surface of the light reflection layer is treated with aminosilane. . [11] The production method according to [10], wherein the light reflecting layer is an aluminum layer, and the aminosilane-treated light reflecting layer is formed by oxidizing the surface of the aluminum layer with ethanol. [Claim 12] The method according to any one of claims 8 to 11, wherein the light reflection layer is formed by vapor deposition of metal. [Claim 13] A plurality of biomolecule probe-immobilized spots having biomolecule probes immobilized on the biomolecule probe immobilization spots of the biomolecule microarray substrate according to any one of claims 1 to 7. Biomolecule 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 probe has the same or different base sequence between the biomolecule probe fixed 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 probe has the same or different residue sequence between the biomolecule probe fixed spots.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A first aspect of the present invention is a biomolecule microarray substrate having a plurality of biomolecule probe immobilization spots. The substrate of the biomolecule microarray substrate may be, for example, a glass substrate, a silicon substrate, or a quartz glass substrate. The biomolecule probe immobilization spot has biotin, avidin, streptavidin and poly-L-lysine, and an amino group, an aldehyde group, a thiol group, a carboxyl group, a succinimide group, a maleimide group, an epoxide group and an isothiocyanate group. It is formed by immobilizing a predetermined amount of at least one biomolecule probe-immobilizing compound selected from the group consisting of compounds. Examples of the compound for immobilizing a biomolecule probe include the following compounds. However, these compounds are merely examples,
It is not intended to be limited to these.

<Compound Having Amino Group> 3-aminopropyltrimethoxysilane N- (2-aminoethyl) -3-aminopropyltrimethoxysilane (EDA) Trimethoxysilylpropyldiethylenetriamine (D
ETA) 3- (2-aminoethylaminopropyl) trimethoxysilane <compound having aldehyde group> glutaraldehyde <compound having thiol group> 4-mercaptopropyltrimethoxysilane (MPT)
S) <Compound having an epoxide group> 3-glycidoxypropyltrimethoxysilane <Compound having an isothiocyanate group> 4-Phenylenediisothiocyanate (PDITC) <Compound having a succinimide and maleimide group> 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)

The amount of the biomolecule probe-immobilizing compound immobilized on the biomolecule probe-immobilizing spot can be appropriately determined according to the kind of the compound. The shape of the biomolecule probe immobilization spot is not particularly limited, but, for example,
It can be circular and its diameter is, for example, 10 to 5
It can be in the range of 00 μm. Further, the shape of the biomolecule probe immobilization spot may be a rectangle or a polygon, and the length of one side of the rectangle or the polygon is
For example, it can be in the range of 10 to 500 μm.

The substrate of the present invention has a light reflecting layer between the biomolecule probe immobilization spot and the substrate surface. Further, it is preferable that the light reflection layer has substantially the same shape as the biomolecule probe immobilization spot provided on the light reflection layer. Accordingly, there is an advantage that the position and area of the biomolecule spot can be automatically recognized by detecting the spot on the light reflection layer.

The substance constituting the light reflecting layer is not particularly limited as long as it is a substance having a light reflecting property. The light reflecting layer may be, for example, a metal layer, and examples of the metal layer may include aluminum, gold, silver, platinum, nickel and the like. Alternatively, the light reflecting layer may be a single-layer or multi-layer reflecting layer made of oxide or the like.

The biomolecule microarray substrate of the present invention may have a water-repellent layer on the surface of the substrate which is not covered with the biomolecule probe immobilization spots. Since the surface of the substrate that is not covered with the biomolecule probe immobilization spot is subjected to the water repellent treatment, 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 agent. 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.

The biomolecule probe immobilized on the biomolecule microarray substrate of the present invention is, for example, oligo DN.
It may be A or cDNA, and the substrate for biomolecule microarray of the present invention includes the substrate for DNA microarray.

The biomolecule microarray substrate of the present invention can be manufactured by the manufacturing method of the present invention including the following steps. A step of providing a light reflection 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,
It includes a step of exposing the photoresist layer through a spot forming mask, a step of developing the photoresist layer, a step of etching a light reflection layer portion not protected by the photoresist layer, and a step of removing the photoresist layer. It can be manufactured by the manufacturing method of the present invention.

The step of providing the light reflection layer on the surface of the substrate can be carried out, for example, by depositing a metal on the surface of the substrate. As the metal vapor deposition method and conditions, known methods and conditions can be used as they are. It is preferable to wash the surface of the substrate before depositing the metal on the surface of the substrate. Then, a biomolecule probe immobilization layer is formed on the light reflection layer. When the light reflection layer is an aluminum layer, the aluminum layer surface is oxidized with ethanol before the biomolecule probe immobilization layer is formed, and further. The aminosilane compound can be used for aminosilane treatment. By treating the light-reflecting layer with aminosilane, biomolecules such as DNA can be chemically fixed instead of physically adsorbed, and thus there is an advantage that loss of biomolecules such as DNA in subsequent operations can be reduced. .

The step of providing the biomolecule probe immobilization layer on the light reflection layer can be carried out by immobilizing the biomolecule probe immobilization compound on the light reflection layer. The compound for immobilizing the biomolecule probe can be appropriately selected from the substances listed for the substrate of the present invention. When the biomolecule probe-immobilizing compound is biotin, the immobilization can be carried out, for example, by reacting biotin succinimide ester with the aminosilane-treated light-reflecting layer surface. When the biomolecule probe-immobilizing compound is glutaraldehyde, which is a compound having an aldehyde group, the immobilization can be carried out by reacting glutaraldehyde with the aminosilane-treated light-reflecting layer surface.

In the step of providing a photoresist layer on the biomolecule probe-immobilized layer, for example, an existing resist material is coated by a conventional method, for example, spin coating,
Then, the resist material can be thermally cured. The type of resist material, coating method, and curing conditions are not particularly limited. For example, a positive resist material is coated with a spin coater to a thickness of about 1 μm and heated in an oven to thermally cure the resist. You can

In the step of exposing the photoresist layer through the spot forming mask, the biomolecule probe immobilizing spots are formed in desired sizes, shapes and intervals depending on whether the resist material is positive or negative. Such a mask is prepared, and this mask is used. The exposure conditions are appropriately determined according to the type of photoresist material.

The step of developing the photoresist layer is carried out by treating the exposed photoresist layer with a developing solution and washing it if necessary. The developing solution can be appropriately selected according to the resist material used.

After the development, the light reflection layer portion not protected by the photoresist layer is etched. This step is performed by appropriately selecting an etching solution capable of etching the light reflecting layer. For example, when the light reflecting layer is a metal layer, the etching solution may be an acid aqueous solution, and the type and concentration of the acid contained in the acid aqueous solution may be appropriately determined in consideration of the type of metal to be etched and the layer thickness. I can decide.

After etching, the photoresist layer is removed. This step can be performed using an organic solvent capable of dissolving the photoresist, for example, acetone. After removing the photoresist layer, if necessary, the substrate is washed to obtain the biomolecule microarray substrate of the present invention.

The manufacturing method of the present invention may include a step of water repellent treatment of the substrate surface between the etching step and the photoresist layer removing step. By performing the 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 biomolecule probe immobilization spots. The water repellent treatment can be appropriately performed depending on the type of the water repellent, and can be performed, for example, by immersing the substrate in the water repellent.

The present invention includes a biomolecule microarray, and the biomolecule microarray of the present invention has a biomolecule probe immobilized on the biomolecule probe immobilization spot of the above-mentioned biomolecule microarray substrate of the present invention. is there. A plurality of biomolecule probe fixing spots are provided on the substrate. The number of biomolecule probe-immobilized spots is not particularly limited and is appropriately determined in consideration of the size of the substrate, the size of the spots, the interval between the spots, and the like.

The immobilized biomolecule 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 fixed spots.
The further immobilized biomolecule probe can also be, for example, a protein or peptide. in this case,
The immobilized biomolecule probe may have the same or different residue sequence between the biomolecule probe-immobilized spots.

The immobilization of the biomolecule probe on the biomolecule probe immobilization spot is carried out by using a biomolecule probe having a substance or a functional group having a binding property with the biomolecule probe immobilization compound forming the biomolecule probe immobilization spot. Have been immobilized in advance, for example, by adding a solution containing this biomolecule probe to the biomolecule probe immobilization spot of the biomolecule microarray substrate of the present invention,
It can be carried out. As the substance having the above-mentioned binding property,
Examples thereof include avidin, streptavidin, and biotin. Examples of the functional group having the above-mentioned binding property include a carboxyl group, an amino group, an aldehyde group, a thiol group, a succinimide group, and a maleimide group. .

[0025]

INDUSTRIAL APPLICABILITY According to the present invention, the shape, area, and position of the biomolecule spots of the biomolecule microarray can be made constant, and a light reflecting layer can be provided under the biomolecule spots to enhance the sensitivity, and the biomolecule spots can be effectively treated. By detecting the light reflection layer having the same planar shape, the position and area of the biomolecule spot can be automatically recognized.
As a result, the fluorescence intensity of the biomolecule spot can be digitally analyzed, and it is possible to significantly reduce the analysis artificial and the analysis time. Since the position and area of the biomolecule spots are recognized, only the fluorescence intensity of the biomolecule spots is measured, and the fluorescence intensity of unnecessary parts (areas other than the biomolecule spots on the microarray) is not measured. It can be shortened more.

EXAMPLES The present invention will be described in more detail with reference to examples. Example 1 Substrate preparation method 1. Cleaning of slide glass 20 scratch-free slide glasses were selected, placed in a slide rack, and ultrasonically cleaned in 200 ml of acetone for 30 minutes.
Rinse with ultrapure water (500 ml x 2,200 ml x 1). Mix hydrogen peroxide and sulfuric acid (1: 1, 200 ml) in an ice bath, soak a slide glass for 30 minutes in ultrasonic cleaning, rinse with ultrapure water (500 ml x 3), and then in 200 ml of ultrapure water. Ultrasonic cleaning was performed for 30 minutes.
After thoroughly rinsing the slide rack with ultrapure water (running water),
It was immersed in 500 ml of ultrapure water and washed. Cover the cleaned slide glass with aluminum foil together with the rack in an oven at 200 ℃ 1
It was dried for 5 minutes, wrapped in aluminum foil, cooled to room temperature in a desiccator, and stored as it was until use.

[0027] 2. Aluminum vapor deposition Set a tungsten board wiped with acetone in a vacuum vapor deposition device, wipe it with acetone and put a 1 cm long aluminum wire (Φ 1.Omm, 99.999%) on it, and set the washed slide glass, 4.0 × The pressure was reduced to 10 ^-3 Pa, and aluminum was vapor-deposited at a speed of 10 nm / sec to a film thickness of 300 to 500 nm.

[0028] 3. Ethanol treatment of aluminum surface Ultrapure water and ethanol were sonicated for 5 minutes and mixed well to prepare 70% ethanol, about 70 ml was added to a Coplin jar containing 5 aluminum vapor-deposited glass slides, and sonicated for 30 minutes. . Each slide glass treated with ethanol was rinsed with ultrapure water (500 ml x 3) one by one, further placed in a slide rack, and the rack was rinsed with ultrapure water (500 ml).

[0029] 4. Aminosilane treatment To 196 ml of ultrapure water, add 4 ml of 3- (2-aminoethylaminopropyl) trimethoxysilane and stir for 30 minutes in a dark room, immerse the slide glass treated with ethanol together with the slide rack in the dark room for 10 minutes. It was made to react. Ultrapure water (200 ml x
After gently rinsing in 2), the slide rack was placed in an oven at 110 ° C, covered with aluminum foil, heated for 45 minutes, wrapped with aluminum foil, and cooled to room temperature in a desiccator.

5. Biotin (Long Arm) NHS-Water Soluble (Vector Labora
tories) in 50 mM Na ₂ C0 ₃ -NaHCO ₃ (pH 8.7) buffer at 1 mg /
A biotinylated buffer was prepared by dissolving to a concentration of ml. Arrange 5 slides of the aminosilane-treated slides in a square dish, and add 25m of biotinylated buffer there.
l was added thereto, the vessel was covered with a lid, and the mixture was gently stirred with a mild mixer overnight. Wash with ultrapure water (500 ml x 3), then in an oven at 60 ° C
Dry for 45 minutes.

6. Patterning Biotinylated slides are spin coated with a positive resist (Chipley Far East Co., Ltd., MP S1813).
Is coated to a thickness of 1 μm (300 rpm, 3 seconds; slop
e, 5 seconds; 3000 rpm, 10 seconds), and heated in an oven at 60 ° C. for 45 minutes. Then, the resist-coated slide glass was exposed to ultraviolet light through a mask having a pattern of spots for immobilizing DNA probes. For the exposure operation, a prototype device capable of scanning and exposing the third harmonic of the YAG laser (wavelength: 355 nm, laser power: 80 mW) was used. The scanning time was determined to be 120 seconds (30 × 80 mm area) by obtaining an appropriate time experimentally. Develop with developing solution (Chipley Far East Co., Ltd., MF CD-26) for 45 seconds with vigorous shaking, rinse with ultrapure water (500 ml x 2), then etch solution
Soak in (phosphoric acid: acetic acid: nitric acid: ultrapure water = 16: 2: 1: 1, 200 ml) for 3
Ultrasonic treatment was performed for a minute. The etched slide glass was thoroughly rinsed with ultrapure water (500 ml x 5). By this operation, the aluminum layer and DNA which are the lower layer of the resist
Only the probe immobilization spot remained on the glass slide.

7. Silicon coating (water repellent treatment) Silicon coating reagent on 195 ml of ultrapure water heated to 70 ℃
(Iwaki Glass Co., Ltd., SIL-COAT 5) (5 ml) was added and stirred for 10 minutes. After soaking the etched slide glass for 10 seconds, wash it thoroughly with ultrapure water (500 ml x 3), cover the slide glass with aluminum foil on the rack, bake at 110 ° C for 30 minutes, wrap in aluminum foil, and store in a desiccator at room temperature. It was cooled to room temperature and stored as it was until use.

8. Dissolving and etching the resist, the glass slides coated with silicon were washed one by one with acetone (70 ml × 3) to dissolve the resist remaining on the spots. After thoroughly rinsing with ultrapure water (500 ml x 2), it was washed in 70 ml of ultrapure water for 30 minutes while sonicating, and then rinsed with 70 ml of ultrapure water.

Example 2 Fabrication of biomolecule microarray 0.05 mg / ml streptoa was prepared on the entire surface of the substrate prepared in Example 1.
Vidin solution (Vector, Lysis buffer: 1XPBS; 137 mM
NaCl, 8.10 mM Na₂HPO_Four, 2.68mM KCl, 1.47mM KH ₂PO_Four, P
H 7.4) was put on the plate, and the mixture was allowed to stand at room temperature for 30 minutes. Then 1XPBS
Wash 3 times for 10 minutes to remove unreacted streptavidin.
I left. Substrates with streptavidin bound with ultrapure water
After rinsing, the water was removed with a centrifuge and dried. this
Streptavidin is applied to the DNA immobilization spot on the substrate.
Are connected. Next, the biotinylated oligo
To immobilize the leotide DNA, a spot device was used.
Accurately on the spot for DNA probe immobilization on this substrate
Stamping biotinylated oligonucleotide DNA solution
did. After stamping to remove unreacted DNA 1
XSSPE buffer (150 mM NaCl, 8.65 mM NaH₂PO_Four, 1.25 mM
Wash with EDTA; pH 7.4) to prepare a DNA microarray.
It was

Example 3 Comparison of Detection Sensitivity Substrates for biomolecule microarrays obtained in Example 1 (DN
A microarray substrate) and a commercially available poly-L-lysine-coated glass substrate for DNA microarray (PLL slide glass) were added to a Cy3-oligo DNA dilution series (0, 0.06) in the same manner as in Example 2. , 0.13, 0.25, 0.50, 1.
(0, 2.0, 4.0, 8.0 pmol / μl) was spotted, and the fluorescence intensity of each spot was measured by the following method without washing. The obtained results are shown in FIG. From the results shown in FIG. 1, Example 1
The substrate for DNA microarray obtained in
A clear fluorescent signal was measured from the spot of / μl, whereas a clear fluorescent signal was measured on a commercially available substrate for a DNA microarray having no light reflecting layer.
It was from 0.50 pmol / μl. That is, it is understood that the substrate for a DNA microarray of the present invention can have a sensitivity about 4 times higher than that of a substrate for a DNA microarray having no light reflection layer.

The measurement of the fluorescent spot was performed by the following method. Fluorescent images of both substrates were obtained using a fluorescent microarray scanner (Japan Laser Electronics Co., Ltd.). At that time, the applied voltage of the detector (photomultiplier tube) of the scanner was measured under the same conditions in the measurement of both substrates. The graph of the intensity of the fluorescent spot was made using image analysis software (NIH image).

Example 4 Identification of Biomolecule Probe Immobilization Spots The biomolecule probe immobilization spots of the DNA microarray obtained in Example 1 above were identified by the following method. Fluorescence microarray scanner- (Japan Laser Electronics Co., Ltd.) Remove the optical cut filter installed on the incident light side of the detector (photomultiplier tube), and the laser light reflected on the surface of the DNA microarray is directly photomultiplier tube. To be incident on. At that time, the applied voltage of the photomultiplier tube is usually
It was set lower than when measuring fluorescence. FIG. 2 shows the measurement of reflected light from the surface of the DNA microarray obtained in Example 1 by this method. The higher the intensity of light incident on the detector, the whiter it is displayed. According to this result, it is understood that the intensity of the reflected light of the laser light is higher in the biomolecule probe immobilization spot having the light reflection layer spot than in the substrate surface not covered with the spot. By utilizing this characteristic, it becomes possible to identify the spot for immobilizing the biomolecule probe.

[Brief description of drawings]

FIG. 1 shows the results obtained in Example 3. The results obtained in Test Example 1 are shown.

FIG. 2 shows the results obtained in Example 4.

Continuation of front page (51) Int.Cl. ⁷ identification code FI theme code (reference) G01N 37/00 102 C12N 15/00 F (72) Inventor Tokuji Tachibana 2-1, Hirosawa, Wako, Saitama Prefectural Institute of Physical and Chemical Research (72) Inventor Kaori Honda, No. 2 Hirosawa, Wako City, Saitama Prefecture, F-Term, RIKEN (Reference) 4B024 AA11 CA09 HA14 HA19 4B029 AA07 AA21 AA23 BB20 CC03 CC08 FA15

Claims (17)

[Claims] 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 reflection layer spot between the biomolecule probe immobilization spot and the substrate surface. The substrate having. 2. The substrate according to claim 1, wherein the light reflection layer spot has substantially the same plane shape as the biomolecule probe immobilization spot. 3. The biomolecule probe-immobilized spot comprises biotin, avidin, streptavidin and poly-L-lysine, and an amino group, an aldehyde group, a thiol group, a carboxyl group, a succinimide group, a maleimide group, an epoxide group and The substrate according to claim 1 or 2, comprising at least one biomolecule probe-immobilizing compound selected from the group consisting of compounds having an isothiocyanate group. 4. The biomolecule probe immobilization spot is circular and has a diameter in the range of 10 to 500 μm.
Or it is rectangular or polygonal, and the length of one side is 10
The substrate according to any one of claims 1 to 3, which has a range of 500 µm. 5. The light reflection layer is a metal layer.
The substrate according to any one of 1. 6. The substrate according to claim 1, wherein the substrate is a glass substrate, a silicon substrate or a quartz glass substrate. 7. The substrate according to claim 1, further comprising a water-repellent layer on the surface of the substrate that is not covered with the biomolecule probe immobilization spot. 8. A step of providing a light reflecting layer on the surface of a substrate, a step of providing a biomolecule probe immobilization layer on the light reflecting layer, a step of providing a photoresist layer on the biomolecule probe immobilization layer, and a photoresist layer. Through the mask for spot formation, the step of developing the photoresist layer, the light reflection layer portion not protected by the photoresist layer is etched to form spots on the biomolecule probe immobilization layer and the light reflection layer. The method for producing a biomolecule microarray substrate according to claim 1, comprising a step of forming and a step of removing the photoresist layer. 9. The method according to claim 8, further comprising a step of subjecting the surface of the substrate to a water repellent treatment between the etching step and the photoresist layer removing step.
The manufacturing method described in. 10. The production method according to claim 8, wherein the biomolecule probe immobilization layer is formed by immobilizing a biomolecule probe immobilization compound after the surface of the light reflecting layer is treated with aminosilane. . 11. The method according to claim 10, wherein the light reflection layer is an aluminum layer, and the aminosilane-treated light reflection layer is formed by oxidizing the surface of the aluminum layer with ethanol. 12. The manufacturing method according to claim 8, wherein the light reflecting layer is formed by vapor deposition of metal. 13. A plurality of biomolecule probe-fixed spots, each having a biomolecule probe immobilized on the biomolecule probe-immobilized spots of the biomolecule microarray substrate according to any one of claims 1 to 7. Biomolecule microarray. 14. The biomolecule microarray according to claim 13, wherein the immobilized biomolecule probe is oligo DNA, cDNA, RNA, or PNA. 15. The biomolecule microarray according to claim 14, wherein the immobilized biomolecule probes have the same or different base sequences between the biomolecule probe fixed spots. 16. The biomolecule microarray according to claim 13, wherein the immobilized biomolecule probe is a protein or a peptide. 17. The biomolecule microarray according to claim 16, wherein the immobilized biomolecule probe has the same or different residue sequence between the biomolecule probe fixed spots.