US20100233429A1 - Substrate for Biochip, Biochip, Method for Manufacturing Substrate for Biochip and Method for Manufacturing Biochip - Google Patents

Abstract

A base plate having a surface on which a plurality of hydroxyl groups can be introduced, a metallic membrane disposed on the base plate and having a plurality of wells reaching the base plate, and a crosslinkable polymer membrane disposed on the metallic membrane are included.

Description

TECHNICAL FIELD
• This invention relates to a technique for detecting a biomolecule, especially to a substrate for a biochip, the biochip, a method for manufacturing the substrate for the biochip, and a method for manufacturing the biochip.
BACKGROUND ART
• “Biochip” is the generic term for devices in which probe biomolecules that react with to-be-detected target biomolecules in a specific manner are fixed at predetermined positions on a chip surface. A deoxyribonucleic acid (DNA) chip that is a typical example of the biochip is used to detect the types and amounts of target DNA included in blood or cell extract. The DNA chip has, for example, a structure in which thousands to tens of thousands of types of probe DNA, each being single-chain DNA having a known sequence, are arranged in an array on a substrate such as a glass slide.
• When a to-be-examined liquid containing fluorescence-marked target DNA is supplied to the DNA chip, only the target DNA which has sequences complementary to the sequences of the probe DNA is bonded to the probe DNA by hydrogen-bonding to form a complementary double chain. As a result, the parts to which the target DNA is fixed is fluorescent-colored. By measuring the position and coloring intensity of the fluorescent-colored parts on the chip, the types and amounts of the target DNA can be detected. Therefore, as described in a published Japanese translations of PCT international publication for patent applications 2002-537869, when the DNA chip is manufactured, the probe DNA having a predetermined sequence should be fixed only on a predetermined portion on a surface of the substrate. However, it is difficult to fix the probe DNA only in the specific location. Therefore, the target DNA bonded to the probe DNA fixed in location other than the specific location becomes background noise in a detection process. It becomes a factor to decrease a detection accuracy of the DNA chip.
DISCLOSURE OF INVENTION
• By a first aspect of present invention, a substrate for a biochip comprising a base plate having a surface on which a plurality of hydroxyl groups can be introduced, a metallic membrane disposed on the base plate and having a plurality of wells reaching the base plate, and a crosslinkable polymer membrane disposed on the metallic membrane is provided.
• By a second aspect of present invention, a biochip comprising a base plate, a metallic membrane disposed on the base plate and having a plurality of wells reaching the base plate, and a plurality of probe biomolecules bonded on a surface of the base plate exposed from the plurality of wells, is provided.
• By a third aspect of present invention, a method for manufacturing a substrate for a biochip including a step for preparing a base plate having a surface on which a plurality of hydroxyl groups can be introduced, a step for forming a metallic membrane on the base plate, a step for forming a crosslinkable polymer membrane on the metallic membrane, a step for selectively removing portions of the polymer membrane, and a step for delineating a plurality of wells reaching the base plate in the metallic membrane, by using the polymer membrane of which the portions were selectively removed as an etching mask is provided.
• By a fourth aspect of present invention, a method for manufacturing a biochip including a step for preparing a base plate, a step for forming a metallic membrane on the base plate, a step for forming a crosslinkable polymer membrane on the metallic membrane, a step for selectively removing portions of the polymer membrane, a step for delineating a plurality of wells reaching the base plate in the metallic membrane, by using the polymer membrane of which the portions were selectively removed as an etching mask, a step for introducing a plurality of hydroxyl groups on a surface of the base plate exposed from the plurality of wells, a step for bonding a plurality of probe biomolecules to the plurality of hydroxyl groups, respectively, and a step for soaking the metallic membrane and the polymer membrane in an alkaline solution to peel off the polymer membrane from the metallic membrane is provided.
• By a fifth aspect of present invention, a method for manufacturing a biochip including a step for preparing a substrate for the biochip comprising a base plate, a metallic membrane disposed on the base plate and having a plurality of wells reaching the base plate, a crosslinkable polymer membrane disposed on the metallic membrane, and a plurality of hydroxyl groups introduced on a surface of the base plate exposed from the plurality of wells, a step for bonding a plurality of probe biomolecules to the plurality of hydroxyl groups, respectively, and a step for soaking the metallic membrane and the polymer membrane in an alkaline solution to peel off the polymer membrane from the metallic membrane is provided.
• By a sixth aspect of present invention, a substrate for a biochip comprising a base plate having a surface on which a plurality of hydroxyl groups can be introduced, and a cover member disposed on the base plate when probe biomolecules are bonded to the plurality of hydroxyl groups, respectively, the cover member having a plurality of through holes to define binding regions where the probe biomolecules are bonded to the surface of the base plate, is provided.
• By a seventh aspect of present invention, a biochip comprising an optical transparency base plate, a light shielding film disposed on the base plate and having a plurality of through holes reaching the base plate, and a plurality of probe biomolecules bonded to the base plate exposed from each of the plurality of through holes is provided.
• By an eighth aspect of present invention, a biochip comprising an optical transparency base plate, a light shielding film disposed on a first surface of the base plate and having a plurality of through holes reaching the first surface, and a plurality of probe biomolecules bonded to a second surface of the base plate opposite to the first surface of the base plate is provided.
• By a ninth aspect of present invention, a biochip comprising an optical transparency base member, a plurality of probe biomolecules bonded on the base member, and a light shielding member disposed around the base member is provided.
BRIEF DESCRIPTION OF DRAWINGS
• FIG. 1 shows a top view of a substrate for a biochip according to a first embodiment of the present invention.
• FIG. 2 shows a sectional view of the substrate for the biochip according to the first embodiment of the present invention.
• FIG. 3 shows chemical formulas of compositions of polymer membrane according to the first embodiment of the present invention.
• FIG. 4 shows a first sectional process drawing of the substrate for the biochip according to the first embodiment of the present invention.
• FIG. 5 shows a second sectional process drawing of the substrate for the biochip according to the first embodiment of the present invention.
• FIG. 6 shows a third sectional process drawing of the substrate for the biochip according to the first embodiment of the present invention.
• FIG. 7 shows a top view of a biochip according to a second embodiment of the present invention.
• FIG. 8 shows a first sectional view of the biochip according to the second embodiment of the present invention.
• FIG. 9 shows an first enlarged sectional view of the biochip according to the second embodiment of the present invention.
• FIG. 10 shows a second sectional view of the biochip according to the second embodiment of the present invention.
• FIG. 11 shows a second enlarged sectional view of the biochip according to the second embodiment of the present invention.
• FIG. 12 shows a third enlarged sectional view of the biochip according to the second embodiment of the present invention.
• FIG. 13 shows a first sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 14 shows a second sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 15 shows a third sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 16 shows a fourth sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 17 shows a chemical formula of nucleoside phosphoramidite according to the second embodiment of the present invention.
• FIG. 18 shows first chemical formulas of bases of the nucleosides according to the second embodiment of the present invention.
• FIG. 19 shows a fifth sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 20 shows a sixth sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 21 shows a seventh sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 22 shows an eighth sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 23 shows a ninth sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 24 shows a tenth sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 25 shows second chemical formulas of the bases of the nucleosides according to the second embodiment of the present invention.
• FIG. 26 shows an eleventh sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 27 shows a twelfth sectional process drawing of the biochip according to the second embodiment of the present invention.
• FIG. 28 shows a first sectional process drawing of the biochip according to a modification of the second embodiment of the present invention.
• FIG. 29 shows a second sectional process drawing of the biochip according to the modification of the second embodiment of the present invention.
• FIG. 30 shows a top view of the substrate for the biochip according to a third embodiment of the present invention.
• FIG. 31 shows a sectional view of the substrate for the biochip according to the third embodiment of the present invention.
• FIG. 32 shows a top view of the biochip according to a fourth embodiment of the present invention.
• FIG. 33 shows a first sectional view of the biochip according to the fourth embodiment of the present invention.
• FIG. 34 shows a second sectional view of the biochip according to the fourth embodiment of the present invention.
• FIG. 35 shows a first sectional process drawing of the biochip according to the fourth embodiment of the present invention.
• FIG. 36 shows a second sectional process drawing of the biochip according to the fourth embodiment of the present invention.
• FIG. 37 shows a third sectional process drawing of the biochip according to the fourth embodiment of the present invention.
• FIG. 38 shows a fourth sectional process drawing of the biochip according to the fourth embodiment of the present invention.
• FIG. 39 shows a fifth sectional process drawing of the biochip according to the fourth embodiment of the present invention.
• FIG. 40 shows a sixth sectional process drawing of the biochip according to the fourth embodiment of the present invention.
• FIG. 41 shows a third sectional view of the biochip according to the fourth embodiment of the present invention.
• FIG. 42 shows a first sectional process drawing of the biochip according to a modification of the fourth embodiment of the present invention.
• FIG. 43 shows a second sectional process drawing of the biochip according to the modification of the fourth embodiment of the present invention.
• FIG. 44 shows a top view of the biochip according to a fifth embodiment of the present invention.
• FIG. 45 shows a first sectional view of the biochip according to the fifth embodiment of the present invention.
• FIG. 46 shows a first sectional process drawing of the biochip according to the fifth embodiment of the present invention.
• FIG. 47 shows a second sectional process drawing of the biochip according to the fifth embodiment of the present invention.
• FIG. 48 shows a second sectional view of the biochip according to the fifth embodiment of the present invention.
• FIG. 49 shows a third sectional view of the biochip according to the fifth embodiment of the present invention.
• FIG. 50 shows a top view of the biochip according to a sixth embodiment of the present invention.
• FIG. 51 shows a first sectional view of the biochip according to the sixth embodiment of the present invention.
• FIG. 52 shows a first sectional process drawing of the biochip according to the sixth embodiment of the present invention.
• FIG. 53 shows a second sectional process drawing of the biochip according to the sixth embodiment of the present invention.
• FIG. 54 shows a third sectional process drawing of the biochip according to the sixth embodiment of the present invention.
• FIG. 55 shows a fourth sectional process drawing of the biochip according to the sixth embodiment of the present invention.
• FIG. 56 shows a second sectional view of the biochip according to the sixth embodiment of the present invention.
• FIG. 57 shows a top view of the biochip according to other embodiment of the present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
• Embodiments of the present invention will be described with reference to the accompanying drawings. It is to be noted that the same or similar reference numerals are applied to the same or similar parts. It should be noted that the drawing are typical ones. Therefore, concrete sizes should be determined by referring the following description, for example. Also, it is a matter of course that portions of which relationship between sizes and ratios of mutual drawings is different are included.
First Embodiment
• With reference toFIG. 1 andFIG. 2 that is a sectional view taken on line II-II, a substrate for a biochip according to a first embodiment of the present invention includes a base plate15 having a surface on which a plurality of hydroxyl groups (—OH) can be introduced, and a metallic membrane13 disposed on the base plate15, wherein a plurality of wells41a,41b,41c,41d,41e,41f,41g,41h,41i,42a,42b,42c,42d,42e,42f,42g,42h,42i,43a,43b,43c,43d,43e,43f,43g,43h,43i,44a,44b,44c,44d,44e,44f,44g,44h,44i,45a,45b,45c,45d,45e,45f,45g,45h,45i,46a,46b,46c,46d,46e,46f,46g,46h,46i,47a,47b,47c,47d,47e,47f,47g,47h,47i,48a,48b,48c,48d,48e,48f,48g,48h,48i,49a,49b,49c,49d,49e,49f,49g,49h,49ithat reach the base plate15 are delineated in the metallic membrane13. The film thickness of thebase plate15 is 550 micrometers, for example. Synthetic quarts (SiO₂) and a silicon substrate having a surface on which a natural oxide film is formed, etc., can be used for the material of thebase plate15. The film thickness of themetallic membrane13 is from 10 nm to 10 micrometers, and preferably 50 nm, for example. Transition metal such as Titan (Ti), platinum (Pt), chrome (Cr), niobium (Nb), tantalum (Ta), tungsten (W), etc., or metal such as aluminum (Al), gold (Au), etc., can be used for a material of themetallic membrane13. Especially, Ti is preferable. In addition, transition metal oxide such as titanium monoxide (TiO), titanium dioxide (TiO₂) etc., transition metal nitride such as titanium nitride (TiN), etc., and transition metal carbide such as titanium carbide (TiC) can be used for the material of themetallic membrane13. Also, ITO (Indium Tin Oxide), etc., can be used for the material of themetallic membrane13.
• Further, the substrate for the biochip according to the first embodiment includes acrosslinkable polymer membrane11 disposed on themetallic membrane13. Thepolymer membrane11 is insoluble in an acid solution such as a mixed solution of hydrofluoric acid (HF), nitric acid (HNO₃), and water (H₂O). The film thickness is micrometers. An epoxy resin that is photosensitive to ultraviolet rays cab be used for a material of thepolymer membrane11. For example, SU-8-3000 series of KAYAKU MICROCHEM Corp. is preferable. As shown inFIG. 3, thepolymer membrane11 is composed of a plurality ofepoxy resins111a,111b,111c,111dthat are cross-linked to each other.
• When the biochip is manufactured by using the substrate for the biochip described above and shown inFIG. 1 toFIG. 3, as mentioned below with reference toFIG. 26, after probe biomolecules are introduced on a surface of thebase plate15 exposed from the plurality of wells41a-41i,42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49iand amino groups in the probe biomolecules are deprotected by an alkaline solution, it is possible to easily peel off thepolymer membrane11 from themetallic membrane13. Especially, by using the SU-8 as the material of thepolymer membrane11, it becomes more easily to peel off it from themetallic membrane13. After the probe biomolecules are introduced, thepolymer membrane11 can be easily peeled off from themetallic membrane13. Therefore, it is possible to inhibit the probe biomolecules from being introduced on the surface ofmetallic membrane13. Accordingly, the probe biomolecules are introduced only on the surface of thebase plate15 exposed from the plurality of wells41a-41i,42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49i, and the probe biomolecules do not adhere to the surface of themetallic membrane13. Consequently, in a process for detecting target biomolecules, it is possible to cancel background noise. By adopting a combination of thepolymer membrane11 and themetallic membrane13, such phenomenon of easy peel-off after the probe biomolecules are introduced can be observed. Therefore, in the substrate for the biochip according to the first embodiment, themetallic membrane13 is disposed on thebase plate15, and thepolymer membrane11 is further disposed on themetallic membrane13.
• Next, with reference toFIG. 4 toFIG. 6, a method for manufacturing the substrate for the biochip according to the first embodiment is described.
• (a) As shown inFIG. 4, thebase plate15 composed of SiO₂, for example, is prepared and themetallic membrane13 composed of Ti, for example, is formed on thebase plate15 by using a sputtering method or a chemical vapor deposition (CVD) method. After themetallic membrane13 is formed, the surface is treated with oxygen (O₂) plasma for 5 minutes. Next, as shown inFIG. 5, a solution including the photosensitive epoxy resin such as SU-8-3000, etc., is spin coated on themetallic membrane13 to form thepolymer membrane11. As to the condition of spin coating, it is accelerated to 300 revolutions per minute by taking 5 seconds, and 300 revolutions per minute is maintained for 10 seconds, for example. Further, it is accelerated to 500 revolutions per minute by taking 5 seconds, and 500 revolutions per minute is maintained for 15 seconds. Thereafter, it is accelerated to 4500 revolutions per minute by taking 5 seconds, and the 4500 revolutions per minute is maintained for 30 seconds. Finally, the spin is halted by taking 5 seconds.
• (b) After thepolymer membrane11 is formed, thepolymer membrane11 is pre-baked. First, thebase plate15 is disposed on a hot plate set to 65 degrees C. After a lapse of 2 minutes, the hot plate is set to 80 degrees C. After a lapse of 20 minutes, the hot plate is set to 95 degrees C. and it is preserved for 15 minutes. After a lapse of 15 minutes, the hot plate is turned off and it is preserved for 1 hour. Thereafter, by using a photomask having a mask pattern corresponding to shapes of the plurality of wells41a-41i,42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49ishown inFIG. 1, portions of thepolymer membrane11 are selectively exposed to the ultraviolet rays. After the exposure, post exposure bake (PEB) for thepolymer membrane11 is performed. Concretely, thebase plate15 is disposed on the hot plate set to 65 degrees C. After a lapse of 2 minutes, the hot plate is set to 95 degrees C. and it is preserved for 6 minutes. After a lapse of 6 minutes, the hot plate is turned off and it is preserved for 1 hour. Thereafter, thepolymer membrane11 is developed by using a SU-8 developer solution, for example. Since thepolymer membrane11 has photosensitivity, the portions of thepolymer membrane11 are selectively removed, as shown inFIG. 6.
• (c) By using thepolymer membrane11 of which the portions were selectively removed as an etching mask, portions of themetallic membrane13 are selectively removed by isotopic wet etching. The mixture solution of hydrofluoric acid (HF), nitric acid (HNO₃), and water (H₂O), for example, can be used for the etching solution. By the selective removal, each of the plurality of wells41a-41i,42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49i, shown inFIG. 2, are formed and the method for manufacturing the substrate for the biochip according to the first embodiment is completed.
• By adopting the method for manufacturing the substrate for the biochip according to the first embodiment described above, thecrosslinkable polymer membrane11 composed of SU-8-3000, etc., has strong solvent resistance against the etching solution. Therefore, there is no need to remove thepolymer membrane11 after the wet etching and to form a new protective film on themetallic membrane13. Consequently, thepolymer membrane11 can be used as the etching mask for delineating each of the plurality of wells41a-41i,42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49iand can be utilized for a protective film of themetallic membrane13 after the etching process. It should be noted that O₂plasma process after formation of themetallic membrane13 can be eliminated.
Second Embodiment
• With reference toFIG. 7 andFIG. 8 that is a sectional view taken on line VIII-VIII, the biochip according to a second embodiment of the present invention includes the base plate15, and the metallic membrane13 disposed on the base plate15, wherein the plurality of wells41a,41b,41c,41d,41e,41f,41g,41h,41i,42a,42b,42c,42d,42e,42f,42g,42h,42i,43a,43b,43c,43d,43e,43f,43g,43h,43i,44a,44b,44c,44d,44e,44f,44g,44h,44i,45a,45b,45c,45d,45e,45f,45g,45h,45i,46a,46b,46c,46d,46e,46f,46g,46h,46i,47a,47b,47c,47d,47e,47f,47g,47h,47i,48a,48b,48c,48d,48e,48f,48g,48h,48i,49a,49b,49c,49d,49e,49f,49g,49h,49ithat reach the base plate15 are delineated in the metallic membrane13. Each film thickness and each material of thebase plate15 and themetallic membrane13 are similar to the substrate for the biochip shown inFIG. 1. So, an explanation is omitted.
• With reference toFIG. 8, the biochip according to the second embodiment further includes a plurality ofbiomaterial films91a,91b,91c,91d,91e,91f,91g,91h,91idisposed on the surface of thebase plate15 exposed from the plurality of wells41a-41i, respectively. In each of the plurality of biomaterial films91a-91i, each functional group of a plurality of probe biomolecules such as a plurality of DNAs, a plurality of ribonucleic acids (RNAs), a plurality of peptide nucleic acids (PNAs), a plurality of proteins, etc., is covalently bonded to the hydroxyl group (—OH) on the surface of the base plate, as shown inFIG. 9. In the case where the probe biomolecules are DNAs, RNAs, or PNAs, each sequence is designed to be complementary to a target biomolecule.
• It should be noted that the second embodiment is not limited to directly disposing each of the plurality of biomaterial films91a-91ion the surface of thebase plate15. In the case shown inFIG. 10,silane coupling films81a,81b,81c,81d,81e,81f,81g,81h,81iare disposed on the portions of the surface of thebase plate15 exposed from the plurality of wells41a-41i, respectively. In each of the silane coupling films81a-81i, as shown inFIG. 11, each methyl group (—CH₃) or each ethyl group (—C₂H₅) of a plurality of silane coupling agents is chemically bounded to the hydroxyl group (—OH) on the surface of thebase plate15 by acid-base reaction.
• 3-Glycidoxypropyltrimethoxysilane, 3-Glycidoxypropylmethyldiethoxysilane, 3-Glycidoxypropyltriethoxysilane, N-(2-Aminoethyl)-3-Aminopropylmethyldimethoxysilane, N-(2-Aminoethyl)-3-Aminopropyltrimethoxysilane, N-(2-Aminoethyl)-3-Aminopropyltriethoxysilane, 3-Aminopropyltrimethoxysilane, 3-Aminopropyltriethoxysilane, etc., can be used for each of the plurality of silane coupling agents.
• Thebiomaterial films91a,91b,91c,91d,91e,91f,91g,91h,91iare disposed on the silane coupling films81a-81i, respectively. In each of the plurality of biomaterial films91a-91i, each phosphoramidite derivative introduced in the plurality of probe biomolecules is covalently bonded to each epoxy group of the silane coupling agents in the silane coupling films81a-81i, by hydrolysis reaction.
• Alternatively, the silane coupling agent and the probe biomolecule may bond via cross linker. For example, a functional group such as amino group (—NH₂) of lysine (Lys), carboxyl group (—COOH) of aspartic acid (Asp) and glutamic acid (Glu), phenolic group (—C₆H₄(OH)) of tyrosine (Tyr), imidazole group (—C₃H₃N₂) of histidine (His), thiol group (—SH) of cysteine (Cys), etc., included in the protein such as a receptor, ligand, antagonist, antibody, antigen, etc., may be bonded to the amino group or the epoxy group of the silane coupling agent by the cross linker. In the case shown inFIG. 12, the amino group (—NH₂) of the silane coupling agent and each amino group (—NH₂) of theantibodies95a,95b,95care bonded by a Disuccinimidyl suberate (DSS) that is the cross linker reacting to the amino groups (—NH₂) at both terminals.
• In addition, Bis[Sulfosuccinimidyl]suberate (BS³), Dimethyl suberimidate HCl DMS), Disuccinimidyl glutarate (DSG), Loman's Reagent, 3,3′-Dithiobis [sulfosuccinimidyl propionate] (DTSSP), and Ethylene glycol bis[succinimidylsuccinate] (EGS) that react to the amino groups at both terminals, and 1-Ethyl-3-[3-Dimethylaminopropyl]carbodiimide Hydrochloride (EDC) that react to the amino group and the carboxyl group can be used for the cross linker.
• In addition, m-Maleimidobenzyl-N-hydroxysuccinimide ester (MBS), Succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC), Succinimidyl 4-[p-maleimidophenyl]-buthrate (SMPB), N-Succinimidyl 3-[2-pyridyldithio]propionate (SPDP), N-[γ-Maleimidobutyloxy]sulfosuccinimide ester (Sulfo-GMBS), Sulfosuccinimidyl 6-[3′ (2-pyridyldithio)-propionamide]hexanoate (Sulfo-LC-SPDP), m-Maleimidebenzoyl-N-hydroroxysulfo-succinimide ester (Sulfo-MBS), Sulfosuccinimidyl 4 [N-maleimidomethyl]-cyclohexane-1-carboxylate (Sulfo-SMCC), and Sulfosuccinimidy 4-[p-maleimidophenyl]-butyrate (Sulfo-SMPB) that react to the amino group and the thiol group can also be used as the cross linker. It should be noted that each sectional view of the other plurality of wells42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49ishown inFIG. 7 is similar toFIG. 8 toFIG. 12. So, an explanation is omitted.
• Next, with reference toFIG. 13 toFIG. 27, a method for manufacturing the biochip according to the second embodiment is described.
• (a) First, the substrate for the biochip shown inFIG. 1 andFIG. 2 is prepared. Next, the substrate for the biochip is left in a stirred sodium hydroxide (NaOH) solution at room temperature for 2 hours. Here, the NaOH solution is a solution obtained by mixing98gof NaOH, 294 ml of distilled water, and 392 ml of ethanol. By leaving it in the NaOH solution, the plurality of hydroxyl groups (—OH) are introduced on the surface ofbase plate15 exposed from each of the wells41a-41i, as shown inFIG. 13. It should be noted that the hydroxyl groups (—OH) may also be introduced by using a UV ozone cleaner, for example.
• (b) For example, the silane coupling agent having the epoxy group as the functional group such as 3-Glycidoxypropylmethyldiethoxysilane 3-Glycidoxypropyltriethoxysilane, etc., or the silane coupling agent having the amine as the functional group such as N-(2-Aminoethyl)-3-Aminopropyltriethoxysilane, 3-Aminopropyltrimethoxysilane, 3-Aminopropyltriethoxysilane, etc., is dropped on the surface of thebase plate15 exposed from each of the wells41a-41ishown inFIG. 2 to form each of thesilane coupling films81a,81b,81c,81d,81e,81f,81g,81h,81ishown inFIG. 14. For example, when the silane coupling agent having the epoxy group is dropped under 15 degrees C. condition, the plurality of epoxy groups are introduced on the surface of thebase plate15, as shown inFIG. 15. In the case where themetallic membrane13 shown inFIG. 14 is opaque, it becomes easy to determine the locations of wells41a-41i, when the silane coupling agent is dropped.
• (c) Unreacted hydroxyl groups (—OH) remaining on the surface of thebase plate15 is acetylated to be capped by treatment with acetic anhydride and 1-methylimidazole (tetrahydrofuran solution). Next, as shown inFIG. 16, the epoxy groups of the silane coupling agent introduced on the surface of thebase plate15 is hydrolyzed and the hydroxyl groups (—OH) are introduced in each of the silane coupling films81a-81i.
• (d) As shown inFIG. 17, a nucleoside of which 5′ terminal is protected by Dimethoxytrityl (DMTr) group and a trivalent phosphoramidite derivative is substituted for hydroxyl group of 3′ terminal is prepared as a first base. Here, as shown inFIG. 18, amino group of adenine (A) and cytosine (C) included in the nucleoside is protected by the benzoyl group, and amino group of guanine (G) is protected by the isobutyl group. Next, the nucleoside as the first base is dropped onto each of the silane coupling films81a-81ishown inFIG. 14. By the dropping, phosphoric cyanoethyl amidite derivative of the nucleoside as the first base is bonded to the hydroxyl group (—OH) of the silane coupling agent, as shown inFIG. 19, by using the base catalyst, for example.
• (e) The unreacted hydroxyl groups (—OH) of the silane coupling agents are acetylated by treatment with acetic anhydride and 1-methylimidazole (tetrahydrofuran solution) and bondings to the nucleosides from a second base onward are inhibited. Next, Dimethoxytrityl (DMTr) groups of the nucleosides as the first bases are deprotected by a 3% trichloroacetic acid/dichloromethane acid solution and 5′ hydroxyl groups (—OH) are introduced in the nucleosides as the first bases, as shown inFIG. 20.
• (f) The nucleosides as the second bases of which trivalent phosphoramidite derivatives are substituted for the 3′ terminal hydroxyl groups are dropped onto the silane coupling films81a-81i, and as shown inFIG. 21, the phosphoric cyanoethyl amidite derivatives of the nucleosides as the second bases are bonded to the 5′ hydroxyl groups (—OH) of the nucleosides as the first bases by condensation reaction, with base catalyst, for example. Then, unreacted5′ hydroxyl groups (—OH) of the nucleosides as the first bases are acetylated to be capped by treatment with acetic anhydride and tetrahydrofuran solution.
• (g) Phosphite triester bonds formed by the condensation reaction are oxidized with iodine and water (pyridine-containing tetrahydrofuran solution) and they change to more stable phosphoric triester bonds, as shown inFIG. 22. Thereafter, Dimethoxytrityl (DMTr) groups of the second bases are removed. Then, the condensation reactions to the nucleoside phosphoramidites are repeated until the desired DNA chain length is obtained, as shown inFIG. 23, and the plurality of biomaterial films91a-91ishown inFIG. 10 are formed.
• (h) After the DNA is elongated, an alkaline solution treatment is performed. Here, thebase plate15 is sunk in the alkaline solution such as ammonia water (NH₄OH) at 55 degrees C. for 30 minutes so that thepolymer membrane11 is soaking. It should be noted that the weight percent concentration is 40% at the preparation time. By the alkaline solution treatment, as shown inFIG. 24, cyanoethyl protecting groups are detached. Also, bases such as adenine (A), cytosine (C), and guanine (G) are deprotected as shown inFIG. 25. Further, by the alkaline solution treatment, adhesion force between themetallic membrane13 and thepolymer membrane11 is diminished. Thereafter, thebase plate15 is took out from the NH₄OH water, and thelateral side115 of thepolymer membrane11 shown inFIG. 26 is blown by gas such as air by using an air gun, for example, to peel off thepolymer membrane11 from themetallic membrane13. Finally, the end terminal Dimethoxytrityl (DMTr) groups are deprotected, as shown inFIG. 27, and the method for manufacturing the biochip according to the second embodiment is completed.
• In an earlier method for manufacturing the biochip, there was no technique for protecting the membrane having the plurality of wells by the polymer membrane during formation of the biomaterial film and then easily peeling off the polymer membrane from the membrane having the plurality of wells. Therefore, if the biomaterial film was formed without protecting the membrane having the plurality of wells, the silane coupling agents were bonded to the surface of the membrane having the plurality of wells, and the probe biomolecules were bonded to the silane coupling agents. Consequently, there was a problem that the fluorescently-labeled target biomolecules also bonded to the surface of the membrane having the plurality of wells and it became the background noise during the detection process. Especially in a micro amount assay, a technique for enhancing contrast in fluorescent assay by trapping the target biomolecules only on the surface of the substrate exposed from each of the plurality of wells and inhibiting the target biomolecule from being trapped on the surface of membrane having the wells was desired.
• However, by the method for manufacturing the biochip according to the second embodiment, as shown inFIG. 13 toFIG. 27, themetallic membrane13 is protected by thepolymer membrane11 during the formation of the plurality of biomaterial films91a-91i, and thepolymer membrane11 is peeled off from themetallic membrane13 after the formation of the plurality of biomaterial films91a-91i. Therefore, the probe biomolecules are not introduced onto themetallic membrane13. Consequently, the target biomolecules are not bonded on themetallic membrane13. Accordingly, by using the biochip according to the second embodiment, it is possible to detect species and concentration of the target biomolecule with considerable accuracy, for example, since the background noise does not generat in the process for detecting the biomolecule. Further, in the process for synthesis of DNA shown inFIG. 17 toFIG. 25, it is possible to secure each depth of the plurality of wells42a-49i, since thepolymer membrane11 is disposed on themetallic membrane13. Therefore, it is possible to drop sufficient synthesis reagents for synthesis of DNA into each of the plurality of wells42a-49i. Further, it is possible to easily peel off thepolymer membrane11 from themetallic membrane13 by immersing it in the NH₄OH water used for the desorption of cyanoethyl protecting group and the deprotection of adenine (A), cytosine (C), guanine (G), for example. Therefore, it is not necessary to prepare a specific stripping solution to peel off thepolymer membrane11. Also, it is not necessary to immerse thepolymer membrane11 into the specific stripping solution. Therefore, it is not feared that the probe biomolecules in the plurality of biomaterial films91a-91iare damaged. The phenomenon of easy peeling off of thepolymer membrane11 can be specifically seen only when thepolymer membrane11 is formed on themetallic membrane13. In earlier, there was no method for manufacturing the biochip including the formation of thepolymer membrane11 as the protecting film on themetallic membrane13. Especially, in the case where thepolymer membrane11 is composed of SU-8-3000 and themetallic membrane13 is composed of Ti, favorable peel-off is obtained. Though the method including forming the plurality of biomaterial films91a-91iafter the silane coupling films81a-81iare formed on thebase plate15 exposed from each of the plurality of wells42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49ishown inFIG. 13 toFIG. 27 is explained, the probe biomolecules can be bonded to the plurality of hydroxyl groups (—OH) shown inFIG. 13 without using the silane coupling films81a-81i.
• (Modification of the Second Embodiment)
• It is possible to synthesize the probe DNAs having different base sequences in each of the plurality of wells41a-41i,42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49i, shown inFIG. 7. A method for synthesizing the probe DNAs having the different base sequences in each of the plurality of wells41a-41i,42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49iis described below.
• (a) As shown inFIG. 14, after each of the silane coupling films81a-81iis formed, the epoxy groups of the silane coupling agents included in each of the silane coupling films81a-81iare hydrolyzed and the hydroxyl groups (—OH) are introduced in each of the silane coupling films81a-81i. Thereafter, the 5 bases of nucleosides having thymines (T) are bonded to the silane coupling agent in series. Next, as shown inFIG. 28, buried resins are embedded in thewells41d,41e,41fto formblock layer130a,130b,130c. Polyhydroxy styrene (PHS) having 20,000 of average molecular weight dissolved in Dimethylsulfoxide (DMSO) at 5% of ratio by weight can be used as a material of the buried resin.
• (b) Dimethoxytrityl (DMTr) groups of the nucleosides in each of thewells41a,41b,41c,41g,41h,41iwhere the buried resins were not embedded are deprotected by the 3% trichloroacetic acid/dichloromethane acid solution. Thereafter, the block layers130a,130b,130care removed by solvent. Next, new nucleosides of which 3′ terminal hydroxyl groups are changed to trivalent phosphoramidite derivatives are dropped into the wells41a-41i. The new nucleosides react and are bonded only to 5′ hydroxyl groups (—OH) of the nucleosides of which Dimethoxytrityl (DMTr) groups were deprotected in thewells41a,41b,41c,41g,41h,41i.
• (c) Thewells41a,41b,41c,41g,41h,41ishown inFIG. 14 are embedded with buried resins, as shown inFIG. 29, to form block layers131a,131b,131c,131d,131e,131f. Next, Dimethoxytrityl (DMTr) groups of the nucleosides in each of thewells41d,41e,41fwhere the buried resins were not embedded are deprotected by the 3% trichloroacetic acid/dichloromethane acid solution. Then, the block layers131a,131b,131c,131d,131e,131fare removed by the solvent. Thereafter, new nucleosides of which 3′ terminal hydroxyl groups are changed to trivalent phosphoramidite derivative are dropped in the wells41a-41i. The new nucleosides react and are bonded to 5′ hydroxyl groups (—OH) of nucleosides of which Dimethoxytrityl (DMTr) groups were deprotected in thewells41d,41e,41f.
• (d) Thereafter, embedding the buried resin in at least any one of the wells41a-41i, deprotection of Dimethoxytrityl (DMTr) group, removal of the buried resin, and polymerization reaction of the nucleosides are repeated and the probe DNAs having the different base sequences in each of the plurality of wells41a-41i,42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49iare synthesized.
• In the process of DNA synthesis shown inFIG. 28 andFIG. 29, it is possible to secure each depth of the plurality of wells42a-49i, since thepolymer membrane11 is disposed on themetallic membrane13. Therefore, it becomes easy to embed the buried resin in each of the plurality of wells42a-49i.
Third Embodiment
• With reference toFIG. 30 andFIG. 31 that is a sectional view taken on line XXXI-XXXI, a substrate for a biochip according to a third embodiment includes the base plate15 having the surface on which the plurality of hydroxyl groups can be introduced, and a cover member211 disposed on the base plate15 when the probe biomolecules are bonded to the plurality of hydroxyl groups, and having a plurality of through holes141a,141b,141c,141d,141e,141f,141g,141h,141i,142a,142b,142c,142d,142e,142f,142g,142h,142i,143a,143b,143c,143d,143e,143f,143g,143h,143i,144a,144b,144c,144d,144e,144f,144g,144h,144i,145a,145b,145c,145d,145e,145f,145g,145h,145i,146a,146b,146c,146d,146e,146f,146g,146h,146i,147a,147b,147c,147d,147e,147f,147g,147h,147i,148a,148b,148c,148d,148e,148f,148g,148h,148i,149a,149b,149c,149d,149e,149f,149g,149h,14′9idefining biding areas of the probe biomolecules on the surface of base plate15.
• During the probe biomolecules are introduced on the surface of thebase plate15 exposed from each of the plurality of through holes141a-149i, thecover member211 is close contact with the upper surface ofbase plate15. A material having an anti peeling property against the nucleic acid synthetic agents such as the resin like SU-8, etc., silicon (Si) rubber, and Poly-dimethyl siloxane (PDMS) can be used as the material of thecover member211. The method for introducing the probe biomolecules onto the surface of thebase plate15 is similar to the method explained withFIG. 15 toFIG. 25. Therefore, an explanation is omitted. After the probe biomolecules are introduced onto thebase plate15 exposed from each of the plurality of through holes141a-149i, thecover member211 is removed from thebase plate15. After thecover member211 is removed from thebase plate15, the probe biomolecules are introduced only on the portions of thebase plate15 exposed form the plurality of through holes141a-149iand the probe biomolecules are not introduced on other portions. Therefore, the target biomolecules are bonded to the portions of thebase plate15 exposed form the plurality of through holes141a-149iand are not bonded to other portions. Consequently, in the process for detecting the target biomolecules, it is possible to cancel the background noise generated by the target biomolecules nonspecifically bonded to the surface of thebase plate15. It should be noted that the metallic membrane may be disposed between thebase plate15 and thecover member211 as explained in the first and second embodiments.
Fourth Embodiment
• With reference toFIG. 32 andFIG. 33 that is a sectional view taken on line XXXIII-XXXIII, a biochip according to a fourth embodiment of the present invention includes an optical transparency base plate15 having a surface on which the plurality of hydroxyl groups (—OH) can be introduced, and a light shielding film113 disposed on the base plate15 and having a plurality of through holes241a,241b,241c,241d,241e,241f,241g,241h,241i,242a,242b,242c,242d,242e,242f,242g,242h,242i,243a,243b,243c,243d,243e,243f,243g,243h,243i,244a,244b,244c,244d,244e,244f,244g,244h,244i,245a,245b,245c,245d,245e,245f,245g,245h,245i,246a,246b,246c,246d,246e,246f,246g,246h,246i,247a,247b,247c,247d,247e,247f,247g,247h,247i,248a,248b,248c,248d,248e,248f,248g,248h,248i,249a,249b,249c,249d,249e,249f,249g,249h,249ithat reach the base plate15. The transition metal such as Titan (Ti), platinum (Pt)), chrome (Cr), niobium (Nb), tantalum (Ta), tungsten (W), etc., and the metal such as aluminum (Al), gold (Au), etc., can be used for the material of thelight shielding film113. In addition, the transition metallic oxide such as titanium monoxide (TiO), titanium dioxide (TiO₂), etc., and the transition metal nitride such as titanium nitride (TiN), etc., and the transition metal carbide such as titanium carbide (TiC), etc., can be used as the material oflight shielding film113, for example. Each diameter of the plurality of through holes241a-249iis above 300 micrometers.
• Further, as shown inFIG. 33, the biochip according to fourth embodiment includes a plurality ofbiomaterial films91a,91b,91c,91d,91e,91f,91g,91h,91idisposed on the surface of thebase plate15 exposed from the plurality of through holes241a-241i, respectively. In each of the plurality of biomaterial films91a-91i, each functional group of the plurality of probe biomolecules such as the plurality of DNAs, the plurality of RNAs, the plurality of PNAs, the plurality of proteins, etc., is covalently bonded to the hydroxyl group (—OH) on the surface of thebase plate15, as explained withFIG. 9.
• It should be noted that the fourth embodiment is not limited to the displacement where the plurality of biomaterial films91a-91idirectly displaced on the surface of thebase plate15, respectively. In a case shown inFIG. 34,silane coupling films81a,81b,81c,81d,81e,81f,81g,81h,81iare disposed on the surface of the portions of thebase plate15 exposed from the plurality of through holes241a-241i, respectively. In each of the silane coupling films81a-81i, as explained withFIG. 11, each methyl group (—CH₃) or each ethyl group (—C₂H₅) of the plurality of silane coupling agents is chemically bonded to the hydroxyl group (—OH) on the surface of thebase plate15 by the acid-base reaction. Alternatively, the silane coupling agent and the probe biomolecule may be coupled via the cross linker. Here, each sectional view of the plurality of throughholes242a-242i,243a-243i,244a-244i,245a-245i,246a-246i,247a-247i,248a-248i,249a-249ishown inFIG. 32 is similar toFIG. 33, so an explanation is omitted.
• As described above, in the biochip according to the fourth embodiment shown inFIG. 32 toFIG. 34, each of the plurality of biomaterial films91a-91iis disposed on the opticaltransparency base plate15 like a spot. Further, thelight shielding film113 is disposed around each of the plurality of biomaterial films91a-91i. When an assay is performed with the biochip according to the fourth embodiment, the target biomolecules labeled with biotins are dropped into the plurality of biomaterial films91a-91i, respectively. After the biochip is left for the time period required for interactions between the target biomolecules and the probe biomolecules, the biochip is washed to remove the unreacted target biomolecules. Then, a solution including streptavidins labeled with Horseradish Peroxidase (HRP) is dropped into each of the plurality of biomaterial films91a-91i. After it is stilly left for 1 hour at room temperature, the biochip is washed to remove the unreacted streptavidin. After the washing, a solution including Tetramethyl benzidine (TMB) is dropped into each of the plurality of biomaterial films91a-91i. When the target biomolecules are trapped in each of the plurality of biomaterial films91a-91i, colors of TMB come out by the HRP of target biomolecule. Therefore, by emitting illuminating light from the back side opposite to the surface of thebase plate15 displaced with thelight shielding film113, it is possible to easily confirm whether the chromogenic reaction occurs in each of the plurality of biomaterial films91a-91iby the transmitted light through thebase plate15, since thelight shielding film113 enhances the contrast. In addition, by setting each diameter of the plurality of through holes241a-249iabove 300 micrometers, it is possible to confirm whether the chromogenic reaction occurs in each of the plurality of biomaterial films91a-91iby the naked eye.
• Next, with reference toFIG. 35 toFIG. 40, the method for manufacturing the biochip according to fourth embodiment is described.
• (a) As shown inFIG. 35, thebase plate15 composed of SiO₂, for example, is prepared and thelight shielding film113 composed of Ti, for example, is formed on thebase plate15 by using the sputtering method or the CVD method, for example. After thelight shielding film113 is formed, the surface is treated with O₂plasma for 5 minutes. Next, as shown inFIG. 36, the solution including the photosensitive epoxy resin such as SU-8-3000 series is spin-coated on thelight shielding film113 to form thepolymer membrane11. After thepolymer membrane11 is formed, prebake is performed for thepolymer membrane11. Thereafter, by using a photomask having a mask pattern corresponding to each shape of the plurality of through holes241a-241i,242a-242i,243a-243i,244a-244i,245a-245i,246a-246i,247a-247i,248a-248i,249a-249ishown inFIG. 32, portions of thepolymer membrane11 are selectively exposed to the ultraviolet rays. After the exposure, the PEB process is performed for thepolymer membrane11. Thereafter, thepolymer membrane11 is developed with SU-8 developer solution, for example, to selectively remove the portions of thepolymer membrane11, as shown inFIG. 37.
• (b) By using thepolymer membrane11 of which the portions are selectively removed as an etching mask, portions of thelight shielding film113 are selectively removed by isotropic wet etching. By the selective removal, each of the plurality of through holes241a-241i,242a-242i,243a-243i,244a-244i,245a-245i,246a-246i,247a-247i,248a-248i,249a-249iis formed as shown inFIG. 38. Then, thebase plate15 is left in the stirred sodium hydroxide (NaOH) solution at room temperature for 2 hours and the plurality of hydroxyl groups (—OH) are introduced on the surface of thebase plate15 exposed from each of the through holes241a-241i, as explained withFIG. 13.
• (c) For example, the silane coupling reagent of which functional group has amine is dropped onto the surface of thebase plate15 exposed from each of the through holes241a-241i, shown inFIG. 38, to form each of thesilane coupling films81a,81b,81c,81d,81e,81f,81g,81h,81ishown inFIG. 39. When the silane coupling agent having the epoxy group is dropped under 15 degrees C. condition, for example, the plurality of epoxy groups are introduced on the surface of thebase plate15, as explained withFIG. 15. It should be noted that the locations of the through holes241a-241ican be easily identified when the silane coupling agent is dropped, because of the contrast between the opticaltransparency base plate15 and thelight shielding film113 shown inFIG. 39.
• (d) The unreacted hydroxyl groups (—OH) remaining on the surface of thebase plate15 are acetylated to be capped. Next, as explained withFIG. 16, the epoxy groups of the silane coupling agents introduced onto the surface of thebase plate15 are hydrolyzed to introduce the hydroxyl groups (—OH) in each of the silane coupling films81a-81i. Thereafter, by the method similar to the explanation ofFIG. 17 toFIG. 23, the plurality of biomaterial films91a-91ishown inFIG. 34 are formed. Thebase plate15 is sunk in the alkaline solution so that thepolymer membrane11 is soaking. As explained withFIG. 24, the cyanoethyl protecting groups bonded to the phosphate group are detached. Also, the bases such as adenine (A), cytosine (C), guanine (G), etc., are deprotected, as explained withFIG. 25. In addition, by the treatment with the alkaline solution, adhesive strength between thelight shielding film113 and thepolymer membrane11 is decreased. Thereafter, thebase plate15 is took out from the NH₄OH solution. And, thelateral side115 of thepolymer membrane11 shown inFIG. 40 is blown by gas such as air by using the air gun, for example, to peel off thepolymer membrane11 from thelight shielding film113. Finally, the terminal Dimethoxytrityl (DMTr) groups are deprotected, as explained withFIG. 27 and the method for manufacturing the biochip according to the fourth embodiment is completed.
• It should be noted that the method for manufacturing the biochip according to the fourth embodiment is not limited to this. For example, it is explained that the light shielding film is formed on thebase plate15 shown inFIG. 35 by using the sputtering method or the CVD method. However, thelight shielding film113 having the plurality of through holes241a-249imay be formed on thebase plate15, by using UV-curable black screen printing ink, for example. Alternatively, thelight shielding film113 having the through holes241a-249imay be prepared in advance, and it may be pasted on thebase plate15 by a bonding agent, for example. Again, the material of thelight shielding film113 is not limited to metals. Resins or insoluble papers also can be used, for example. In addition, the light shielding film may be formed by using an ink-jet printer, for example. Also, as shown inFIG. 41, thebase plate15 of the biochip according to the fourth embodiment may have a plurality ofwells51a,51b,51c,51d,51e,51f,51g,51h,51ithat open to the plurality of through holes241a-241iof thelight shielding film113, respectively. The plurality of biomaterial films91a-91iare disposed at each bottom of the plurality of wells51a-51i. When the biochip shown inFIG. 41 is manufactured, the plurality of through holes241a-241iare formed in thelight shielding film113, as shown inFIG. 38, and then, thebase plate15 may be selectively removed by the dry etching method by using thelight shielding film113 as the etching mask, for example. In the case where it is desired to secure the volume of the specimen solution, for example, the plurality of wells51a-51iare formed.
• (Modification of the Fourth Embodiment)
• It is possible to synthesize the probe DNAs having the different base sequences on the surface of thebase plate15, shown inFIG. 32, exposed from the plurality of through holes241a-241i,242a-242i,243a-243i,244a-244i,245a-245i,246a-246i,247a-247i,248a-248i,249a-249i, respectively. A method for synthesizing the probe DNAs having the different base sequences in the plurality of through holes241a-241i,242a-242i,243a-243i,244a-244i,245a-245i,246a-246i,247a-247i,248a-248i,249a-249i, respectively, is described below.
• (a) As shown inFIG. 39, after each of the silane coupling films81a-81iis formed, the epoxy groups of the silane coupling agents included in each of the silane coupling films81a-81iare hydrolyzed to introduce the hydroxyl groups (—OH) in each of the silane coupling films81a-81i. Thereafter, the 5 bases of nucleosides having thymines (T) are bonded to the silane coupling agent in series. Next, the buried resins are embedded into the throughholes241d,241e,241f, as shown inFIG. 42, to form the block layers130a,130b,130c.
• (b) Dimethoxytrityl (DMTr) groups of the nucleosides in each of the throughholes241a,241b,241c,241g,241h,241iwhere the buried resins were not embedded are deprotected by the 3% trichloroacetic acid/dichloromethane acid solution. Thereafter, the block layers130a,130b,130care removed by the solvent. Next, new nucleosides of which 3′ terminal hydroxyl groups are changed to the trivalent phosphoramidite derivatives are dropped into the through holes241a-241i, and the new nucleosides only react and are bonded to the 5′ hydroxyl groups (—OH) of the nucleosides of which Dimethoxytrityl (DMTr) groups were deprotected.
• (c) As shown inFIG. 43, the buried resins are embedded in the throughhole241a,241b,241c,241g,241h,241ishown inFIG. 39 to form the block layers131a,131b,131c,131d,131e,131f. Next, Dimethoxytrityl (DMTr) groups of the nucleosides in each of the throughholes241d,241e,241fwhere the buried resins were not embedded are deprotected by the 3% trichloroacetic acid/dichloromethane acid solution, and the block layers131a,131b,131c,131d,131e,131fare removed by the solvent. Thereafter, when new nucleosides of which 3′ terminal hydroxyl groups were changed to the trivalent phosphoramidite derivatives are dropped in the through holes241a-241i, the new nucleosides only react and are bonded to the 5′ hydroxyl groups (—OH) of the nucleosides of which Dimethoxytrityl (DMTr) groups were deprotected in the throughholes241d,241e,241f.
• (d) Thereafter, embedding the buried resin in any of the through holes241a-241i, deprotction of the Dimethoxytrityl (DMTr) group, removal of the buried resins, and polymerization reaction of the nucleosides are repeated, and the probe DNAs having the different base sequences in the plurality of through holes241a-241i,242a-242i,243a-243i,244a-244i,245a-245i,246a-246i,247a-247i,248a-248i,249a-249iare synthesized.
Fifth Embodiment
• With reference toFIG. 44 andFIG. 45 that is a sectional view taken on line XLV-XLV, a biochip according to a fifth embodiment includes an optical transparency base plate15, a light shielding film113 disposed on a first surface10 of the base plate15 and having a plurality of through holes241a,241b,241c,241d,241e,241f,241g,241h,241ithat reach the first surface10, and a plurality of biomaterial films91a,91b,91c,91d,91e,91f,91g,91h,91i,92a,92b,92c,92d,92e,92f,92g,92h,92i,93a,93b,93c,93d,93e,93f,93g,93h,93i,94a,94b,94c,94d,94e,94f,94g,94h,94i,95a,95b,95c,95d,95e,95f,95g,95h,95i,96a,96b,96c,96d,96e,96f,96g,96h,96i,97a,97b,97c,97d,97e,97f,97g,97h,97i,98a,98b,98c,98d,98e,98f,98g,98h,98i,99a,99b,99c,99d,99e,99f,99g,99h,99ihaving a plurality of probe biomolecules, respectively, that are bonded to a second surface20 of the base plate15 opposite to the first surface10 of the base plate15. Each enlarged sectional view of the plurality of biomaterial films91a-99iis similar toFIG. 9, so an explanation is omitted. Also, as shown inFIG. 11 andFIG. 12, the plurality of probe biomolecules may be bonded to thebase plate15 via the silane coupling agent or the cross linking agent, for example. After a specimen solution including the biotin labeled target biomolecules is dropped into each of the plurality of biomaterial films91a-99ishown inFIG. 44 andFIG. 45, a solution including HRP labeled streptavidin is dropped into each of the plurality of biomaterial films91a-99i. After it is stilly left and washed, a solution including TMP is dropped into each of the plurality of biomaterial films91a-91i. Here, if the target biomolecules are trapped in each of the plurality of biomaterial films91a-91i, the colors of TMB come out by the HRP of the target biomolecule. Therefore, when the illuminating light is emitted from thefirst surface10, it is possible to easily confirm whether the chromogenic reaction occurs by the transmitted light incident on thebase plate15 from the through holes241a-241i.
• Next, a method for manufacturing the biochip according to the fifth embodiment is described. By using the method explained withFIG. 35 toFIG. 38, thelight shielding film113 having the plurality of through holes241a-241iare formed on thefirst surface10 of thebase plate15. InFIG. 46, apolymer membrane311 having a plurality of openings is formed on the second surface of thebase plate15, by using a lithography method, for example. Here, locations of the plurality of formed openings of thepolymer membrane311 confront locations of the plurality of formed through holes241a-241iof thelight shielding film113, respectively. InFIG. 47, the plurality of biomaterial films91a-91iare formed on thesecond surface20 of thebase plate15 exposed from the plurality of openings of thepolymer membrane311, respectively. Thereafter, thepolymer membrane311 is peeled off from thesecond surface20 and the biochip according to the fifth embodiment is achieved.
• It should be noted that the shape of the biochip according to the fifth embodiment is not limited toFIG. 45. For example, as shown inFIG. 48,wells61a,61b,61c,61d,61e,61f,61g,61h,61imay be delineated in thebase plate15, and the plurality of biomaterial films91a-99imay be disposed on the bottoms of the wells61a-61i, respectively. Alternatively, abiomaterial film191 may be disposed on thesecond surface20, as shown inFIG. 49. When the illuminating light is emitted from thefirst surface10 of thebase plate15, thelight shielding film113 restricts regions where the illuminating light penetrates. Therefore, even if thebiomaterial film191 are evenly disposed on thesecond surface20, the contrast between presence and absence of chromogenic reaction becomes clear.
Sixth Embodiment
• With reference toFIG. 50 andFIG. 51 that is a sectional view taken on line LI-LI, a biochip according to a sixth embodiment includes a plurality of opticaltransparency base members215a,215b,215c,215d,215e,215f,215g,215h,215i, a plurality ofbiomaterial films291a,291b,291c,291d,291e,291f,291g,291h,291ihaving a plurality of probe biomolecules bonded to the plurality of base members215a-215i, respectively, and alight shielding member213 disposed around each of the base members215a-215i. SiO₂can be used as a material of each of the plurality of base members215a-215i, for example. The metals and the resins can be used as a material of thelight shielding member213, for example. Also, the base members are disposed on the bottoms of the plurality ofbiomaterial films292a,292b,292c,292d,292e,292f,292g,292h,292i,293a,293b,293c,293d,293e,293f,293g,293h,293i,294a,294b,294c,294d,294e,294f,294g,294h,294i,295a,295b,295c,295d,295e,295f,295g,295h,295i,296a,296b,296c,296d,296e,296f,296g,296h,296i,297a,297b,297c,297d,297e,297f,297g,297h,297i,298a,298b,298c,298d,298e,298f,298g,298h,298i,299a,299b,299c,299d,299e,299f,299g,299h,299i, shown inFIG. 50, respectively. Each enlarged sectional view of the plurality of biomaterial films291a-299iis similar to the enlarged sectional view of thebiomaterial film91ashown inFIG. 9. So, an explanation is omitted. As a matter of course, the plurality of probe biomolecules may be bonded to the plurality of base members215a-215ivia the silane coupling agents or the cross coupling agents, respectively, as shown inFIG. 11 andFIG. 12, for example. After the specimen solution including the biotin labeled target biomolecules is dropped onto each of the plurality of biomaterial films291a-299ishown inFIG. 50 andFIG. 51, the solution including HRP labeled streptavidin is dropped onto each of the plurality of biomaterial films291a-291i. After it is stilly left and washed, the solution including TMP is dropped into each of the plurality of biomaterial films291a-291i. In the case where the target biomolecules are trapped in each of the plurality of biomaterial films291a-291i, the colors of TMB come out by the HRP of the target biomolecule. Therefore, when the illuminating light is emitted from the side where the plurality of biomaterial films291a-299iare not disposed, it is possible to easily confirm the presence or absence of the chromogenic reaction, because of the contrast between the transmitted light of the illuminating light incident on each of the plurality of base members215a-215iand thelight shielding member213.
• Next, a method for manufacturing the biochip according to the sixth embodiment is described. First, as shown inFIG. 52, thelight shielding member213 having the plurality of through holes71a,71b,71c,71d,71e,71f,71g,71h,71iis prepared. InFIG. 53, the plurality of base members215a-215iare inserted into the plurality of through holes71a-71i, respectively. InFIG. 54, thepolymer membrane311 having the plurality of openings exposing the plurality of base members215a-215iis formed on thelight shielding member213, by the lithography method. InFIG. 55, the plurality of biomaterial films91a-91iare formed on the plurality of base members215a-215i, respectively. Thereafter, thepolymer membrane311 is removed and the biochip according to the sixth embodiment is achieved.
• It should be noted that the shape of the biochip according to the sixth embodiment is not limited toFIG. 51. For example, as shown inFIG. 56, each thickness of the plurality of base members215a-215imay be different from the thickness of thelight shielding member213. By setting the thickness of thelight shielding member213 thicker than each thickness of the plurality of base members215a-215i, a plurality of wells of which side walls are thelight shielding member213 and bottoms are the surfaces of the plurality of base members215a-215i, respectively, are provided. Thebiomaterial films291a,291b,291c,291d,291e,291f,291g,291h,291imay be disposed on the bottoms of the plurality of wells, respectively.
Other Embodiment
• Although the invention has been described above by reference to the embodiment of the present invention, the present invention is not limited to the embodiment described above. Modifications and variations of the embodiment described above will occur to those skilled in the art, in the light of the above teachings. For example, as shown inFIG. 57, by delineatinggrooves120a,120b,120c,120dbetween the plurality of wells41a-41i,42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49iin themetallic membrane13, it becomes easy to determine the locations where the plurality of wells41a-41i,42a-42i,43a-43i,44a-44i,45a-45i,46a-46i,47a-47i,48a-48i,49a-49iare present, by naked eye. In addition, by delineating the grooves120a-120din themetallic membrane13, it becomes easy to peel off thepolymer membrane11 from themetallic membrane13 in the process for manufacturing the biochip. As described above, the present invention includes many variations of embodiments that are not described here. Therefore, the scope of the invention is defined with reference to the following claims that are appropriate from this disclosure.
INDUSTRIAL APPLICABILITY
• The substrate for the biochip, the biochip, the method for manufacturing the substrate for the biochip, the method for manufacturing the biochip according to the present invention can be utilized in a healthcare industry, a household industry, and a cosmetic industry, for example.

Claims (44)

1. A substrate for a biochip comprising:

a base plate having a surface on which a plurality of hydroxyl groups can be introduced;

a metallic membrane disposed on the base plate and having a plurality of wells reaching the base plate; and

a crosslinkable polymer membrane disposed on the metallic membrane.

2. The substrate for the biochip ofclaim 1, wherein the base plate is composed of a silicon oxide.

3. The substrate for the biochip ofclaim 1, wherein the metallic membrane is composed of a Titan.

4. The substrate for the biochip ofclaim 1, wherein the metallic membrane is composed of a transition metallic oxide.

5. The substrate for the biochip ofclaim 1, wherein the metallic membrane is composed of a transition metal nitride.

6. The substrate for the biochip ofclaim 1, wherein the metallic membrane is composed of a transition metal carbide.

7. The substrate for the biochip ofclaim 1, wherein the polymer membrane is insoluble in an acid solution.

8. The substrate for the biochip ofclaim 1, wherein the polymer membrane has a photosensitivity.

9. The substrate for the biochip ofclaim 1, wherein the polymer membrane is composed of an epoxy resin.

10. The substrate for the biochip ofclaim 1, wherein the polymer membrane is composed of SU8.

11. A biochip comprising:

a base plate;

a metallic membrane disposed on the base plate and having a plurality of wells reaching the base plate; and

a plurality of probe biomolecules bonded on a surface of the base plate exposed from the plurality of wells.

12. The biochip ofclaim 11, wherein the base plate is composed of a silicon oxide.

13. The biochip ofclaim 11, wherein the metallic membrane is composed of a Titan.

14. The biochip ofclaim 12, wherein the metallic membrane is composed of a transition metallic oxide.

15. The biochip ofclaim 12, wherein the metallic membrane is composed of a transition metal nitride.

16. The biochip ofclaim 11, wherein the metallic membrane is composed of a transition metal carbide.

17. The biochip ofclaim 16, wherein the plurality of probe biomolecules are bonded to the surface via a plurality of hydroxyl groups introduced on the surface.

18. A method for manufacturing a substrate for a biochip including:

a step for preparing a base plate having a surface on which a plurality of hydroxyl groups can be introduced;

a step for forming a metallic membrane on the base plate;

a step for forming a crosslinkable polymer membrane on the metallic membrane;

a step for selectively removing portions of the polymer membrane; and

a step for delineating a plurality of wells reaching the base plate in the metallic membrane, by using the polymer membrane of which the portions were selectively removed as an etching mask.

19. The method for manufacturing the substrate for the biochip ofclaim 18, wherein the metallic membrane is composed of a Titan.

20. The method for manufacturing the substrate for the biochip ofclaim 18, wherein the metallic membrane is composed of a transition metallic oxide.

21. The method for manufacturing the substrate for the biochip ofclaim 18, wherein the metallic membrane is composed of a transition metal nitride.

22. The method for manufacturing the substrate for the biochip ofclaim 18, wherein the metallic membrane is composed of a transition metal carbide.

23. The method for manufacturing the substrate for the biochip ofclaim 22, wherein the polymer membrane is composed of an epoxy resin.

24. The method for manufacturing the substrate for the biochip ofclaim 18, wherein the polymer membrane is composed of SU8.

25. A method for manufacturing a biochip including:

a step for preparing a base plate;

a step for forming a metallic membrane on the base plate;

a step for forming a crosslinkable polymer membrane on the metallic membrane;

a step for selectively removing portions of the polymer membrane;

a step for delineating a plurality of wells reaching the base plate in the metallic membrane, by using the polymer membrane of which the portions were selectively removed as an etching mask;

a step for introducing a plurality of hydroxyl groups on a surface of the base plate exposed from the plurality of wells;

a step for bonding a plurality of probe biomolecules to the plurality of hydroxyl groups, respectively; and

a step for soaking the metallic membrane and the polymer membrane in an alkaline solution to peel off the polymer membrane from the metallic membrane.

26. A method for manufacturing a biochip including:

a step for preparing a substrate for the biochip comprising a base plate, a metallic membrane disposed on the base plate and having a plurality of wells reaching the base plate, a crosslinkable polymer membrane disposed on the metallic membrane, and a plurality of hydroxyl groups introduced on a surface of the base plate exposed from the plurality of wells;

a step for bonding a plurality of probe biomolecules to the plurality of hydroxyl groups, respectively; and

a step for soaking the metallic membrane and the polymer membrane in an alkaline solution to peel off the polymer membrane from the metallic membrane.

27. The method for manufacturing the biochip ofclaim 25, wherein the base plate is composed of a silicon oxide.

28. The method for manufacturing the biochip ofclaim 26, wherein the metallic membrane is composed of a Titan.

29. The method for manufacturing the biochip ofclaim 27, wherein the metallic membrane is composed of a transition metallic oxide.

30. The method for manufacturing the biochip ofclaim 25, wherein the metallic membrane is composed of a transition metal nitride.

31. The method for manufacturing the biochip ofclaim 26, wherein the metallic membrane is composed of a transition metal carbide.

32. The method for manufacturing the biochip ofclaim 25, wherein the polymer membrane is composed of an epoxy resin.

33. The method for manufacturing the biochip ofclaim 31, wherein the polymer membrane is composed of SU8.

34. The method for manufacturing the biochip ofclaim 25, further including a step for deprotecting amino groups included in the plurality of probe biomolecules by the alkaline solution.

35. The method for manufacturing the biochip ofclaim 34, wherein the step for peeling off the polymer membrane from the metallic membrane includes a step for blowing the polymer membrane with an air.

36. A substrate for a biochip comprising:

a base plate having a surface on which a plurality of hydroxyl groups can be introduced; and

a cover member disposed on the base plate when probe biomolecules are bonded to the plurality of hydroxyl groups, respectively, the cover member having a plurality of through holes to define binding regions where the probe biomolecules are bonded to the surface of the base plate.

37. A biochip comprising:

an optical transparency base plate;

a light shielding film disposed on the base plate and having a plurality of through holes reaching the base plate; and

a plurality of probe biomolecules bonded to the base plate exposed from each of the plurality of through holes.

38. The biochip ofclaim 37, wherein a plurality of wells that open to the plurality of through holes are delineated in the base plate.

39. A biochip comprising:

an optical transparency base plate;

a light shielding film disposed on a first surface of the base plate and having a plurality of through holes reaching the first surface; and

a plurality of probe biomolecules bonded to a second surface of the base plate opposite to the first surface of the base plate.

40. The biochip ofclaim 39, wherein the base plate is composed of a silicon oxide.

41. The biochip ofclaim 39, wherein the plurality of probe biomolecules are bonded to the base plate via a plurality of hydroxyl groups introduced on the base plate.

42. A biochip comprising:

an optical transparency base member;

a plurality of probe biomolecules bonded on the base member; and

a light shielding member disposed around the base member.

43. The biochip ofclaim 42, wherein the base member is composed of a silicon oxide.

44. The biochip ofclaim 42, wherein the plurality of probe biomolecules are bonded to the base member via a plurality of hydroxyl groups introduced on the base member.

Images