JP4346017B2 - Manufacturing method of microarray chip - Google Patents

Description

  The present invention relates to a method for producing a microarray chip in which a large number of specific biopolymers that specifically bind to specific DNA or protein are arranged.

  In recent years, DNA chips, DNA arrays, protein chips, capillary arrays, μTAS, MEMS, etc. that can perform high-throughput biological detection of many types or functions (biological interact), such as nucleic acid and amino acid sequences on the substrate A microarray chip having a specific biopolymer immobilized thereon has been proposed and put into practical use. In the production of these microarray chips, wafer processing technology, microcontact printing technology, ink jet technology, etc., which are also used in semiconductor production, have been incorporated, and various specific biopolymers can be accurately arranged in minute areas. By fixing, it is possible to detect from a very small amount of sample in a short time.

  For example, a DNA chip in which an oligonucleotide or cDNA (DNA complementary to mRNA (a nucleic acid encoding protein sequence information)) that is part of deoxyribonucleic acid (DNA) is arrayed and immobilized as a specific biopolymer on a substrate And a microarray chip called a DNA array. Examples of nucleic acids arranged on such a DNA chip or DNA array include short chain (20 to 30 bases) and long chain (50 bases or more) oligonucleotides and cDNA. As a method for arranging and immobilizing these nucleic acids, a photopolymer synthesis method is used when the nucleic acid is a short oligonucleotide, and when cDNA is used as the nucleic acid, microcontact printing or inkjet technology is used. A method using the used array device is generally used. When long-chain oligonucleotides are used as nucleic acids, both are used for a while.

  Here, the manufacturing method developed by Affymetrix is the most well-known method for manufacturing DNA arrays and the like using a photosensitive polymer synthesis method used when arranging and immobilizing short oligonucleotides (patents). Reference 1). In this method, a protective group that is selectively removed by light irradiation and a photolithographic technique used in semiconductor manufacturing are used, and oligonucleotides or the like are densely provided for each rectangular pattern of several tens of μm on a substrate. A specific biopolymer is synthesized.

  Specifically, an immobilization layer having a photosensitive protective group is first formed on a substrate, and pattern exposure is performed on the immobilization layer by photolithography. In this exposed portion, the photosensitive protecting group is eliminated, and the monomer filled in the chamber reacts with the exposed selected residue. Furthermore, the desired polymer sequence can be synthesized by performing arbitrary pattern exposure and performing the same reaction in multiple stages.

  Here, the microarray chip such as the DNA array as described above is required to have fineness in order to obtain high throughput, the amount and density of the specific biological material is large, and contrast with different specific biological materials. Is required to be high. The sensitivity of the microarray chip increases as the amount, density, contrast, and the like of the arraying substance increase. However, as the pattern for arranging the specific biomaterial becomes finer, the density of the specific biomaterial is required.

  Here, when many kinds of specific biological materials are adjacent to each other on the microarray chip, it is important that adjacent specific biological materials are not mixed in order to increase the contrast with the adjacent specific biological materials. Therefore, when the specific biopolymer is formed by the photosensitive polymer synthesis method as described above, a chamber optical member / filling monomer solvent is devised so that light is not diffused in the chamber as much as possible, and an optical path (substrate, chamber) Attempts have been made to minimize the gap) and to optimize and minimize the exposure time. However, depending on the degree of monomer diffusion within the exposure time, alignment accuracy, etc., a portion other than the target region may be exposed to react with the monomer, or the monomer or the like may be exposed to the target region due to the diffusibility of the monomer. May not be able to react. As a result, for example, a monomer reacts in a space portion between adjacent specific biological materials to form a polymer or the like, or a specific biological material may be mixed in a region where the specific biological material is formed. . In addition, a specific biological material different from the target specific biological material may be formed.

  However, when different specific biological substances are mixed in this way, or when a polymer is formed in the space portion, it is difficult to remove or modify the portion. For this reason, non-specific adsorption of the object to be inspected causes a decrease in the contrast of each specific biological substance and a decrease in accuracy.

Japanese Patent Publication No. 2000-508542

  Therefore, when a defective part occurs, it can be corrected and removed, and the specific biopolymer is arranged in a fine pattern without causing non-specific adsorption of the test object. There is a need to provide a method for manufacturing a fixed microarray chip.

The present invention provides an immobilization layer forming step of forming an immobilization layer having a photosensitive protective group on a substrate,
A specific biopolymer immobilization step of immobilizing a specific biopolymer to a specific biopolymer immobilization part on the immobilization layer, comprising:
There is provided a method for producing a microarray chip, comprising an organic group removing step of removing an organic group present in a space part between the specific biopolymer immobilization parts.

  According to the present invention, since the organic group in the space portion between each specific biopolymer immobilization portion is removed by the organic group removal step, the organic group of the immobilization layer and the specific biomolecule height are included in the space portion. A microarray chip to which no molecule or the like is attached can be obtained. As a result, the object to be inspected cannot be non-specifically adsorbed on the space portion of the microarray chip manufactured according to the present invention, and the microarray chip has high contrast and high accuracy. It can be done.

The present invention also includes an immobilization layer forming step of forming an immobilization layer having a photosensitive protective group on a substrate,
A specific biopolymer immobilization step of immobilizing a specific biopolymer to a specific biopolymer immobilization part on the immobilization layer, comprising:
A method of manufacturing a microarray chip, comprising: a defective portion removing step of removing the specific biopolymer and the immobilization layer in a defective portion when a defect occurs in the specific biopolymer immobilizing step. provide.

  According to the present invention, in the specific biopolymer immobilization step, when the different specific biomaterial is formed, or when different specific biopolymers are mixed, the defective portion These defective portions can be removed by the removing step. Therefore, it is possible to obtain a high quality microarray chip in which the object to be inspected is not non-specifically adsorbed on the defective portion.

  In the above invention, the second fixing layer forming step of forming a second fixing layer having a photosensitive protective group on the defective portion removing portion from which the defective portion has been removed by the defective portion removing step, and the second fixing And a second specific biopolymer immobilization step of immobilizing the specific biopolymer on the formation layer. This is because the target specific biopolymer can be immobilized again on the defective portion removing portion from which the defective portion has been removed, and a high-quality microarray chip can be obtained.

  In the above invention, the organic group removing step or the defective portion removing step may include the step of removing the photocatalyst-containing layer of the photocatalyst-containing layer-side substrate having the photocatalyst-containing layer and the substrate. It is preferably performed by irradiating energy after the biopolymer is placed with a gap. According to this method, since the organic group removal step and the defective portion removal step can be performed by the action of a photocatalyst accompanying energy irradiation, the immobilization layer or the specific biological body is used, for example, when using a photolithography method. There is no need to use chemicals that adversely affect polymers. In addition, by irradiating the space portion and the defective portion with energy using the photocatalyst-containing layer side substrate, the organic group or the like can be easily decomposed and removed, so that the above process can be performed efficiently. Have.

  The present invention also includes a substrate and an immobilization layer formed on the substrate and containing a photosensitive protective group, and the immobilization layer is a region for immobilizing a specific biopolymer. Provided is a microarray chip substrate which is formed in a pattern of specific biopolymer immobilization parts.

  According to the present invention, since the immobilization layer is formed in the pattern of the specific biopolymer immobilization part, when the specific biopolymer is immobilized on the microarray chip substrate, there is a specific area in this gap. It can be assumed that no adherent biopolymer or other deposits are attached. Therefore, it can be set as the microarray chip | tip board | substrate which can form the microarray chip | tip without a to-be-detected object adsorb | sucking nonspecifically in a clearance gap.

  The present invention also provides a microarray chip, wherein a specific biopolymer is immobilized on the immobilization layer of the microarray chip substrate described above.

  According to the present invention, since the specific biopolymer is formed on the immobilization layer formed in the pattern, the specific biopolymer is formed in the region where the immobilization layer is not formed. In other words, the object to be detected is not non-specifically adsorbed in the gap, and a high-quality microarray chip can be obtained.

  According to the present invention, a microarray chip can be obtained in which the organic group of the immobilization layer, the specific biopolymer, and the like are not attached to the space between the specific biopolymer immobilization units. As a result, the object to be inspected cannot be non-specifically adsorbed on the space portion of the microarray chip manufactured according to the present invention, and the microarray chip has high contrast and high accuracy. There is an effect that it is possible.

  The present invention can correct even when a defective part is produced during production, and the specific biopolymer is arranged and fixed in a fine pattern without non-specific adsorption of the test object. The present invention relates to a microarray chip manufacturing method, a microarray chip substrate used for the microarray chip, and a microarray chip. Each will be described in detail below.

A. First, a method for manufacturing a microarray chip of the present invention will be described. The microarray chip manufacturing method of the present invention has two embodiments. Hereinafter, each embodiment will be described separately.

1. First Embodiment First, a first embodiment in the method for manufacturing a microarray chip of the present invention will be described. The manufacturing method of the microarray chip of this embodiment includes an immobilization layer forming step of forming an immobilization layer having a photosensitive protective group on a substrate,
A specific biopolymer immobilization step of immobilizing a specific biopolymer to a specific biopolymer immobilization part on the immobilization layer, comprising:
It has an organic group removing step of removing an organic group present in the space between the specific biopolymer immobilization parts.

  For example, as shown in FIG. 1, the microarray chip manufacturing method of this embodiment includes an immobilization layer forming step (FIG. 1A) for forming an immobilization layer 2 on a substrate 1, and the immobilization layer 2. A specific biopolymer immobilization step (FIG. 1 (b)) for immobilizing the specific biopolymer 3 to the specific biopolymer immobilization part a, and fixing the specific biopolymer For example, an organic group removing step (FIG. 1C) for removing organic groups such as the specific biopolymer 3 and the immobilization layer 2 existing in the space portion b between the portions a is included. Here, the specific biopolymer immobilization part is a region where the specific biopolymer is finally immobilized in the microarray chip. The space portion refers to a region between the specific biopolymer immobilization portions adjacent to each other.

  In general, when a specific biopolymer is formed using a photosensitive polymer synthesis method in a pattern of a fine specific biopolymer immobilization part, depending on the alignment accuracy at the time of exposure, for example, FIG. As shown in FIG. 2, the specific biopolymer 3 may be formed in a region wider than the specific biopolymer fixing part a, that is, in the space part b. In this way, when the specific biopolymer is immobilized or the organic group of the immobilized layer is present on the space part, the object to be inspected is detected on the space part when performing detection using the microarray chip. Non-specific adsorption may occur and detection may not be performed with high accuracy.

  Therefore, in this embodiment, an organic group removal step for removing the organic group on the space portion is performed, and the microarray chip is configured so that nonspecific adsorption does not occur between the space portion and the test object. is there.

In the present embodiment, the organic group removal step may be performed at any time during the production of the microarray chip. For example, as shown in FIG. It may be performed after the molecular immobilization step. For example, as shown in FIG. 2, first, an immobilization layer forming step (FIG. 2A) for forming the immobilization layer 2 on the substrate 1 is performed, and then the immobilization layer 2 present in the space portion b. After performing the organic group removing step (FIG. 2B) for removing the organic group, the specific biopolymer 3 is immobilized on the specific biopolymer immobilization portion a where the immobilization layer 2 remains. A specific biopolymer immobilization step (FIG. 2C) may be performed. In this case, since the organic group in the immobilization layer on the space portion is removed by the organic group removing step, the specific biopolymer cannot be fixed in the space portion.
Hereafter, each process in the manufacturing method of the microarray chip | tip of this embodiment as mentioned above is demonstrated.

(Fixed layer forming process)
First, the immobilization layer forming process of this embodiment will be described. The immobilization layer forming step in this embodiment is a step of forming an immobilization layer having a photosensitive protective group on a substrate.

  The immobilization layer formed by this step has a photosensitive protective group, and this photosensitive protective group is detached by exposure in the specific biopolymer immobilization step described later, and the specific biopolymer is removed. For example, functional groups that react with, for example, amino acids and bases are exposed.

  As a compound which has the said functional group and has adhesiveness with a base material, the compound which adheres, for example by a base material, a carbon-carbon bond, etc., Preferably by a siloxane bond is mentioned. Examples of the compound having a siloxane bond with such a substrate include a compound having a trichlorosilyl group or a trialkoxysilyl group. Further, a compound having an amine, hydroxyl, thiol, carboxylic acid, ester, amide, epoxide, isocyanate, or isothiocyanate as a functional group that is exposed when the photosensitive protective group is eliminated is preferable. Specific examples of such compounds include aminoalkyltrialkoxysilanes, aminoalkyltrichlorosilanes, hydroxyalkyltrialkoxy-silanes, hydroxyalkyltrichlorosilanes, carboxyalkyltrialkoxysilanes, polyethylene glycols, triethoxysilanes, epoxyalkyltrialkylsilanes. Examples include alkoxysilanes and combinations thereof.

  In this embodiment, among the above, a silane coupling agent containing an amino group is preferably used. Examples of the silane coupling agent containing an amino group include N-β (aminoethyl) γ-aminopropyltrimethoxysilane, N-β (aminoethyl) γ-aminopropylmethyldimethoxysilane, γ-aminopropyltriethoxysilane, N -Phenyl-γ-aminopropyltrimethoxysilane and the like can be mentioned.

  In addition, as the above-described photosensitive protective group, when exposed in a specific biopolymer immobilization step described later, it is easily detached from the functional group and stable in an unexposed region. It is preferable to prevent the functional group from reacting. The removal rate of such a photosensitive protecting group depends on the incident wavelength and irradiation intensity of the energy irradiated in the specific biopolymer immobilization step, and the physical and chemical characteristics of the photosensitive protecting group itself. . In this step, in particular, those that can be removed quickly with low energy are preferred, and it is generally a photosensitive protective group that undergoes photolysis with an energy of a wavelength of 300 nm to 450 nm.

  Examples of such photosensitive protecting groups include ortho- (o-) nitrobenzyl derivatives, o-nitrobenzyloxycarbonyl derivatives, aromatic amine N-oxide derivatives, o-nitrobenzyl carbamate derivatives, allylformamide derivatives, N-benzylamide derivatives, benzylcarbamate derivatives, benzylsulfonamide derivatives, bis (o-nitrophenyl) methyl ester derivatives, S, S-dibenzylacetal derivatives and ketal derivatives, 3,5-dimethoxybenzylcarbamate derivatives, 4 -(4 ', 8'-dimethoxynaphthylmethyl) benzenesulfonamide derivatives, 3,4-dimethoxy-6-nitrobenzylcarbamate derivatives, α- (3,5-dimethoxyphenyl) phenacyl ester derivatives, N, N -Dimethylhydrazone derivative, di-2-nitro Benzyl acetal derivative, 2- (9,10-dioxo) anthrylmethyl ester derivative, 4,5-diphenyl-3-oxazolin-2-one derivative, 1,3-dithiolene derivative, 9-fluorenecarboxylate ester derivative, p -Methoxybenzyl ether derivatives, phenacyl derivatives, p-methoxyphenacyl ester derivatives, N-methylamine derivatives, 1-methyl-1- (3,5-dimethoxyphenyl) ethylcarbamate derivatives, α-methylphenacyl ester derivatives, nitro Rate ester derivatives, nitroamide derivatives, o-nitroanilide derivatives, N-o-nitrobenzylamine derivatives, o-nitrobenzyl carbonate derivatives, o-nitrobenzyl ester derivatives, o-nitrobenzyl ether derivatives, o-nitrobenzylidene acetal derivatives N-7-nitroindolylamide derivatives, m-nitrophenylcarbamate derivatives, 4-o-nitrophenyl-1,3-dioxolane derivatives, N-8-nitro-1,2,3,4-tetrahydroquinolylamide derivatives , 1,3-oxothiolane derivatives, phenyl (o-nitrophenyl) methylcarbamate derivatives, 1-pyrenylmethyl ester derivatives, toluenesulfonamide derivatives, toluenesulfonate derivatives, xanthenecarboxylate ester derivatives, nitrophenylsulfenyl Specifically, for example, o-nitrobenzyl group, α-methyl-nitropiperonyl group, α-methyl-nitropiperonyloxycarbonyl group, 1-pyrenylmethyloxycarbonyl group, α, α- Dimethyl-3,5-dimethoxybenzyloxycarbo Group, 4-nitropyridine N-oxide group, m-nitrophenyl carbamate group, 3,5-dimethoxybenzyl carbamate group, o-nitrobenzyloxycarbonyl group, 3,4-dimethoxy-6-nitrobenzyl carbamate group, phenyl (o-nitrophenyl) methyl carbamate group, p-methoxyphenacyl group, 1,3-dithiolene group, 1,3-oxathiolene group, p-methoxybenzyl group, tosyl group, benzenesulfonamide group, etc. Can do.

  Examples of the above functional groups and photosensitive protective groups include, for example, “Protective groups in organic synthesis” (Second Edition), by Theodora W. Greene and Peter GM Wuts, John Wiley & Sons, 1991), JP 2000-508542 A. What was described in the gazette etc. can also be used.

  The method for forming the immobilization layer having the functional group and the photosensitive protective group in this step is particularly limited as long as the method can form an immobilization layer in which the functional group is protected by the photosensitive protective group. For example, a material in which the above functional group is protected with the above-described photosensitive protective group is dissolved in a solvent to form a coating solution for forming an immobilization layer, and spin coating, flexographic coating, or gravure coating is applied to the substrate. And a method of forming a thin film by a coating method such as offset coating. For example, when the molecule having a functional group protected by the photosensitive protective group is a low-molecular material, a method of forming a thin film on a substrate by a vapor deposition method may be used. Furthermore, a method of forming a self-assembled monolayer may be used. For this self-assembly method (self-assembled monolayer), see, for example, Abraham Ulman, “Formation and structure of self-assembled monolayers”, Chemical Review, vol. 96, 1533-1554 (1996). be able to.

  Alternatively, a method of forming a thin film (LB film) on a substrate by the Langmuir-Blodgett method (LB method) may be used, and the molecule having a functional group protected with the above-mentioned photosensitive protective group is a polymer electrolyte. In some cases, a thin film can be formed by an adsorption method and an alternate adsorption method.

  Here, in this step, for example, after fixing the functional group-containing material on the substrate by the above-described method, a method for protecting the above-described functional group by a photosensitive protective group by a general method is used. May be.

  Moreover, as a base material used for this process, it is possible to use the base material normally used for a microarray chip, for example, a silicon wafer, a metal, quartz, glass, a diamond, diamond-like carbon (DLC), an alumina, high Molecular materials and the like can be used, and these are appropriately selected and used according to the use of the microarray chip to be manufactured. Moreover, as a shape of the base material to be used, the thing on a flat plate is generally used, However It does not restrict | limit in particular, It can be set as a suitable shape according to a use.

(Specific biopolymer immobilization process)
Next, the specific biopolymer immobilization step in this embodiment will be described. This step is a step of immobilizing a specific biopolymer to the specific biopolymer immobilization part on the immobilization layer formed by the specific biopolymer immobilization step, and the specific biopolymer is immobilized on the immobilization layer. The specific biopolymer immobilization method and the like are not particularly limited as long as the specific biopolymer can be immobilized along the pattern of the specific biopolymer immobilization portion.

  Here, the specific biopolymer immobilization part is a region where the specific biopolymer is finally immobilized as described above. Usually, the specific biopolymer immobilization part is different for each specific biopolymer immobilization part. The molecule is to be immobilized. The shape of the specific biopolymer immobilization part is appropriately selected depending on the use of the microarray chip, and may be, for example, a circle or the like, but is generally a rectangular pattern.

  Further, although depending on the use of the microarray chip, etc., usually about 100 to 500,000 specific biopolymer immobilization parts are provided on one microarray chip. When the molecule is immobilized by a photosensitive polymer synthesis method, 1,000 to 200,000, particularly 10,000 to 100,000 are provided. In addition, the number of the specific biopolymer immobilization parts is the number in one microarray chip, and the production is not limited to the above number when the microarray chip is produced using a large-sized base material in the manufacturing process. When the specific biopolymer is immobilized by a photosensitive polymer synthesis method, the length of one side of the rectangular specific biopolymer immobilization part is 1 μm to 2,000 μm, and more preferably 5 μm to 200 μm. In particular, the thickness is preferably 10 μm to 100 μm. In this case, the space part, which is a region between adjacent specific biopolymer immobilization parts, is usually 1 μm to 2,000 μm, more preferably 2 μm to 150 μm, and particularly preferably 5 μm to 75 μm.

  Here, the specific biopolymer refers to a biopolymer that can specifically bind to a predetermined biopolymer, and the specific biopolymer used in this step has such properties. It is not particularly limited as long as it has a biopolymer.

  Examples of such a combination of a specific biopolymer and a biopolymer include, for example, a combination of a nucleic acid (eg, oligonucleotide or polynucleotide) and a complementary nucleic acid, an antigen and an antibody (or antibody fragment) Combinations, combinations of receptors and their ligands (eg, hormones, cytokines, neurotransmitters, or lectins), combinations of enzymes and their ligands (eg, enzyme substrate analogs, coenzymes, modulators, or inhibitors) And a combination of an enzyme analog and a substrate of the enzyme that is the base of the enzyme analog, or a combination of a lectin and a sugar. The “enzyme analog” means a substance having a high specific affinity with a substrate for the original enzyme but showing no catalytic activity. In addition, each of the compounds in each of the above combinations is a “specific biopolymer” and the other is a substance to be detected. For example, in the “combination of antigen and antibody”, when the antigen is “a substance to be detected”, the antibody can be “specific biopolymer”, and conversely, the antibody is “detected” In the case of “substance”, the antigen becomes “specific biopolymer”.

  For example, when the microarray chip produced according to this embodiment is applied to a nucleic acid hybridization assay, the specific biopolymer is complementary to a nucleic acid (for example, an oligonucleotide or a polynucleotide) that is a substance to be detected. Oligonucleotides or polynucleotides that can be bound to each other can be used. As used herein, “oligonucleotide” or “polynucleotide” includes 2′-deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and peptide nucleic acid (PNA). PNA is an artificial nucleic acid obtained by converting a DNA phosphodiester bond into a peptide bond. The chain length of the oligonucleotide or polynucleotide bound to the insoluble particle can be appropriately selected depending on the purpose of analysis, and is based on, for example, the chain length of the complementary sequence of DNA, RNA, or PNA to be captured. Can be determined. In addition, by arranging oligomers of confirmed base sequences in different combinations on the array and determining the complementary binding position with a part of the specific biopolymer that is sufficiently longer than that, the sequence can be determined by a computer. It is also possible to perform a wide range of sequencing by mining common points.

Moreover, when the microarray chip of this embodiment is applied to an immunological assay, an antigen (including a hapten) or an antibody that specifically binds to a substance to be detected may be used as the specific biopolymer. it can. In this case, the substance to be detected is not particularly limited as long as it is a component generally contained in a test sample and can be detected immunologically. For example, various proteins, polysaccharides, lipids, fungal bodies, various environmental substances, and the like can be given. More specifically, an immunoglobulin (eg, IgG, IgM, or IgA), an infection-related marker (eg, HBs antigen, HBs antibody, HIV-1 antibody, HIV-2 antibody, HTLV-1 antibody, or treponema antibody) Tumor associated antigens (eg, AFP, CRP, or CEA), coagulation fibrinolytic markers (eg, plasminogen, antithrombin-III, D-dimer, TAT, or PPI), antiepileptic drugs (eg, hormones), Examples include various drugs (for example, digoxin), bacterial cells (for example, O-157 or Salmonella) or their endotoxins or exotoxins, microorganisms, enzymes, residual agricultural chemicals, environmental hormones, and the like. As the antibody used as the specific biopolymer, either a polyclonal antibody or a monoclonal antibody obtained by a well-known method can be used. Further, the antibody may be a protein [for example, an enzyme (for example, pepsin or papain)] [for example, an antibody fragment (for example, Fab, Fab ′, F (ab ′) ₂ , or Fv)]. it can.

  A method for immobilizing such a specific biopolymer to the specific biopolymer immobilization part of the immobilization layer is not particularly limited, but the specific biopolymer is immobilized in a fine pattern. It is preferable to carry out by a photosensitive polymer synthesizing method from the point that it is possible to make it.

  The formation of a specific biopolymer by the photopolymer synthesis method is performed by reacting each low molecular block such as a monomer constituting the specific biopolymer, and repeating these reactions to achieve the target specific biopolymer. Synthesize polymers. Such small molecule blocks include, for example, L-amino acids, D-amino acids, natural or synthetic amino acids, and sets of nucleotides (ribonucleotides and deoxyribonucleotides, both natural and non-natural), and pentoses and hexoses. Can be mentioned. For example, when the specific biopolymer is a nucleotide, it is formed using blocks of four types of bases A, T (or U), G, and C, respectively.

  As a specific method for forming a specific biopolymer, for example, energy is irradiated only on an immobilization layer of a specific biopolymer immobilization part to cause a reaction using, for example, a photomask, and the above-described immobilization. The photosensitive protective group of the chemical layer is removed. A target monomer or the like is brought into contact with a region where the photosensitive protecting group is removed and the functional group is exposed, and the functional group and the monomer or the like are reacted. After that, the substituent of the monomer that has reacted with the functional group is protected again by the above-described photosensitive protective group or the like, and when the specific biopolymer immobilization part in another region is reacted with the monomer, It is assumed that no reaction takes place in the area protected by the photosensitive protecting group. By repeating such exposure and reaction with a monomer or the like, different specific biopolymers can be immobilized on each specific biopolymer immobilization part.

  The light source used for such exposure is not particularly limited as long as it has energy of a wavelength that does not damage the obtained polymer arrangement and can remove the above-mentioned photosensitive protective group. Generally, energy having a wavelength longer than 340 nm is used. For example, an Hg-Arc lamp using an air cut-off filter can be used.

  In addition, the monomer or the like constituting the specific biopolymer is appropriately selected according to the target specific biopolymer, and for example, those described in JP-T-2000-508542 are used. Can do.

(Organic group removal process)
Next, the organic group removal step of this embodiment will be described. The organic group removal step in this embodiment is a step of removing organic groups present in the space portion between the specific biopolymer immobilization portions. This step may be performed after the immobilization layer forming step and before the specific biopolymer immobilization step, or may be performed after the specific biopolymer immobilization step. . Further, it may be performed during the specific biopolymer immobilization step.

  For example, when the organic group removal step is performed after the above-described immobilization layer formation step and before the specific biopolymer immobilization step, the immobilization present in the space between the specific biopolymer immobilization portions by the organic group removal step The organic group of the layer will be removed. Thereby, when immobilizing the specific biopolymer in the specific biopolymer immobilization step, since there is no organic group of the immobilization layer in this space portion, the specific biopolymer does not adhere, In the space part of the finally manufactured microarray chip, it is possible that the organic group is not attached. In addition, for example, when the organic group removal step is performed after the specific biopolymer immobilization step, the specific biopolymer is attached to the space portion by the specific biopolymer immobilization step or Even if the organic group of the immobilization layer is present, the specific biopolymer and the organic group of the immobilization layer can be removed, and the organic group is present in the space portion of the manufactured microarray chip. It can be non-attached. The same applies to the case where it is performed during the specific biopolymer immobilization step.

  Here, the organic group removed by this step is the functional group or the specific biopolymer of the immobilization layer, for example, the silane compound in which the immobilization layer is bonded to the substrate, etc. In the case of having a portion made of an inorganic material, the portion made of the inorganic material may be removed simultaneously with the organic group, or the portion made of the inorganic material may remain on the substrate surface.

  The method for removing the organic group in this step is not particularly limited as long as it is a method capable of removing the organic group present in the space portion. For example, it may be performed by a commonly performed photolithography method or the like, or may be a method of decomposing an organic group by irradiating, for example, short wavelength ultraviolet light or the like in a pattern. .

  In this embodiment, for example, as shown in FIG. 3, the photocatalyst-containing layer 11 of the photocatalyst-containing layer side substrate 13 having the photocatalyst-containing layer 11 and the substrate 12 is replaced with the specific biopolymer 3 (or It is preferable to perform the irradiation by irradiating energy 15 using, for example, a photomask 14 or the like after being arranged with a gap from the fixing layer 2).

According to this method, the organic group can be removed by the action of a photocatalyst accompanying energy irradiation, so that it adversely affects a specific biopolymer such as an alkali developer used in a photolithography method. There is no need to use special chemicals. In addition, by irradiating the space portion with energy using the above-mentioned photocatalyst-containing layer side substrate, it is possible to easily decompose and remove organic groups, etc., so that organic groups can be efficiently removed without using high energy light. This is because it also has the advantage that it can be performed.
The photocatalyst containing layer side substrate, energy, etc. used in the method for removing organic groups using such a photocatalyst containing layer side substrate will be described below.

a. Photocatalyst containing layer side substrate First, the photocatalyst containing layer side substrate used in this step will be described. The photocatalyst containing layer side substrate used in this step has a photocatalyst containing layer containing a photocatalyst and a substrate. Thus, the photocatalyst-containing layer side substrate used in this step has at least a photocatalyst-containing layer and a substrate, and usually a thin-film photocatalyst-containing layer formed by a predetermined method is formed on the substrate. It will be. Moreover, the thing in which the photocatalyst containing layer side light shielding part formed in pattern shape was formed can also be used for this photocatalyst containing layer side board | substrate. Hereinafter, each structure of the photocatalyst containing layer side substrate will be described.

(1) Photocatalyst-containing layer The photocatalyst-containing layer used in this step is not particularly limited as long as the photocatalyst in the photocatalyst-containing layer can decompose and remove the target organic group. The photocatalyst and the binder may be used, or the photocatalyst may be formed into a single film. Further, the wettability of the surface may be particularly lyophilic or lyophobic.

  The photocatalyst-containing layer used in this step may be formed on the entire surface of the substrate 12 as shown in FIG. 3, for example, but for example, as shown in FIG. 11 may be formed in a pattern.

  By forming the photocatalyst-containing layer in a pattern in this way, it is not necessary to irradiate energy in a pattern using a photomask or the like at the time of energy irradiation, which will be described later. This is because part of the organic group can be removed.

  In addition, since only the organic groups that actually face the photocatalyst-containing layer are decomposed and removed, the energy irradiation direction is such that the energy is directed to the part where the photocatalyst-containing layer and the specific biopolymer etc. face each other. As long as it irradiates, you may irradiate from what direction, and also has the advantage that the irradiated energy is not limited to parallel things, such as parallel light.

  In such a photocatalyst-containing layer, the mechanism of action of a photocatalyst represented by titanium dioxide as described later is not necessarily clear, but a carrier generated by light irradiation reacts directly with a nearby compound, or It is considered that the active oxygen species generated in the presence of oxygen and water change the chemical structure of organic matter. In the present invention, it is considered that this carrier acts on the organic group in the space portion arranged in the vicinity of the photocatalyst containing layer.

Examples of the photocatalyst used in the present invention include titanium dioxide (TiO ₂ ), zinc oxide (ZnO), tin oxide (SnO ₂ ), strontium titanate (SrTiO ₃ ), tungsten oxide (WO ₃ ), which are known as photo semiconductors. Bismuth oxide (Bi ₂ O ₃ ) and iron oxide (Fe ₂ O ₃ ) can be mentioned, and one or a mixture of two or more selected from these can be used.

  In the present invention, titanium dioxide is particularly preferably used because it has a high band gap energy, is chemically stable, has no toxicity, and is easily available. Titanium dioxide includes an anatase type and a rutile type, and both can be used in the present invention, but anatase type titanium dioxide is preferred. Anatase type titanium dioxide has an excitation wavelength of 380 nm or less.

  Examples of such anatase type titanium dioxide include hydrochloric acid peptizer type anatase type titania sol (STS-02 manufactured by Ishihara Sangyo Co., Ltd. (average particle size 7 nm), ST-K01 manufactured by Ishihara Sangyo Co., Ltd.), nitric acid solution An anatase type titania sol (TA-15 manufactured by Nissan Chemical Co., Ltd. (average particle size 12 nm)) and the like can be mentioned.

  Further, a visible light responsive type may be used as the titanium oxide. Visible light responsive titanium oxide is also excited by the energy of visible light. Examples of such a visible light responsive method include a method of nitriding titanium oxide.

When titanium oxide (TiO ₂ ) is subjected to nitriding treatment, a new energy level is formed inside the band gap of titanium oxide (TiO ₂ ), and the band gap is narrowed. As a result, the excitation wavelength of titanium oxide (TiO ₂ ) is usually 380 nm, but it can be excited even by visible light having a longer wavelength than the excitation wavelength. As a result, the wavelength in the visible light region of energy irradiation from various light sources can also contribute to the excitation of titanium oxide (TiO ₂ ), so that it is possible to further increase the sensitivity of titanium oxide. .

Here, the nitriding treatment of titanium oxide referred to in the present invention is a treatment for replacing part of the oxygen sites of the titanium oxide (TiO ₂ ) crystal with nitrogen atoms, or between the lattices of the titanium oxide (TiO ₂ ) crystal. A treatment of doping nitrogen atoms or a treatment of arranging nitrogen atoms at the grain boundaries of a polycrystalline aggregate of titanium oxide (TiO ₂ ) crystals.

The method of nitriding titanium oxide (TiO ₂ ) is not particularly limited. For example, crystalline titanium oxide fine particles are doped with nitrogen by heat treatment at 700 ° C. in an ammonia atmosphere, and the nitrogen is doped. Examples thereof include a method of forming a dispersion using fine particles and an inorganic binder, a solvent, or the like.

  Here, the smaller the particle size of the photocatalyst, the more effective the photocatalytic reaction occurs. The average particle size is preferably 50 nm or less, and it is particularly preferable to use a photocatalyst having a particle size of 20 nm or less.

  The photocatalyst-containing layer used in this step may be formed by the photocatalyst alone as described above, or may be formed by mixing with a binder.

  In the case of a photocatalyst-containing layer consisting only of a photocatalyst, the efficiency for decomposing and removing organic groups is improved, which is advantageous in terms of cost such as shortening of processing time. On the other hand, in the case of a photocatalyst-containing layer comprising a photocatalyst and a binder, there is an advantage that the formation of the photocatalyst-containing layer is easy.

  Examples of a method for forming a photocatalyst-containing layer composed only of a photocatalyst include a method using a vacuum film forming method such as a sputtering method, a CVD method, or a vacuum deposition method. By forming the photocatalyst-containing layer by vacuum film-forming method, it is possible to obtain a photocatalyst-containing layer that is a uniform film and contains only the photocatalyst, which enables uniform decomposition and removal of organic groups, etc. It is.

  Examples of a method for forming a photocatalyst-containing layer composed only of a photocatalyst include a method in which amorphous titania is formed on a substrate when the photocatalyst is titanium dioxide, and then the phase is changed to crystalline titania by firing. As the amorphous titania used here, for example, hydrolysis, dehydration condensation, tetraethoxytitanium, tetraisopropoxytitanium, tetra-n-propoxytitanium, tetrabutoxytitanium, titanium inorganic salts such as titanium tetrachloride and titanium sulfate, An organic titanium compound such as tetramethoxytitanium can be obtained by hydrolysis and dehydration condensation in the presence of an acid. Next, it can be modified to anatase titania by baking at 400 ° C. to 500 ° C. and modified to rutile type titania by baking at 600 ° C. to 700 ° C.

  Moreover, when using a binder, what has the high bond energy that the main frame | skeleton of a binder is not decomposed | disassembled by photoexcitation of said photocatalyst is preferable, for example, organopolysiloxane etc. can be mentioned.

  When organopolysiloxane is used as a binder in this way, the photocatalyst-containing layer is prepared by dispersing the photocatalyst and the binder organopolysiloxane in a solvent together with other additives as necessary. The coating solution can be formed by coating on a substrate. As the solvent to be used, alcohol-based organic solvents such as ethanol and isopropanol are preferable. The application can be performed by a known application method such as spin coating, spray coating, dip coating, roll coating, or bead coating. When an ultraviolet curable component is contained as a binder, the photocatalyst-containing layer can be formed by irradiating with ultraviolet rays and performing a curing treatment.

Moreover, an amorphous silica precursor can be used as a binder. This amorphous silica precursor is represented by the general formula SiX _4, X is a halogen, a methoxy group, an ethoxy group or a silicon compound an acetyl group or the like, and silanol or average molecular weight of 3,000 or less, their hydrolysates Polysiloxane is preferred.

  Specific examples include tetraethoxysilane, tetraisopropoxysilane, tetra-n-propoxysilane, tetrabutoxysilane, and tetramethoxysilane. In this case, the amorphous silica precursor and the photocatalyst particles are uniformly dispersed in a non-aqueous solvent, hydrolyzed with moisture in the air on the substrate to form silanol, and then at room temperature. A photocatalyst-containing layer can be formed by dehydration condensation polymerization. If dehydration condensation polymerization of silanol is carried out at 100 ° C. or higher, the degree of polymerization of silanol increases and the strength of the film surface can be improved. Moreover, these binders can be used individually or in mixture of 2 or more types.

  When the binder is used, the content of the photocatalyst in the photocatalyst containing layer can be set in the range of 5 to 60% by weight, preferably 20 to 40% by weight. The thickness of the photocatalyst containing layer is preferably in the range of 0.05 to 10 μm.

  In addition to the photocatalyst and the binder, the photocatalyst containing layer can contain a surfactant. Specifically, hydrocarbons such as NIKKOL BL, BC, BO, BB series manufactured by Nikko Chemicals Co., Ltd., ZONYL FSN, FSO manufactured by DuPont, Surflon S-141, 145 manufactured by Asahi Glass Co., Ltd., Dainippon Megafac F-141, 144 manufactured by Ink Chemical Industry Co., Ltd., Footgent F-200, F251 manufactured by Neos Co., Ltd., Unidyne DS-401, 402 manufactured by Daikin Industries, Ltd., Fluorard FC-170 manufactured by 3M Co., Ltd. Fluorine-based or silicone-based nonionic surfactants such as 176, and cationic surfactants, anionic surfactants, and amphoteric surfactants can also be used.

  In addition to the above surfactants, the photocatalyst-containing layer includes polyvinyl alcohol, unsaturated polyester, acrylic resin, polyethylene, diallyl phthalate, ethylene propylene diene monomer, epoxy resin, phenol resin, polyurethane, melamine resin, polycarbonate, Polyvinyl chloride, polyamide, polyimide, styrene butadiene rubber, chloroprene rubber, polypropylene, polybutylene, polystyrene, polyvinyl acetate, polyester, polybutadiene, polybenzimidazole, polyacrylonitrile, epichlorohydrin, polysulfide, polyisoprene, oligomers, polymers, etc. It can be included.

(2) Substrate The photocatalyst-containing layer side substrate used in this step has at least a base 12 and a photocatalyst-containing layer 11 formed on the base 12 as shown in FIG.
At this time, the material constituting the substrate to be used is appropriately selected depending on the energy irradiation direction, which will be described later, and whether the microarray chip has transparency.

  That is, for example, when the microarray chip is opaque, the energy irradiation direction is necessarily from the photocatalyst containing layer side substrate side, and for example, as shown in FIG. It is necessary to irradiate with energy. As will be described later, the photocatalyst-containing layer side light-shielding portion is formed in advance in a predetermined pattern on the photocatalyst-containing layer side substrate, and the photocatalyst-containing layer side light-shielding portion is used to form a pattern. It is necessary to irradiate energy from the side substrate side. In such a case, the substrate needs to be transparent.

  On the other hand, if the microarray chip is transparent, a photomask can be arranged on the microarray chip side to irradiate energy. In such a case, the transparency of the substrate is not particularly required.

  Such a substrate may be flexible, such as a resin film, or may not be flexible, such as a glass substrate. This is appropriately selected depending on the energy irradiation method.

As described above, the material used for the substrate used for the photocatalyst-containing layer side substrate is not particularly limited, but in this step, the photocatalyst-containing layer side substrate is used repeatedly, so that the predetermined substrate is used. A material having strength and having a surface with good adhesion to the photocatalyst-containing layer is preferably used.
Specific examples include glass, ceramic, metal, and plastic.

  In order to improve the adhesion between the substrate surface and the photocatalyst containing layer, an anchor layer may be formed on the substrate. Examples of such an anchor layer include silane-based and titanium-based coupling agents.

(3) Photocatalyst containing layer side light-shielding part As the photocatalyst containing layer side light shielding part used for this process, you may use what the photocatalyst containing layer side light shielding part formed in pattern shape was formed. By using the photocatalyst containing layer side substrate having the photocatalyst containing layer side light shielding portion in this way, it is not necessary to use a photomask or perform drawing irradiation with a laser beam at the time of exposure. Therefore, since alignment between the photocatalyst-containing layer side substrate and the photomask is not necessary, it is possible to use a simple process, and an expensive apparatus necessary for drawing irradiation is also unnecessary, so that the cost is reduced. Has the advantage of being advantageous.

  The photocatalyst-containing layer side substrate having such a photocatalyst-containing layer side light-shielding part can have the following two modes depending on the formation position of the photocatalyst-containing layer side light-shielding part.

  For example, as shown in FIG. 5, a photocatalyst containing layer side light shielding portion 16 is formed on a substrate 12, and a photocatalyst containing layer 11 is formed on the photocatalyst containing layer side light shielding portion 16 to form a photocatalyst containing layer side substrate. It is an aspect to make. For example, as shown in FIG. 6, the photocatalyst containing layer 11 is formed on the substrate 12, and the photocatalyst containing layer side light-shielding portion 16 is formed thereon to form a photocatalyst containing layer side substrate.

  In any embodiment, the photocatalyst-containing layer-side light-shielding portion is disposed in the vicinity of the portion where the photocatalyst-containing layer and the specific biopolymer etc. are located with a gap as compared with the case where a photomask is used. Therefore, since the influence of energy scattering in the substrate or the like can be reduced, the energy pattern irradiation can be performed very accurately.

  Furthermore, in the embodiment in which the photocatalyst containing layer side light shielding portion is formed on the photocatalyst containing layer, when the photocatalyst containing layer and the specific biopolymer are arranged with a predetermined gap, the photocatalyst containing layer side By making the film thickness of the light shielding portion coincide with the width of the gap, there is an advantage that the photocatalyst containing layer side light shielding portion can be used as a spacer for making the gap constant.

  That is, when the photocatalyst-containing layer and the specific biopolymer etc. are arranged with a predetermined gap, the photocatalyst-containing layer side light-shielding portion and the specific biopolymer etc. are arranged in close contact with each other. Thus, the predetermined gap can be made accurate, and by irradiating energy from the photocatalyst-containing layer side substrate in this state, the organic group can be decomposed and removed with high accuracy.

  The method for forming the photocatalyst-containing layer side light-shielding part used in this step is not particularly limited, and is appropriately determined according to the characteristics of the formation surface of the photocatalyst-containing layer side light-shielding part, the shielding property against the required energy, and the like. Selected and used.

  For example, it may be formed by forming a metal thin film of chromium or the like having a thickness of about 1000 to 2000 mm by a sputtering method, a vacuum deposition method, or the like, and patterning the thin film. As this patterning method, a normal patterning method such as sputtering can be used.

  Alternatively, a method may be used in which a layer containing light-shielding particles such as carbon fine particles, metal oxides, inorganic pigments, and organic pigments in a resin binder is formed in a pattern. As the resin binder to be used, polyimide resin, acrylic resin, epoxy resin, polyacrylamide, polyvinyl alcohol, gelatin, casein, cellulose, or a mixture of one or more kinds, photosensitive resin, or O / A W emulsion type resin composition, for example, an emulsion of a reactive silicone can be used. The thickness of the resin photocatalyst-containing layer side light-shielding part can be set within a range of 0.5 to 10 μm. As a method for patterning the light shielding part on the resin photocatalyst-containing layer side, a commonly used method such as a photolithography method or a printing method can be used.

  In the above description, the two positions of the photocatalyst containing layer side light shielding portion between the substrate and the photocatalyst containing layer and the surface of the photocatalyst containing layer have been described as the formation position of the photocatalyst containing layer side. It is also possible to adopt a mode in which the photocatalyst-containing layer side light shielding portion is formed on the surface that is not provided. In this aspect, for example, a case where the photomask is brought into close contact with the surface so as to be detachable can be considered, and it can be suitably used when the energy irradiation region is changed in a small lot.

(4) Primer layer Next, the primer layer used for a photocatalyst containing layer side substrate is explained. In this step, when the photocatalyst-containing layer side light-shielding portion is formed in a pattern on the substrate as described above, and the photocatalyst-containing layer is formed thereon to form a photocatalyst-containing layer side substrate, the photocatalyst-containing layer A primer layer may be formed between the side light shielding part and the photocatalyst containing layer.

  The action and function of this primer layer are not always clear, but by forming a primer layer between the photocatalyst containing layer side light shielding part and the photocatalyst containing layer, the primer layer decomposes and removes organic groups by the action of the photocatalyst. Impurities from the openings present between the photocatalyst containing layer side light shielding part and the photocatalyst containing layer side light shielding part, which are factors that hinder, etc., in particular, residues generated when patterning the photocatalyst containing layer side light shielding part, metal, metal It is considered that it has a function of preventing diffusion of impurities such as ions. Therefore, by forming the primer layer, processing such as decomposition and removal of the organic group proceeds with high sensitivity, and as a result, the organic group in the space portion can be removed with a high resolution pattern.

  In addition, since the said primer layer prevents that the impurity which exists in the opening formed between not only the photocatalyst containing layer side light shielding part but between the photocatalyst containing layer side light shielding parts influences the effect | action of a photocatalyst, primer The layer is preferably formed over the entire surface of the photocatalyst containing layer side light shielding part including the opening.

  The primer layer is not particularly limited as long as the primer layer is formed so that the photocatalyst containing layer side light-shielding portion of the photocatalyst containing layer side substrate is not in contact with the photocatalyst containing layer.

The material constituting the primer layer is not particularly limited, but an inorganic material that is not easily decomposed by the action of the photocatalyst is preferable. Specific examples include amorphous silica. When such amorphous silica is used, the precursor of the amorphous silica is represented by the general formula SiX ₄ and X is a silicon compound such as halogen, methoxy group, ethoxy group, or acetyl group, Silanol which is a hydrolyzate thereof or polysiloxane having an average molecular weight of 3000 or less is preferable.

  The thickness of the primer layer is preferably in the range of 0.001 μm to 1 μm, particularly preferably in the range of 0.001 μm to 0.1 μm.

b. Energy Irradiation Next, an energy irradiation method will be described. In this step, the photocatalyst-containing layer and the specific biopolymer or the immobilization layer are arranged with a gap between them, and the organic groups present in the space part are formed by irradiating energy from a predetermined direction. Can be removed. Here, when the specific biopolymer is formed, the photocatalyst-containing layer side substrate is disposed with a gap between the specific biopolymer and before the specific biopolymer immobilization step. When this step is performed, the fixing layer is disposed with a gap.

  The above arrangement in this step means a state in which the action of the photocatalyst substantially extends to the organic group, and the photocatalyst-containing layer and the specific biopolymer are in close contact with each other. In addition, the photocatalyst-containing layer and the specific biopolymer are arranged at a predetermined interval. This gap is preferably 200 μm or less.

  In the present invention, the gap is preferably in the range of 0.2 μm to 10 μm, preferably in the range of 1 μm to 5 μm, considering that the photocatalyst is highly sensitive and the organic group is efficiently decomposed. preferable. Such a gap range is particularly effective for a small-area microarray chip that can control the gap with high accuracy.

  On the other hand, when processing a microarray chip having a large area of, for example, 300 mm × 300 mm or more, it is extremely difficult to form a fine gap as described above between the photocatalyst-containing layer side substrate and the specific biopolymer. Have difficulty. Therefore, when the microarray chip has a relatively large area, the gap is preferably in the range of 10 to 100 μm, particularly in the range of 50 to 75 μm. By setting the gap within such a range, there is no problem such as a decrease in accuracy of the pattern for removing the organic group or a problem that the sensitivity of the photocatalyst is deteriorated and the efficiency for decomposing and removing the organic group is deteriorated. Moreover, it is because there is an effect that unevenness does not occur in the removal of the organic group.

  Thus, when irradiating energy to a relatively large area microarray chip, the setting of the gap in the positioning device between the photocatalyst containing layer side substrate and the transparent base material in the energy irradiation device is within a range of 10 μm to 200 μm, particularly It is preferable to set within a range of 25 μm to 75 μm. This is because by setting the set value within such a range, it is possible to arrange the photocatalyst without significantly deteriorating the sensitivity of the photocatalyst.

  Thus, by disposing the photocatalyst-containing layer and the specific biopolymer etc. at a predetermined interval, oxygen, water, and active oxygen species generated by the photocatalytic action are easily desorbed. That is, when the interval between the photocatalyst containing layer and the specific biopolymer is made narrower than the above range, it becomes difficult to desorb the active oxygen species, resulting in a slower rate of decomposition and removal of the organic group. If it is placed at a distance from the above range, it is difficult for the generated active oxygen species to reach the specific biopolymer, etc., and in this case also the rate of decomposition and removal of organic groups, etc. This is not preferable because there is a possibility of slowing down.

  As a method for uniformly forming such a very narrow gap and arranging the photocatalyst-containing layer and the specific biopolymer etc., for example, a method using a spacer can be mentioned. By using the spacer in this way, a uniform gap can be formed, and the portion in contact with the spacer does not have an action of the photocatalyst on the organic group. By having this pattern, the organic group can be decomposed and removed on the pattern of the space portion.

  In this step, such a spacer may be formed as one member. However, for simplification of the process, as described in the column of the photocatalyst containing layer side substrate, the photocatalyst of the photocatalyst containing layer side substrate is used. It is preferable to form on the surface of the containing layer. In the above description of the photocatalyst-containing layer side substrate, the photocatalyst-containing layer side light-shielding portion has been described. However, in the present invention, such a spacer does not act on the surface of a specific biopolymer or the like. Since it only needs to have a function of protecting the surface, it may be made of a material that does not have a function of shielding the energy to be irradiated.

  In the case of using a photocatalyst-containing layer side substrate formed on a flexible substrate such as a resin film in which the photocatalyst-containing layer is flexible, it is difficult to provide the gap as described above. From the viewpoint of efficiency and the like, it is preferable that the photocatalyst-containing layer and the specific biopolymer are arranged so as to contact each other.

  In the present invention, such an arrangement state with a gap need only be maintained at least during energy irradiation.

  Next, energy irradiation is performed in a state where the arrangement state as described above is maintained. The energy irradiation (exposure) as used in the present invention is a concept including irradiation of any energy beam capable of decomposing and removing an organic group by the photocatalyst containing layer, and is limited to visible light irradiation. is not.

  Usually, the wavelength of light used for such exposure is set in the range of 400 nm or less, preferably in the range of 380 nm or less. This is because, as described above, the preferred photocatalyst used in the photocatalyst-containing layer is titanium dioxide, and light having the above-described wavelength is preferable as the energy for activating the photocatalytic action by the titanium dioxide.

  Examples of light sources that can be used for such exposure include mercury lamps, metal halide lamps, xenon lamps, excimer lamps, and various other light sources.

  In addition to the method of performing pattern irradiation through a photomask using the light source as described above, it is also possible to use a method of drawing and irradiating in a pattern using a laser such as excimer or YAG. In this case, there is an advantage that a photomask is not necessary.

In addition, the amount of energy applied upon exposure is set to an amount necessary to decompose and remove the organic groups present in the space portion by the action of the photocatalyst in the photocatalyst-containing layer.
At this time, it is preferable in that the photocatalyst-containing layer is exposed while being heated, so that the sensitivity can be increased and the characteristic can be changed efficiently. Specifically, it is preferable to heat within a range of 30 ° C to 80 ° C.

  The energy irradiation direction in this step is determined by whether the photocatalyst-containing layer side substrate or the manufactured microarray chip is transparent as described above.

  That is, when the photocatalyst containing layer side light-shielding portion is formed, it is necessary to perform exposure from the photocatalyst containing layer side substrate side, and in this case, the photocatalyst containing layer side substrate is transparent to the energy irradiated. There must be.

  In addition, the exposure direction when the photocatalyst-containing layer is formed in a pattern is irradiated from any direction as long as energy is applied to the portion where the photocatalyst-containing layer and the property change layer are in contact as described above. May be.

  Similarly, in the case of using the above-described spacer, irradiation may be performed from any direction as long as energy is applied to the contact portion.

  In the case of using a photomask, energy is irradiated from the side where the photomask is arranged. In this case, the substrate on the side where the photomask is arranged, that is, either the photocatalyst-containing layer side substrate or the microarray chip needs to be transparent.

  After the energy irradiation as described above is completed, the organic group in the space part can be removed by removing the photocatalyst-containing layer side substrate from the arrangement position.

2. Second Embodiment Next, a second embodiment of the microarray chip manufacturing method of the present invention will be described. The manufacturing method of the microarray chip in this embodiment is as follows:
An immobilization layer forming step of forming an immobilization layer having a photosensitive protective group on a substrate;
A specific biopolymer immobilization step of immobilizing a specific biopolymer to a specific biopolymer immobilization part on the immobilization layer, comprising:
When a defect occurs in the specific biopolymer immobilization step, the method has a defective portion removal step of removing the specific biopolymer in the defective portion and the immobilization layer.

  For example, as shown in FIG. 7, the microarray chip manufacturing method of the present embodiment includes an immobilization layer forming step (FIG. 7A) for forming an immobilization layer 2 on a substrate 1, and the immobilization layer 2. A specific biopolymer immobilization step (FIG. 7 (b)) in which the specific biopolymer 3 (3 ′, 3 ″, 3 ″, etc.) is fixed to the specific biopolymer immobilization part a It is what has. In this embodiment, in the specific biopolymer immobilization step, for example, when the specific biopolymer 3 in the adjacent region is mixed and a defective portion 4 ′ is generated, or the target specific biopolymer is formed. When a defective portion 4 ″ formed with something different from the above occurs, a defective portion removing step (FIG. 7C) for removing the specific biopolymer 3 and the immobilization layer 2 of the defective portion 4 is performed. It is what you have.

  According to this embodiment, since the defective portion generated during the specific biopolymer immobilization step can be removed, in the manufactured microarray chip, for example, the defective portion and the test object are nonspecific. Can be prevented. In addition, when something different from the target specific biopolymer has been formed, only the defective portion is removed, and an immobilization layer is formed again on that portion to immobilize the specific biopolymer. It becomes possible. Thereby, a microarray chip without defects can be manufactured.

  Here, the defective portion removal step may be performed after the specific biopolymer immobilization step, or may be performed during the specific biopolymer immobilization step. Here, in this embodiment, for example, when a defective part occurs in a part of the target specific biopolymer immobilization part, if the defective part is removed, there is no problem when a microarray chip is obtained. In this case, it is only necessary to perform a defective part removing step for removing only the defective part. However, for example, the defective part occupies most of the specific biopolymer fixing part, and the specific biopolymer of the part is missing. Therefore, in the case where trouble is caused when the microarray chip is used, after performing the defective portion removing step for removing the defective portion, the defective portion removing portion from which the defective portion is removed, for example, as shown in FIG. A second immobilization layer forming step (FIG. 8A) for forming the second immobilization layer 6 on c is performed, and the target specific biopolymer 7 is immobilized on the second immobilization layer 6. Second specific biological height It is preferred to carry out the child fixing step (Figure 8 (b)).

  In addition, it is preferable that the microarray chip manufactured in this embodiment also has the organic group removal step of the first embodiment described above. As a result, there is no organic group on the space part of the manufactured microarray chip, and the test object can be made a high-quality microarray chip without nonspecific adsorption or the like on the space part. is there.

  Here, since the immobilization layer forming step and the specific biopolymer immobilization step in this embodiment are the same as those in the first embodiment described above, description thereof will be omitted, and in this step, hereinafter. The defective part removing step, the second immobilized layer forming step, and the second specific biopolymer immobilizing step will be described.

(Bad part removal process)
First, the defective part removing step in this step will be described. The defective portion removing step in this step is a step of removing the specific biopolymer and the immobilization layer in the defective portion generated in the above-described specific biopolymer immobilization step. As described above, this step may be performed during the specific biopolymer immobilization step, or may be performed after the specific biopolymer immobilization step. For example, when the second immobilized layer forming step and the second specific biopolymer immobilization step described later are not performed, such as when removing a portion where a small amount of the adjacent specific biopolymer is mixed, It may be performed after the specific biopolymer immobilization step or during the specific biopolymer immobilization step.

  On the other hand, when it is necessary to perform the second immobilization layer forming step and the second specific biopolymer immobilization step described later, such as when the defective portion occupies most of the specific biopolymer immobilization portion. It can be said that it is preferable to perform a defective portion removing step when loss is found to occur, with little loss.

  Here, the removal of the specific biopolymer and the immobilization layer in the defective portion removal step of the present embodiment includes not only the case where the specific biopolymer and the immobilization layer are completely removed, but also one of the immobilization layers. It also includes the case where the part remains. For example, as described above, when the portion where the immobilization layer is in close contact with the substrate is made of an inorganic material, the portion of the inorganic material may be completely removed or the inorganic material may remain. .

  The method for removing the defective portion in this step is not particularly limited as long as it is a method capable of removing the defective portion, and is performed by a commonly performed photolithography method or the like. For example, the specific biopolymer or the immobilization layer may be removed by irradiating ultraviolet light having a short wavelength in a pattern.

  In this embodiment, in particular, for example, as shown in FIG. 3, the photocatalyst containing layer 11 of the photocatalyst containing layer side substrate 13 having the photocatalyst containing layer 11 and the base 12 containing the photocatalyst is used as the specific biopolymer 3. It is preferable to perform the irradiation by irradiating energy 15 using, for example, a photomask 14 or the like after being arranged with a gap.

  This eliminates the need to use chemicals that adversely affect other parts of specific biopolymers, such as alkaline developers used in photolithography, for example. Moreover, by irradiating the defective portion with energy using the photocatalyst-containing layer side substrate, it is possible to easily decompose and remove organic groups, etc. This is because the molecule and the immobilization layer can be removed.

  Here, the photocatalyst-containing layer side substrate used in the present embodiment and the energy to be irradiated are the same as those used in the organic group removal step of the first embodiment described above, and thus the description thereof is omitted here. To do.

(Second immobilized layer forming step)
Next, the 2nd fixed layer formation process in this embodiment is demonstrated. The second immobilization layer forming step in the present embodiment is the same as the second immobilization layer forming step formed in the immobilization layer forming step in the defective portion removing portion from which the defective portion has been removed after the defective portion removing step. This is a step of forming an immobilization layer. By forming the second immobilization layer in this step, a specific biopolymer can be newly attached on the defective portion removal portion.

  The second fixing layer formed by the second fixing layer forming step is the same as the above-described fixing layer, and the formation method can be the same, and detailed description here. Is omitted.

(Second specific biopolymer immobilization step)
Next, the second specific biopolymer immobilization step in this embodiment will be described. In the second specific biopolymer immobilization step in this embodiment, the specific immobilization in the specific biopolymer immobilization step is performed on the second immobilization layer formed by the second immobilization layer formation step. This is a step of immobilizing a specific biopolymer similar to the biopolymer.

  The method for immobilizing the specific biopolymer in this step is not particularly limited as long as it can immobilize the specific biopolymer on the second immobilization layer. Since it can be the same as that of the biopolymer immobilization step, detailed description here is omitted.

B. Next, the microarray chip substrate of the present invention will be described. The substrate for microarray chip of the present invention has a base material and an immobilization layer formed on the base material and containing a photosensitive protective group, and the immobilization layer immobilizes a specific biopolymer. It is formed in a pattern of a specific biopolymer immobilization part which is a region.

  The microarray chip substrate of the present invention has an immobilization layer 2 on a base material 1 as shown in FIG. 9, for example, and this immobilization layer 2 has a specificity for immobilizing a specific biopolymer. The biopolymer fixing part a is formed in a pattern.

  In a general microarray chip substrate, since an immobilization layer is formed between adjacent specific biopolymer immobilization parts, when immobilizing a specific biopolymer by a photosensitive polymer polymerization method, There may be a case where a specific biopolymer adheres to this part or a case where adjacent specific biopolymers are mixed. In such a case, when the microarray chip is used between the adjacent specific biopolymer immobilization parts or in a part where different specific biopolymers are mixed, the test object is nonspecifically adsorbed. There are problems that the accuracy of the microarray chip is lowered and the contrast is lowered. In addition, when the organic group of the immobilization layer is present, there is a problem that the test object similarly adsorbs nonspecifically.

  Therefore, in the present invention, the immobilization layer is formed in the shape of the specific biopolymer immobilization part to which such a specific biopolymer is attached. Therefore, for example, when the specific biopolymer is immobilized on the immobilization layer by a photosensitive polymer polymerization method or the like, the immobilization layer is not formed in a portion other than the specific biopolymer immobilization portion. The specific biopolymer does not adhere and the organic group of the immobilization layer does not exist. As a result, it is possible to obtain a high-quality microarray chip that does not have a decrease in accuracy or a decrease in contrast as described above.

  Here, the specific biopolymer immobilization part is an area for immobilizing the specific biopolymer as described above, and a different specific biopolymer is usually immobilized for each specific biopolymer immobilization part. It will be

  Here, the gap between adjacent specific biopolymer immobilization parts is preferably 1 μm to 2,000 μm, more preferably 2 μm to 150 μm, and particularly preferably 5 μm to 75 μm. By setting the gap between adjacent specific biopolymer immobilization parts like this, when specific biopolymers are immobilized by a photosensitive polymer polymerization method, etc., different specific biopolymers may be mixed together. This is because no defective portion can be generated. Moreover, since it is in such a range, it can also be said that the advantage of this invention can be utilized.

  As a method for forming such an immobilization layer, for example, an immobilization layer can be formed on the entire surface of a substrate, and this immobilization layer can be patterned. Such a patterning method may be, for example, a general photolithography method or irradiation with light having high energy. In the present invention, in particular, the photocatalyst-containing layer containing at least a photocatalyst is provided. It is preferable that the photocatalyst-containing layer side substrate is used to irradiate energy to decompose and remove the fixed layer in the target region by the action of the photocatalyst accompanying energy irradiation. This eliminates the need to use a material that adversely affects the immobilization layer or the specific biopolymer, such as an alkali developer in a photolithography method. Another reason is that the immobilization layer can be efficiently removed without using light having high energy.

  In addition, about the base material used for this embodiment, an immobilization layer, the formation method of the immobilization layer, the patterning method of an immobilization layer, etc. which were demonstrated by "A. manufacturing method of a microarray chip" mentioned above. The description here is omitted.

C. Next, the microarray chip of the present invention will be described. The microarray chip of the present invention is obtained by immobilizing a specific biopolymer on the immobilization layer of the above-described microarray chip substrate.

  According to the present invention, since the immobilization layer is formed in the shape of the specific biopolymer immobilization part as described above, the specific biopolymer is interposed between the adjacent specific biopolymer immobilization parts. It can be a microarray chip that is not attached.

  As a result, when the microarray chip is used, it is possible to obtain a high-quality microarray chip without nonspecific adsorption due to organic substances or the like attached between the specific biopolymer immobilization parts.

  Here, the specific biopolymer used in the microarray chip of the present invention, the formation method thereof, and the like are the same as those described in the above-mentioned “A. Microarray chip manufacturing method”. Is omitted.

  The present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.

  The following examples illustrate the present invention more specifically.

(Production of microarray chip)
First, surface organic substances were removed by piranha reagent and washing, and a 3-glycidoxypropyltrimethoxysilane solution dissolved in isopropyl alcohol to 1% was coated on a cleaned glass substrate at a spinner of 1500 rpm for 3 minutes. What was dried at 10 ° C. for 10 minutes was rinsed in a 0.1 M hydrochloric acid aqueous solution for 2 hours. Then, it washed with water and dried at 110 degreeC for 30 minutes, and was set as the fixed layer.
Next, hexaethylene glycol-cyanoethyl phosphoramidide prepared by reacting hexaethylene glycol with a phosphoramidide reagent in acetonitrile was adjusted to 100 mM, the substrate was rinsed and coupled for 30 minutes, washed with water, and dried. I let you.
Subsequently, as a photosensitive protecting group, rinveratyloxycarbonyl 100 mM prepared using vanillin as a starting material and pyridine 100 mM in tetrahydrofuran were rinsed and coupled for 2 minutes to protect the hydroxyl group on the substrate, followed by washing with ethanol and drying.
This substrate was again immersed in tetrahydrofuran, half of the substrate was masked, and ultraviolet light of 365 nm was irradiated with 20,000 μW / cm ² / nm for 5 minutes. Next, the substrate was replaced with a pyridine solution of 100 mM of protected adenosine phosphoramidide reagent and 5 mM of allenesulfonyltriazole, and this substrate was rinsed for 2 minutes, washed with ethanol and dried.
Further, the adenosine protecting group on the substrate was rinsed and removed with a 5% trichloroacetic acid aqueous solution for 10 minutes, washed with water and dried, and then rinsed with ethanol in a pyridine solution adjusted to 100 mM fluorescein phosphoramidide reagent and 5 mM allenesulfonyltriazole. Washed and dried to prepare a substrate in which a nucleic acid and a fluorescent reagent were linked by phosphoramidide synthesis and photosensitive polymer synthesis. When this substrate was irradiated with UV and fluorescein was excited to emit light, fluorescence was observed only on the half surface of the substrate where the photosensitive protecting group was deprotected.

(Preparation of photocatalyst-containing layer side substrate)
5 g of trimethoxymethylsilane (GE Toshiba Silicone Co., Ltd., TSL8113) and 2.5 g of 0.5N hydrochloric acid were mixed and stirred for 8 hours. This was diluted 10 times with isopropyl alcohol to obtain an anchor composition.
The anchor layer composition was applied onto a 20 μm line & space photomask substrate by a spin coater and dried at 150 ° C. for 10 minutes to form a transparent anchor layer (thickness 0.2 μm). .
Next, 30 g of isopropyl alcohol, 3 g of trimethoxymethylsilane (manufactured by GE Toshiba Silicone Co., Ltd., TSL8113) and 20 g of ST-K03 (manufactured by Ishihara Sangyo Co., Ltd.), which is a photocatalytic inorganic coating agent, are mixed at 100 ° C. Stir for 20 minutes. This was diluted 3 times with isopropyl alcohol to obtain a composition for a photocatalyst-containing layer.
A transparent photocatalyst-containing layer (thickness: 0.15 μm) is formed by applying the photocatalyst-containing layer composition on a photomask substrate on which an anchor layer is formed by a spin coater and performing a drying treatment at 150 ° C. for 10 minutes. Formed.

(Organic group removal)
The substrate and the photocatalyst-containing layer of the photocatalyst substrate were opposed to each other with a gap of 50 μm, and then ultraviolet light with a wavelength of 255 to 350 nm was irradiated for 30 seconds with an illuminance of 20,000 μW / cm ² / nm. Washed with water and dried.

(Evaluation)
When this substrate was subjected to excitation emission of fluorescein by UV irradiation, no excitation emission was seen from the exposed location.
In addition, this substrate was subjected to 45 ° incident reflection under a dark place microscope and observed at 90 °. As a result, scattered light was observed from an unexposed location, and the exposed location was equivalent to the untreated clean glass surface. A state without scattered light was observed.

It is process drawing which shows an example of the manufacturing method of the microarray chip | tip of this invention. It is process drawing which shows the other example of the manufacturing method of the microarray chip | tip of this invention. It is a schematic sectional drawing which shows an example of the organic group removal process in the manufacturing method of the microarray chip | tip of this invention. It is a schematic sectional drawing which shows an example of the photocatalyst containing layer side board | substrate used for the organic group removal process in the manufacturing method of the microarray chip | tip of this invention. It is a schematic sectional drawing which shows the other example of the photocatalyst containing layer side board | substrate used for the organic group removal process in the manufacturing method of the microarray chip | tip of this invention. It is a schematic sectional drawing which shows the other example of the photocatalyst containing layer side board | substrate used for the organic group removal process in the manufacturing method of the microarray chip | tip of this invention. It is process drawing which shows the other example of the manufacturing method of the microarray chip | tip of this invention. It is process drawing which shows an example of the 2nd fixed layer formation process and the 2nd specific biopolymer fixation process in the manufacturing method of the microarray chip | tip of this invention. It is a schematic sectional drawing which shows an example of the board | substrate for microarray chips of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Base material 2 ... Immobilization layer 3 ... Specific biopolymer 4 ... Defect part 6 ... 2nd immobilization layer 7 ... Specific biopolymer 11 ... Photocatalyst containing layer 12 ... Base | substrate 13 ... Photocatalyst containing layer side board | substrate 15 …energy

Claims (3)

An immobilization layer forming step of forming an immobilization layer having a photosensitive protective group on a substrate;
A specific biopolymer immobilization step of immobilizing a specific biopolymer to a specific biopolymer immobilization part on the immobilization layer, comprising:
Have a organic group removing step of removing only the organic groups present in the space portion between the specific biopolymers fixed part,
After the organic group removal step, the photocatalyst containing layer of the photocatalyst containing layer side substrate having a photocatalyst containing layer and a substrate containing a photocatalyst is disposed with a gap from the immobilization layer or the specific biopolymer, A method of manufacturing a microarray chip, which is performed by irradiating energy . An immobilization layer forming step of forming an immobilization layer having a photosensitive protective group on a substrate;
A specific biopolymer immobilization step of immobilizing a specific biopolymer to a specific biopolymer immobilization part on the immobilization layer, comprising:
When a defect in the specific biopolymer immobilization process occurs, it has a defective portion removing step of removing only the specific biopolymer and the fixed layer of the defective portion,
After the defective portion removing step, the photocatalyst-containing layer of the photocatalyst-containing layer side substrate having a photocatalyst-containing layer and a substrate containing the photocatalyst is disposed with a gap from the immobilized layer or the specific biopolymer, A method of manufacturing a microarray chip, which is performed by irradiating energy . A second immobilization layer forming step of forming a second immobilization layer having a photosensitive protective group on the defective portion removal portion from which the defective portion has been removed by the defective portion removal step;
The method for producing a microarray chip according to claim 2, further comprising a second specific biopolymer immobilization step of immobilizing the specific biopolymer on the second immobilization layer.

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