JP2008209130A - Color reaction test kit and color reaction test method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a kit of inspecting color developing reaction for stabilizing color developing reaction using a biochip. <P>SOLUTION: The kit includes a substrate 10, the biochip 100 arranged on the substrate 10 to carry a probe biomolecule, the frame 20 arranged on the substrate 10 to surround the biochip 100 and the lid plate 21 arranged on the frame 20 to cover the biochip 100. The space surrounded by the substrate 10, the frame 20 and the lid plate 21 can be filled with a reagent solution containing a color developing reagent for detecting the target biomolecule bonded to the probe biomolecule. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a biomolecule detection technique, and more particularly to a color reaction test kit and a color reaction test method.

The biochip is a general term for elements in which probe biomolecules that specifically bind to target biomolecules to be examined are fixed at specific positions on the substrate surface. In a DNA chip, for example, several thousand to several tens of thousands of probe DNAs that are single-stranded DNAs each having a known sequence are arranged in an array on a substrate such as a slide glass plate. When a solution containing target DNA fluorescently labeled is dropped onto a DNA chip, only the target DNA whose sequence is complementary to the probe DNA is bonded to the probe DNA by hydrogen bonding to form a complementary double strand. Therefore, it can be confirmed whether or not the target DNA complementary to the probe DNA exists in the solution based on the presence or absence of fluorescence (see, for example, Patent Document 1). Therefore, the DNA chip is useful as an inspection tool for diseases such as genetic diseases, and is expected to be widely used in medical institutions and homes. However, there has been a problem that the detection of fluorescence requires an expensive detection device. For this reason, it has been studied to apply a color development reaction that can be visually confirmed to the biochip, but there is a problem that the color development reaction is unstable.
Special Table 2002-537869

  The present invention provides a color reaction test kit and a color reaction test method for stabilizing a color reaction using a biochip.

  In order to achieve the above object, the present invention is characterized by (a) a substrate, (b) a biochip disposed on the substrate and having a probe biomolecule, and (c) disposed on the substrate and surrounding the biochip. And (d) a target living body that binds to the probe biomolecule in a space surrounded by the substrate, the frame, and the cover plate. The gist of the present invention is a color reaction test kit filled with a reagent solution containing a color reagent for detecting molecules.

  According to the color reaction test kit according to the feature of the present invention, the reagent solution is filled in the space surrounded by the substrate, the frame, and the cover plate. Therefore, the balance of the interfacial tension between the reagent solution and the substrate, the interfacial tension between the reagent solution and the frame, and the interfacial tension between the reagent solution and the cover plate is not easily lost, and the flow of the reagent solution can be suppressed. . Therefore, it is possible to suppress the destabilization of the color development reaction due to the flow of the reagent solution.

  Other features of the present invention include: (a) a substrate provided with a recess; (b) a biochip having a probe biomolecule disposed in the recess of the substrate; and (c) a lid plate covering the recess of the substrate. And (d) a color reaction test kit in which a concave portion of a substrate covered with a cover plate is filled with a reagent solution containing a color reagent for detecting a target biomolecule that binds to a probe biomolecule. And

  According to the color reaction test kit according to another aspect of the present invention, the reagent solution is filled in the concave portion of the substrate covered with the cover plate. Therefore, the balance between the interfacial tension between the reagent solution and the substrate and the interfacial tension between the reagent solution and the cover plate is not easily lost, and the flow of the reagent solution can be suppressed. Therefore, it is possible to suppress the destabilization of the color development reaction due to the flow of the reagent solution.

  Still another feature of the present invention includes: (a) a substrate provided with a recess; (b) a probe biomolecule bonded to the bottom surface of the recess of the substrate; and (c) a lid plate covering the recess of the substrate. (D) a color reaction test kit in which a concave portion of a substrate covered with a cover plate is filled with a reagent solution containing a color reagent for detecting a target biomolecule that binds to a probe biomolecule; To do.

  According to the color reaction test kit according to another aspect of the present invention, the reagent solution is filled in the concave portion of the substrate covered with the cover plate. Therefore, the balance between the interfacial tension between the reagent solution and the substrate and the interfacial tension between the reagent solution and the cover plate is not easily lost, and the flow of the reagent solution can be suppressed. Therefore, it is possible to suppress the destabilization of the color development reaction due to the flow of the reagent solution.

  Still another feature of the present invention is that (a) a biochip having a probe biomolecule is disposed on a substrate, (b) a frame surrounding the biochip is disposed on the substrate, and (c) ) Dropping a test solution containing a target biomolecule that binds to a probe biomolecule into the frame; (d) dropping a reagent solution containing a coloring reagent for detecting the target biomolecule into the frame; E) Covering the frame filled with the reagent solution with a lid plate, and (f) Detecting the color reaction of the color reagent contained in the reagent solution filled in the space surrounded by the substrate, the frame, and the lid plate And a coloring reaction inspection method comprising:

  Still another feature of the present invention is that (a) a biochip having a probe biomolecule is disposed in a recess of a substrate provided with a recess, and (b) a test including a target biomolecule that binds to the probe biomolecule. Dropping the solution into the recess; (c) dropping the reagent solution containing a coloring reagent for detecting the target biomolecule into the recess; and (d) using the lid plate for the recess of the substrate filled with the reagent solution. The gist of the invention is to provide a color reaction test method comprising: covering and (e) detecting a color reaction of a color reagent contained in a reagent solution filled in a recess of a substrate covered with a cover plate.

  Still another feature of the present invention is that (a) a target biomolecule that binds to a probe biomolecule is disposed in a recess of a biochip that includes a substrate provided with a recess and a probe biomolecule bonded to the bottom surface of the recess of the substrate. Dropping a test solution containing, (b) dropping a reagent solution containing a coloring reagent for detecting a target biomolecule into the recess, and (c) a recess of the substrate filled with the reagent solution with a lid plate The gist of the present invention is a color reaction test method comprising: covering and (d) detecting a color reaction of a color reagent contained in a reagent solution filled in a recess of a substrate covered with a cover plate.

  According to the present invention, it is possible to provide a color reaction test kit and a color reaction test method for stabilizing a color reaction using a biochip.

  Embodiments of the present invention will be described below. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined in light of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.

(First embodiment)
The color reaction test kit according to the first embodiment of the present invention stores the biochip 100 and the biochip 100 so as to be visible as shown in FIG. 1 and FIG. 2 which is a cross-sectional view seen from the AA direction. And a case 50 filled with the solution.

  The biochip 100 includes a translucent substrate 15 capable of introducing a plurality of hydroxyl (-OH) groups on its surface, and a plurality of reaction layers 91a, 91b, 91c including probe biomolecules bonded to the surface of the substrate 15. 91d, 91e, 91f, 91g, 91h, 91i are provided.

On the substrate 15, as shown in FIG. 1, a plurality of through holes 41a, 41b, 41c, 41d, 41e, 41f, 41g, 41h, 41i, 42a, 42b, 42c, 42d, 42e, 42f reaching the substrate 15 , 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, 49i are disposed. As a material for the substrate 15, synthetic quartz (SiO ₂ ) or the like can be used. The material of the light shielding film 13 is transition metal such as titanium (Ti), platinum (Pt), chromium (Cr), niobium (Nb), tantalum (Ta), and tungsten (W), or aluminum (Al) and gold ( Metals such as Au) can be used. Other materials for the light shielding film 13 include transition metal oxides such as titanium monoxide (TiO) or titanium dioxide (TiO ₂ ), transition metal nitrides such as titanium nitride (TiN), and titanium carbide (TiC). These transition metal carbides can be used. Each of the plurality of through holes 41a to 49i has a diameter of 300 μm or more.

  As shown in FIG. 2, a plurality of reaction layers 91a, 91b, 91c, 91d, 91e, 91f, 91g, 91h, 91i are formed on the surface of the base 15 exposed through each of the plurality of through holes 41a to 41i. Is arranged. In each of the plurality of reaction layers 91a to 91i, each of a plurality of probe biomolecules such as a plurality of deoxyribonucleic acids (DNA), a plurality of ribonucleic acids (RNA), a plurality of peptide nucleic acids (PNA), or a plurality of proteins. The functional group is covalently bonded to a hydroxyl group (—OH) group on the surface of the substrate 15 as shown in FIG. When the probe biomolecule is DNA, RNA, or PNA, each sequence is designed to be complementary to the target biomolecule to be tested.

Note that the first embodiment is not limited to disposing each of the reaction layers 91a to 91i directly on the surface of the substrate 15. In the example shown in FIG. 4, the silane coupling layers 81a, 81b, 81c, 81d, 81e, 81f, 81g, 81h are formed on the surface of the portion exposed through each of the plurality of through holes 41a to 41i of the base 15. , 81i are arranged. In each of the silane coupling layer 81a~81i, as shown in FIG. 5, each of the methyl groups of the plurality of silane coupling agent (-CH ₃₎ or ethyl (-C ₂ H ₅₎ is the substrate 15 surface It is chemically bonded to the hydroxyl (-OH) group by an acid-base reaction.

  Each of the silane coupling agents includes 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, N-2 (aminoethyl) 3 -Aminopropylmethyldimethoxysilane, N-2 (aminoethyl) 3-aminopropyltrimethoxysilane, N-2 (aminoethyl) 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, and 3-aminopropyl Triethoxysilane or the like can be used.

  On each of the silane coupling layers 81a to 81i, reaction layers 91a, 91b, 91c, 91d, 91e, 91f, 91g, 91h, 91i are arranged. In each of the reaction layers 91a to 91i, the phosphate amidite derivative introduced into each of the plurality of probe biomolecules is covalently bonded to the epoxy group of the silane coupling agent of the silane coupling layers 81a to 81i through a hydrolysis reaction. Yes.

Alternatively, the silane coupling agent and the probe biomolecule may be bonded via a crosslinking agent. For example, receptors, ligands, antagonists, antibodies, amino of a lysine contained in the protein antigen such as (Lys) (-NH ₂₎ group, a carboxyl (-COOH) group of aspartic acid (Asp) and glutamic acid (Glu), tyrosine Functional groups such as (Tyr) phenol (-C ₆ H ₄ (OH)) group, histidine (His) imidazole (-C ₃ H ₃ N ₂ ) group, cysteine (Cys) thiol (-SH) group, etc. The amino group or epoxy group of the silane coupling agent may be bonded with a crosslinking agent. In the example shown in FIG. 6, the amino silane coupling agent (-NH ₂₎ group and antibodies 95a, 95b, an amino (-NH ₂₎ group 95c, is reacted with an amino (-NH ₂₎ group at both ends The cross-linking agent is bonded with disuccinimidyl suberate (DSS).

Other crosslinking agents include bissulfosuccinimidyl suberate (Bis [Sulfosuccinimidyl] suberate: BS ³ ), dimethyl suberimidate (HCl: DMS), and disuccicinimid, which react with amino groups at both ends. Disuccinimidyl glutarate (DSG), Loman's Reagent, 3, 3'-dithiobissulfosuccinimidyl propionate (3,3'-Dithiobis [sulfosuccinimidyl propionate]: DTSSP), ethylene glycol bis Succinimidyl succinate (Ethylene glycol bis [succinimidylsuccinate]: EGS), 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (1-Ethyl-3-[3 -Dimethylaminopropyl] carbodiimide Hydrochloride (EDC) can be used.

  Furthermore, as cross-linking agents, m-maleimidobenzyl-N-hydroxysuccinimide ester (MBS), which reacts with amino group and thiol group, succinimidyl 4- [N-maleimidomethyl] -cyclohexane- 1-Carboxylate (Succinimidyl 4-[N-maleimidophenyl]-cyclohexane-1-carboxylate: SMCC), Succinimidyl 4-[p-Maleimidophenyl]-Butyrate (Succinimidyl 4-[p-maleimidophenyl]-buthrate: SMPB), N -Succinimidyl 3- (2-pyridyldithio) propionate (N-Succinimidyl 3- [2-pyridyldithio] propionate: SPDP), N- (γ-maleimidobutyryloxy) sulfosuccinimide ester (N- [γ-Maleimidobutyloxy] sulfosuccinimide ester: Sulfo-GMBS), Sulfosuccinimidyl 6-[3 '(2-Pyridyldithio)-propionamide] Hexanoate (Sulfosuccinimidyl 6-[3 '(2-pyridyldithio)-propionamide] hexanoate: Sulfo-LC-SPDP), m-Maleimidebenzoyl-N-hydoroxysulfo-succinimide ester: Sulfo-MBS) Sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfosuccinimidyl 4 [N-maleimidomethyl]-cyclohexane-1-carboxylate: Sulfo-SMCC), Sulfosuccinimidyl-4- ( p-maleimidophenyl) butyrate (Sulfosuccinimidy 4-[p-maleimidophenyl] -butyrate: Sulfo-SMPB) can be used. Each of the other through holes 42a to 42i, 43a to 43i, 44a to 44i, 45a to 45i, 46a to 46i, 47a to 47i, 48a to 48i, and 48a to 48i, 49a to 49i shown in FIG. The description is omitted because it is similar to FIGS.

  The case 50 shown in FIG. 2 is disposed on the transparent substrate 10 on which the biochip 100 is disposed, on the substrate 10, on the frame 20 that surrounds the biochip 100, and on the frame 20. A transparent cover plate 21 is provided. The substrate 10 is made of synthetic quartz or the like, and a slide glass or the like can be used. The frame 20 has oxygen permeability and adsorbs the substrate 10 and the cover plate 21. Therefore, the solution does not leak from the contact surface between the frame 20 and the substrate 10. Further, the solution does not leak from the contact surface between the frame 20 and the cover plate 21. For example, the frame 20 is made of silicone rubber such as polydimethylsiloxane (PDMS). The cover plate 21 is made of, for example, silicone rubber or synthetic quartz, and a cover glass or the like can be used. Each of the substrate 10 and the cover plate 21 may have oxygen permeability.

  Next, the color reaction test method according to the first embodiment using the color reaction test kit shown in FIGS. 1 and 2 will be described.

  (a) As shown in FIG. 7, the biochip 100 is placed on the substrate 10. Next, as shown in FIG. 8, a frame 20 is arranged on the substrate 10 so as to surround the biochip 100, and the substrate 10 and the adsorbing frame 20 are brought into close contact with each other. Thereafter, a test solution containing a target biomolecule labeled with biotin is dropped into a space surrounded by the substrate 10 and the frame 20, and the frame 20 is filled with the test solution. Thereafter, the biochip 100 is left for a time required for the interaction between the target biomolecule and the probe biomolecules of the plurality of reaction layers 91a to 91i. While left untreated, the cover plate 21 may be disposed on the frame 20 as shown in FIG. Thereafter, as shown in FIG. 8, the cover plate 21 is removed, the test solution is discarded, the biochip 100 is washed, and unreacted target biomolecules are removed.

  (b) A first reagent solution containing horseradish peroxidase (HRP) -labeled streptavidin is dropped into the space surrounded by the substrate 10 and the frame 20, and the inside of the frame 20 is filled with the first reagent solution. Fulfill. Thereafter, the biochip 100 is left for a time required for interaction with biotin for labeling the target biomolecule. While left untreated, the cover plate 21 may be disposed on the frame 20 as shown in FIG. Thereafter, as shown in FIG. 8, the cover plate 21 is removed, the first reagent solution is discarded, the biochip 100 is washed, and unreacted HRP-labeled streptavidin is removed.

  (c) A second reagent solution containing tetramethylbenzidine (TMB) is dropped into a space surrounded by the substrate 10 and the frame 20, and the inside of the frame 20 is filled with the second reagent solution. Next, as shown in FIG. 2, the lid plate 21 is disposed on the frame 20, and the adsorbing frame 20 and the lid plate 21 are brought into close contact with each other. As a result, the space surrounded by the substrate 10, the frame 20, and the lid plate 21 is filled with the second reagent solution. If the target biomolecule is trapped in each of the plurality of reaction layers 91a to 91i, the TMB is oxidized by the HRP bonded to the target biomolecule and colored when left for 30 minutes thereafter. The color reaction is confirmed from above the transparent cover plate 21, and the color reaction test method according to the first embodiment is completed.

According to the color reaction test kit and the color reaction test method according to the first embodiment described above, the case 50 storing the biochip 100 has a test solution, a first reagent solution, or a second reagent. Filled with solution. Therefore, it is possible to reduce the amounts of the test solution, the first reagent solution, and the second reagent solution. Even when the color reaction test kit is carried around, the case 50 is filled with the test solution, the first reagent solution, or the second reagent solution, so that the test solution, the first reagent solution, or the second reagent solution is filled. This reagent solution does not spill from each surface of the plurality of reaction layers 91a to 91i. In addition, oxygen (O ₂ ) is required for color development of TMB by HRP. On the other hand, the frame 20 is impermeable to the solution but has oxygen permeability, so it does not interfere with the color development reaction of TMB by HRP.

  Further, in the biochip 100 according to the first embodiment, each of the plurality of reaction layers 91a to 91i is arranged in a spot shape on the translucent substrate 15, and further around each of the plurality of reaction layers 91a to 91i. A light shielding film 13 is disposed on the surface. Therefore, if illumination light is irradiated from the back surface opposite to the surface on which the light shielding film 13 of the substrate 15 is disposed, the contrast is improved by the light shielding film 13, so the presence or absence of a color reaction in each of the reaction layers 91a to 91i. Can be easily confirmed by the transmitted light of the substrate 15. In addition, by setting the diameters of the plurality of through holes 41a to 49i to 300 μm or more, it is possible to visually confirm the presence or absence of a color reaction in each of the reaction layers 91a to 91i. Therefore, it is not necessary to use an expensive scanner. Of course, the coloring reaction may be confirmed from the transparent substrate 10 side.

  Here, the test kit according to the comparative example shown in FIG. 9 includes a substrate 300 having a hydrophobic surface and a biochip 200. The biochip 200 includes a substrate 15 and a plurality of reaction layers 91a to 91i. When an inspection method is performed using the inspection kit according to the comparative example, a reagent solution 72 containing a coloring reagent is dropped on the biochip 200, and the dropped reagent solution is biodegraded by the water-repellent action of the hydrophobically treated substrate 300. It is held on the chip 200. In this case, when an impact is applied to the test kit according to the comparative example by carrying it, the interfacial tension between the biochip 200 and the reagent solution 72, the interfacial tension between the substrate 300 and the reagent solution 72, the reagent solution 72 and the surrounding gas The surface tension between the substrate 300 and the surface tension between the substrate 300 and the surrounding gas is lost, and the reagent solution 72 flows as shown in FIG. When the reagent solution 72 flows, for example, a coloring reaction occurring on part or all of the reaction layer 91a may move from the reaction layer 91a. Therefore, it is difficult to confirm in which reaction layer the color development reaction has occurred. In particular, if the diameter of each of the reaction layers 91a to 91i is set to 300 μm or more so that the presence or absence of a color reaction can be confirmed with the naked eye, the area of the biochip 200 also increases, so that the reagent solution 72 flows more. It becomes easy. Therefore, the fluidity of the reagent solution 72 cannot be ignored. That is, if the diameter of each of the plurality of reaction layers 91a to 91i is set to 300 μm or more so as to be confirmed with the naked eye, the fluidity of the reagent solution 72 is increased, and it is difficult to confirm the color development of TMB by HRP.

  On the other hand, in the color reaction test kit according to the first embodiment shown in FIG. 2, the biochip 100 is stored in the case 50, and the case 50 is filled with the reagent solution. Therefore, even when an impact is applied from the outside, the balance of the interfacial tension between the case 50 and the reagent solution is not easily broken. Therefore, the flow of the reagent solution on the biochip 100 can be suppressed, and the color development reaction can be stabilized. In addition, the thickness of the solution in the direction perpendicular to the biochip surface can be forcibly reduced by the case 50. Therefore, it is possible to reduce the number of solutions required for the inspection, reduce the inspection cost, and make it easy and useful to implement the color reaction test method.

  Next, a method for manufacturing the biochip 100 according to the first embodiment will be described with reference to FIGS.

(a) As shown in FIG. 11, a base 15 made of synthetic quartz or the like is prepared, and a light shielding film 13 made of Ti or the like is formed on the base 15 using a sputtering method or a chemical vapor deposition method (CVD) method. Form. After the formation of the light shielding film 13, the surface is treated with oxygen (O ₂ ) plasma for 5 minutes. Next, as shown in FIG. 12, a polymer film 11 is formed on the light-shielding film 13 by spin-coating a photosensitive epoxy resin-containing solution such as Syu 8 (SU-8) 3000 series manufactured by Kayaku Microchem Corporation. As conditions for spin coating, for example, acceleration is performed to 300 revolutions per minute over 5 seconds, and 300 revolutions per minute is maintained for 10 seconds. It further accelerates to 500 revolutions per minute over 5 seconds and maintains 500 revolutions per minute for 15 seconds. Then it accelerates to 4500 revolutions per minute over 5 seconds and maintains 4500 revolutions per minute for 30 seconds. Finally, stop spinning for 5 seconds.

  (b) After the polymer film 11 is formed, the polymer film 11 is pre-cured (pre-baked). First, the substrate 15 is placed on a hot plate set at 65 ° C., and after 2 minutes, the hot plate is set at 80 ° C. After 20 minutes, set the hot plate at 95 ° C and leave it for 15 minutes. After 15 minutes, turn off the hot plate and leave it for 1 hour. Thereafter, the shapes of the plurality of through holes 41a to 41i, 42a to 42i, 43a to 43i, 44a to 44i, 45a to 45i, 46a to 46i, 47a to 47i, 48a to 48i, 49a to 49i shown in FIG. A part of the polymer film 11 is selectively exposed with ultraviolet rays using a photomask having a mask pattern corresponding to the above. After the exposure, the polymer film 11 is subjected to a post-exposure bake (PEB) process. Specifically, the substrate 15 is placed on a hot plate set at 65 ° C., and after 2 minutes, the hot plate is set at 95 ° C. and left for 6 minutes. After 6 minutes, turn off the hot plate and leave it for 1 hour. Thereafter, when the polymer film 11 is developed using SU-8 developer or the like, the polymer film 11 has photosensitivity, so that a part of the polymer film 11 is selectively removed as shown in FIG. .

(c) A part of the light shielding film 13 is selectively removed by isotropic wet etching using the polymer film 11 from which part has been selectively removed as an etching mask. As the etching solution, a mixed solution of hydrofluoric acid (HF), nitric acid (HNO ₃ ), water (H ₂ O), or the like can be used. By selective removal, each of the plurality of through holes 41a to 41i, 42a to 42i, 43a to 43i, 44a to 44i, 45a to 45i, 46a to 46i, 47a to 47i, 48a to 48i, 49a to 49i shown in FIG. Is formed.

  (d) Next, the substrate 15 is left in a stirred sodium hydroxide (NaOH) solution at room temperature for 2 hours. Here, the NaOH solution is a solution in which 98 g of NaOH, 294 ml of distilled water, and 392 ml of ethanol are mixed. By leaving it in the NaOH solution, as shown in FIG. 15, a plurality of hydroxyl (—OH) groups are introduced onto the surface of the base 15 exposed from each of the through holes 41a to 41i. The introduction of a hydroxyl (—OH) group may be performed with a UV ozone cleaner or the like.

  (e) Silane coupling agents having an epoxy group on the functional group such as 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, or N-2 (aminoethyl) 3-aminopropyltri Silane coupling agents having amines in functional groups such as ethoxysilane, 3-aminopropyltrimethoxysilane, and 3-aminopropyltriethoxysilane, etc., are exposed from each of the through holes 41a to 41i shown in FIG. It is dropped on the surface to form silane coupling layers 81a, 81b, 81c, 81d, 81e, 81f, 81g, 81h, 81i shown in FIG. For example, when a silane coupling agent having an epoxy group is dropped in an environment of 15 ° C., a plurality of epoxy groups are introduced on the surface of the substrate 15 as shown in FIG. Note that the position of the through holes 41a to 41i can be easily determined when the silane coupling agent is dropped by the contrast between the translucent substrate 15 and the light shielding film 13 shown in FIG.

  (f) Unreacted hydroxyl (—OH) groups remaining on the surface of the substrate 15 are acetylated by, for example, treating with acetic anhydride and 1-methylimidazole (tetrahydrofuran solution), and capping. Next, as shown in FIG. 18, the epoxy group of the silane coupling agent introduced onto the surface of the substrate 15 is hydrolyzed to introduce a hydroxyl group (—OH) into each of the silane coupling layers 81a to 81i.

  (g) As shown in FIG. 19, a first base nucleoside is prepared in which the 5 'end is protected with a dimethoxytrityl (DMTr) group and the 3' end hydroxyl group is a trivalent phosphate amidite derivative. As shown in FIG. 20, the amino groups of adenine (A) and cytosine (C) contained in the nucleoside are protected with a benzoyl group, and the amino group of guanine (G) is protected with an isobutyl group. Next, the first base nucleoside is dropped onto each of the silane coupling layers 81a to 81i shown in FIG. By dropping, as shown in FIG. 21, the cyanoethyl amidite derivative of the first base nucleoside is bound to the hydroxyl group (—OH) of the silane coupling agent using a base catalyst or the like.

  (h) The unreacted hydroxyl (—OH) group of the silane coupling agent is acetylated by treating with acetic anhydride and 1-methylimidazole (tetrahydrofuran solution) to inhibit the binding of the second and subsequent nucleosides. Next, the dimethoxytrityl (DMTr) group of the first base nucleoside was deprotected with 3% trichloroacetic acid / dichloromethane acidic solvent, and the 5 ′ hydroxyl (-OH) group was removed from the first base nucleoside as shown in FIG. To introduce.

  (i) The second base nucleoside in which the hydroxyl group at the 3 ′ end is a trivalent phosphate amidite derivative is dropped onto the silane coupling layers 81a to 81i, and as shown in FIG. 'The cyanoethylamidite derivative of the second base nucleoside is bonded to the hydroxyl group (-OH) by a condensation reaction using a base catalyst or the like. Note that the unreacted 5 'hydroxyl (-OH) group of the first base nucleoside is acetylated by treatment with acetic anhydride and tetrahydrofuran solution and capped.

  (j) The phosphite triester bond generated by the condensation reaction is oxidized with iodine and water (pyridine-containing tetrahydrofuran solution) and converted to a more stable phosphate triester bond as shown in FIG. Thereafter, the second base dimethoxytrityl (DMTr) group was removed, and the condensation reaction with the nucleoside phosphoramidite was repeated until the target DNA chain length was reached as shown in FIG. 25, and reaction layers 91a to 91a shown in FIG. 91i is formed.

(k) After the DNA elongation, an alkaline solution treatment is performed in which the substrate 15 is immersed in an alkaline solution such as ammonia (NH ₄ OH) water at 55 ° C. for 30 minutes so that the polymer film 11 is immersed. The weight percent concentration of NH ₄ OH water is 40% at the time of adjustment. By the alkaline solution treatment, the cyanoethyl protecting group bonded to the phosphate group is eliminated as shown in FIG. In addition, bases such as adenine (A), cytosine (C), and guanine (G) are deprotected as shown in FIG. Furthermore, the adhesion between the light shielding film 13 and the polymer film 11 is reduced by the alkaline solution treatment. Thereafter, the substrate 15 is taken out from the NH ₄ OH water, and a gas such as air is blown onto the side surface 115 of the polymer film 11 shown in FIG. Finally, the terminal dimethoxytrityl (DMTr) group is deprotected as shown in FIG. 29, and the manufacturing method of the biochip 100 according to the first embodiment is completed.

  Note that the method of manufacturing the biochip 100 according to the first embodiment is not limited to this. For example, it has been described in FIG. 11 that the light shielding film is formed on the substrate 15 by using a sputtering method or a chemical vapor deposition method (CVD) method. On the other hand, the light-shielding film 13 having the plurality of through holes 41a to 49i may be formed on the base 15 using, for example, a UV curable black screen ink. Alternatively, the light-shielding film 13 having a plurality of through holes 41a to 49i may be prepared in advance and pasted on the substrate 15 with an adhesive or the like. Also in this case, the material of the light shielding film 13 is not limited to metals, and resin, insoluble paper, or the like may be used. In addition, a light shielding film may be formed using an inkjet printer or the like.

(Second embodiment)
As shown in FIG. 31, the case 150 of the color reaction test kit shown in FIG. 30 according to the second embodiment of the present invention is provided with a recess 111, and the biochip 100 is arranged inside the recess 111. A cover plate 121 shown in FIG. 30 covering the substrate 110 and the recess 111 of the substrate 110 is provided. The substrate 110 is made of quartz or the like. The cover plate 121 has oxygen permeability. The cover plate 121 is made of, for example, silicone rubber such as polydimethylsiloxane, and has an adsorptivity to the substrate 110. The other components of the color reaction test kit according to the second embodiment are the same as those in FIG.

  Next, a color reaction test method according to the second embodiment using the color reaction test kit shown in FIG. 30 will be described.

  (a) As shown in FIG. 32, the biochip 100 is placed in the recess 111 of the substrate 110. Next, a test solution containing a target biomolecule labeled with biotin is dropped into the recess 111 of the substrate 110 to fill the recess 111 with the test solution. Thereafter, the biochip 100 is left for a time required for the interaction between the target biomolecule and the probe biomolecule included in each of the reaction layers 91a to 91i. While left untreated, the recess 111 of the substrate 110 may be covered with a cover plate 121 as shown in FIG. Thereafter, as shown in FIG. 32, the cover plate 121 is removed, the test solution is discarded, the biochip 100 is washed, and unreacted target biomolecules are removed.

  (b) A first reagent solution containing HRP-labeled streptavidin is dropped into the recess 111 of the substrate 110, and the recess 111 is filled with the first reagent solution. Thereafter, the biochip 100 is left for a time required for interaction with biotin for labeling the target biomolecule. While left untreated, the recess 111 of the substrate 110 may be covered with a cover plate 121 as shown in FIG. Thereafter, as shown in FIG. 32, the cover plate 121 is removed, the first reagent solution is discarded, the biochip 100 is washed, and unreacted HRP-labeled streptavidin is removed.

  (c) A second reagent solution containing TMB is dropped into the recess 111 of the substrate 110 to fill the recess 111 with the second reagent solution. Next, as shown in FIG. 30, the cover plate 121 covers the recess 111 of the substrate 110, and the adsorbent cover plate 121 and the substrate 110 are brought into close contact with each other. As a result, the concave portion 111 of the substrate 110 covered with the cover plate 121 is filled with the second reagent solution. If the target biomolecule is trapped in each of the plurality of reaction layers 91a to 91i, the TMB is oxidized by the HRP bonded to the target biomolecule and colored when left for 30 minutes thereafter. The color reaction is confirmed from above the transparent cover plate 121, and the color reaction test method according to the second embodiment is completed.

  According to the color reaction test kit and the color reaction test method according to the second embodiment described above, the recess 111 of the substrate 110 storing the biochip 100 includes the test solution, the first reagent solution, and the second It is filled with one of the reagent solutions and covered with a cover plate 121. Therefore, even when the color reaction test kit is carried around, the flow of the test solution, the first reagent solution, or the second reagent solution on the biochip 100 can be suppressed. In addition, since the lid plate 121 has oxygen permeability, it does not hinder the color development reaction of TMB by HRP.

(Third embodiment)
As shown in FIG. 33, the biochip 120 according to the third embodiment includes a substrate 25 provided with a recess 122, and a plurality of reactions each including a probe biomolecule bonded to the bottom surface of the recess 122 of the substrate 25. Layers 91a-91i are provided. The color reaction test kit according to the third embodiment includes a biochip 120 and a cover plate 121 that covers the recess 122 of the base 25 of the biochip 120, as shown in FIG. As a material for the transparent substrate 25, synthetic quartz or the like can be used. The cover plate 121 has oxygen permeability and adsorbability with respect to the base 25. The lid plate 121 is made of, for example, silicone rubber such as polydimethylsiloxane and has an adsorptivity to the substrate 110. The other components of the color reaction test kit according to the third embodiment are the same as those shown in FIG.

  Next, a color reaction test method according to the third embodiment using the color reaction test kit shown in FIG. 34 will be described.

  (a) A test solution containing a target biomolecule labeled with biotin is dropped into the recess 122 of the substrate 25 of the biochip 120 shown in FIG. 33, and the recess 122 is filled with the test solution. Thereafter, the biochip 120 is left for the time required for the interaction between the target biomolecule and the probe biomolecule included in each of the reaction layers 91a to 91i. While left untreated, the recess 122 of the base body 25 may be covered with a cover plate 121 as shown in FIG. Thereafter, as shown in FIG. 33, the cover plate 121 is removed, the test solution is discarded, the biochip 120 is washed, and unreacted target biomolecules are removed.

  (b) A first reagent solution containing HRP-labeled streptavidin is dropped into the concave portion 122 of the substrate 25 to fill the concave portion 122 with the first reagent solution. Thereafter, the biochip 120 is left for a time required for interaction with biotin for labeling the target biomolecule. While left untreated, the recess 122 of the base body 25 may be covered with a cover plate 121 as shown in FIG. Thereafter, as shown in FIG. 33, the cover plate 121 is removed, the first reagent solution is discarded, the biochip 120 is washed, and unreacted HRP-labeled streptavidin is removed.

  (c) A second reagent solution containing TMB is dropped into the inside of the recess 111 of the substrate 25, and the inside of the recess 122 is filled with the second reagent solution. Next, as shown in FIG. 34, the cover plate 121 covers the concave portion 111 of the base body 25 so that the adsorbent cover plate 121 and the base body 25 are brought into close contact with each other. As a result, the recess 111 of the base body 25 covered with the cover plate 121 is filled with the second reagent solution. If the target biomolecule is trapped in each of the plurality of reaction layers 91a to 91i, the TMB is oxidized by the HRP bonded to the target biomolecule and colored when left for 30 minutes thereafter. The color reaction is confirmed from above the transparent cover plate 121, and the color reaction test method according to the third embodiment is completed.

  According to the color reaction test kit and color reaction test method according to the third embodiment described above, the recess 122 of the base 25 of the biochip 120 is the test solution, the first reagent solution, and the second reagent solution. And covered with a cover plate 121. Therefore, even when the color reaction test kit is carried around, the flow of the test solution, the first reagent solution, or the second reagent solution on the biochip 120 can be suppressed. In addition, since the lid plate 121 has oxygen permeability, it does not hinder the color development reaction of TMB by HRP.

(Other embodiments)
As described above, the present invention has been described according to the embodiment. However, it should not be understood that the description and drawings constituting a part of this disclosure limit the present invention. From this disclosure, various alternative embodiments, examples and operational techniques should be apparent to those skilled in the art. For example, the light shielding film 13 provided with a plurality of through holes 41a to 41i may be disposed on the bottom surface of the recess 122 of the base body 25 shown in FIG. 33, as shown in FIG. In this case, the plurality of reaction layers 91a to 91i are arranged on the surface of the substrate 15 exposed through each of the plurality of through holes 41a to 41i. Since the contrast is improved by the light shielding film 13, it is possible to easily confirm the color development reaction in each of the reaction layers 91a to 91i from above the cover plate 121 shown in FIG. Thus, it should be understood that the present invention includes various embodiments and the like not described herein. Therefore, the present invention is limited only by the invention specifying matters in the scope of claims reasonable from this disclosure.

It is a top view of the color reaction test kit according to the first embodiment of the present invention. It is sectional drawing of the color reaction test kit which concerns on the 1st Embodiment of this invention. It is a 1st expanded sectional view of the biochip which concerns on the 1st Embodiment of this invention. 1 is a first cross-sectional view of a biochip according to a first embodiment of the present invention. It is a 2nd expanded sectional view of the biochip which concerns on the 1st Embodiment of this invention. It is a 3rd expanded sectional view of the biochip which concerns on the 1st Embodiment of this invention. It is a 1st process sectional view of the coloring reaction inspection method concerning a 1st embodiment of the present invention. It is a 2nd process sectional view of the coloring reaction inspection method concerning a 1st embodiment of the present invention. It is the 1st sectional view of the biochip concerning a comparative example of the present invention. It is 2nd sectional drawing of the biochip which concerns on the comparative example of this invention. It is 1st process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is 2nd process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is 3rd process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is a 4th process sectional view of the biochip concerning a 1st embodiment of the present invention. It is 5th process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is 6th process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is 7th process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is 8th process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. 1 is a chemical formula of a nucleoside phosphoramidite according to the first embodiment of the present invention. 1 is a first chemical formula of a base of a nucleoside according to a first embodiment of the present invention. It is a 9th process sectional view of the biochip concerning a 1st embodiment of the present invention. It is a 10th process sectional view of the biochip concerning a 1st embodiment of the present invention. It is 11th process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is a 12th process sectional view of the biochip concerning the 1st embodiment of the present invention. It is a 13th process sectional view of the biochip concerning the 1st embodiment of the present invention. It is 14th process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is 2nd chemical formula of the base of the nucleoside which concerns on the 1st Embodiment of this invention. It is a 15th process sectional view of the biochip concerning the 1st embodiment of the present invention. It is 16th process sectional drawing of the biochip which concerns on the 1st Embodiment of this invention. It is 1st sectional drawing of the kit for color reaction test based on the 2nd Embodiment of this invention. It is 2nd sectional drawing of the color reaction test kit based on the 2nd Embodiment of this invention. It is process sectional drawing of the kit for color reaction test based on the 2nd Embodiment of this invention. It is sectional drawing of the biochip which concerns on the 3rd Embodiment of this invention. It is sectional drawing of the kit for color reaction test based on the 3rd Embodiment of this invention. It is sectional drawing of the biochip which concerns on other embodiment of this invention. It is sectional drawing of the kit for color reaction test which concerns on other embodiment of this invention.

Explanation of symbols

10, 110… Board
11 ... polymer membrane
13 ... Light shielding film
15, 25… Base
20 ... Frame
21, 121 ... Lid
41a-49i ... through hole
50, 150 ... case
71a-71i ... solution
81a-81i ... Silane coupling layer
91a-91i ... Reaction layer
95a, 95b, 95c… antibody
100, 120, 200… Biochip
111, 122 ... recess
115 ... side

Claims (10)

A substrate,
A biochip disposed on the substrate and having a probe biomolecule;
A frame disposed on the substrate and surrounding the biochip;
A lid plate disposed on the frame and covering the biochip,
A kit for color reaction test, wherein a space surrounded by the substrate, the frame, and the cover plate is filled with a reagent solution containing a color reagent for detecting a target biomolecule that binds to the probe biomolecule.   The color reaction test kit according to claim 1, wherein either the frame or the cover plate has oxygen permeability.   The color reaction test kit according to claim 1, wherein the frame has adsorptivity. A substrate provided with a recess;
A biochip disposed in a recess of the substrate and having a probe biomolecule;
A cover plate covering the concave portion of the substrate,
A color reaction test kit in which a concave portion of the substrate covered with the cover plate is filled with a reagent solution containing a color reagent for detecting a target biomolecule that binds to the probe biomolecule. A substrate provided with a recess;
A probe biomolecule bound to the bottom surface of the recess of the substrate;
A cover plate covering the concave portion of the base,
A color reaction test kit in which a concave portion of the substrate covered with the cover plate is filled with a reagent solution containing a color reagent for detecting a target biomolecule that binds to the probe biomolecule.   The color reaction test kit according to claim 4 or 5, wherein the cover plate has oxygen permeability.   The color reaction test kit according to any one of claims 4 to 6, wherein the lid plate has adsorptivity. Placing a biochip having a probe biomolecule on a substrate;
Disposing a frame surrounding the periphery of the biochip on the substrate;
Dropping a test solution containing a target biomolecule that binds to the probe biomolecule into the frame;
Dropping a reagent solution containing a coloring reagent for detecting the target biomolecule into the frame;
Covering the frame filled with the reagent solution with a cover plate;
Detecting a color reaction of the color reagent contained in the reagent solution filled in the space surrounded by the substrate, the frame, and the cover plate. Arranging a biochip having a probe biomolecule in the recess of the substrate provided with the recess;
Dropping a test solution containing a target biomolecule that binds to the probe biomolecule into the recess;
Dropping a reagent solution containing a coloring reagent for detecting the target biomolecule into the recess;
Covering the concave portion of the substrate filled with the reagent solution with a cover plate;
Detecting a color reaction of the color reagent contained in the reagent solution filled in the concave portion of the substrate covered with the lid plate. Dropping a test solution containing a target biomolecule that binds to the probe biomolecule into the recess of a biochip including a substrate provided with a recess and a probe biomolecule bonded to the bottom surface of the recess of the substrate;
Dropping a reagent solution containing a coloring reagent for detecting the target biomolecule into the recess;
Covering the recess of the substrate filled with the reagent solution with a lid plate;
A color reaction test method comprising: detecting a color reaction of the color reagent contained in the reagent solution filled in the recess of the substrate covered with the cover plate.

Images