KR20130020402A - Biochip using the insulated membrane of metal oxide - Google Patents

Abstract

PURPOSE: A bio-chip using an insulating film of a metallic oxide is provided to obtain a bio-chip which does not generate changes in the measurement by a sensor. CONSTITUTION: A bio-chip(1) using an insulating film of a metallic oxide comprises one or more wells(131a) for arranging and fixing probe substances and a substrate(10). One or more electrode wires are formed on the substrate and coated with the insulating film of the metallic oxide. One or more holes reaching a surface of an electrode are formed on the insulating film on each area of the electrode wires.

Description

Biochip using the insulated membrane of metal oxide

The present invention relates to a biochip using an insulating film of a metal oxide. More specifically, the present invention relates to a biochip having an insulating film of a metal oxide in which a plurality of wells are formed and having improved detection sensitivity.

In recent years, in the fields of new drug development processes, medical diagnostics, and the like, biochips have been attracting attention, and research and development are actively progressing.

Biochips are immobilized nucleic acids such as DNA, proteins such as enzymes and antibodies, biomolecule arrays such as peptides, or cells on solid surfaces (such as silicon substrates, glass substrates, polymers, and gold substrates). The specific reaction generated when a specific target substance is bound to a probe substance such as an immobilized biomolecule array is detected (Patent Documents 1 and 2).

In addition, biochips using liposomes, β-amyloid oligomers, and reducing sugars have also been proposed as probe materials [Patent Documents 3 and 4].

Various probe materials are available, and the usefulness of biochips is increasing. In particular, since a large amount of samples can be used to detect and interpret high throughput at high throughput, a large amount of simultaneous processing is required. Biochip-related technologies are becoming essential for the functional analysis technology of biomolecule arrays in the post-genome era.

The biochip of patent documents 1-4 forms one or several electrode wirings on a board | substrate, coat | covers the one or several electrode wirings with an insulating film, and the insulating film in each area | region of each of these 1 or several electrode wirings. One or more wells are formed by forming one or a plurality of holes reaching the surface of the electrode at the bottom, and then, by fabricating the probe material at the bottom of the one or more wells.

The top view of such a biochip is shown in FIG. Here, eight electrode wirings 13 are formed on the glass substrate 10, and the biochip 1 which can measure 8 points at a time is formed.

At one end of the electrode wiring 13, a biomolecule array region 131 is formed, and at the other end, a pad 132 for taking out an electrical signal detected in the biomolecule array region is formed.

2 shows an enlarged top view of the biomolecule array region 131. An insulating resist film 14 is formed on the biomolecule array region 131, and one or a plurality of wells 131a for immobilizing the biomolecules are formed by forming one or a plurality of holes in the insulating film.

When the diameter of the well 131a is 5 µm or more in the biomolecular array region 131, a single well can be formed as shown in FIG. 2A, and the diameter of the well 131a is about 20 nm or less in submicron range. If so, a plurality of wells can be arranged as shown in Fig. 2B.

A cross-sectional view of the biomolecule array region 131 is shown in FIG. 3A. Here, on the glass substrate 10, first, the Ti thin film 13a was formed and the Au thin film 13b was formed on it, and the electrode wiring 13 which consists of Au / Ti laminated thin films was formed. Although the Au thin film can be formed directly on the glass substrate, since the adhesive strength of the Au thin film is weak, the Ti thin film was used to prevent peeling of the Au thin film and to improve the reliability of the biochip. Cr may be used in addition to Ti.

As this figure shows, the hole formed in the insulating resist film reaches the surface of the electrode wiring of a lower layer. That is, the side surface of the well formed in the biomolecule array region is determined by the insulating resist film 14, and the bottom of the well is defined by the surface of the electrode wiring 13.

By this structure, as shown in FIG. 3B, the biomolecule which becomes a probe is immobilized only on the exposed electrode surface of the bottom part of the well 131a. In this figure, the lipid double layer 4 is immobilized on the electrode surface, and the probe protein 2 is immobilized thereon.

In order to manufacture such a biochip, photofabrication technology, which is one of semiconductor manufacturing technologies, may be used. Here, the term "photo fabrication technique" means a technique which combines a photolithography technique, a vapor deposition technique, an etching technique, and the like.

Since photofabrication technology has already been established as one of IC and LSI manufacturing technology, miniaturization and automation of manufacturing processes are possible as with conventional DNA chips, leading to mass production and low cost of chips.

4-9 is a schematic diagram which shows the process of manufacturing a biochip by photo fabrication technique.

First, the process of manufacturing a single well of 5 micrometers or more in diameter is demonstrated. The photoresist 11 which is a photosensitive material is apply | coated on the board | substrate 10 using a spin coater, and it bakes at 90 degreeC for 2 minutes (FIGS. 4A and 4B). Although a glass substrate was used in this description, a substrate made of any material such as an alumina substrate, a silicon substrate, or a resin substrate such as a silicone resin can be used.

Subsequently, the photomask is exposed to ultraviolet light 12a for 20 seconds (FIG. 4C), and the photoresist 11 is developed to form a wiring pattern for the electrode wiring 13 (FIG. 4D). The photoresist may be a bond that is decomposed by exposure to be dissolved in a developer (positive type), and a polymer is not polymerized to be dissolved (negative type). In this case, the photoresist 11 is a positive resist (AZ1500: Clariant Japan Corporation).

Thereafter, a metal thin film serving as an electrode material is formed on the substrate by a vapor deposition technique such as vacuum deposition or RF sputtering (FIG. 4E).

Moreover, this is immersed in the organic solvent like acetone, and the resist 11 is peeled off, and the electrode wiring 13 is formed on the glass substrate 10 (FIG. 4F). 6A shows a schematic top view of a chip on which electrode wiring 13 is formed. In Fig. 6, a circular biomolecule array region 131 is formed at one end, and electrode wirings having a square pad 132 for extracting electrical signals are shown at the other end. As an example, it is not limited to this shape. Those skilled in the art can modify any shape that can be used as a biochip mounted on a biosensor.

Moreover, the number of electrode wirings can also be suitably adapted to the form of use, and can increase and decrease suitably.

Next, an insulating resist 14 (AZ1500) is applied onto the substrate on which the electrode wiring 13 is formed using a spin coater, and baked at 90 ° C. for 2 minutes (FIG. 5A). Subsequently, the photoresist 12b is exposed to ultraviolet light 12a for 20 seconds (FIG. 5B), and the electrode surface is exposed by developing the resist 14 to expose the well 131a on the biomolecule array region 131. To form (FIG. 5C). By baking this at 150 degreeC for 5 minutes, the resist 14 is fixed (FIG. 5D). A schematic top view of a chip with wells formed is shown in FIG. 6B.

Next, a process for producing an array of wells of submicron diameter or less will be described. An electrode wiring was formed in the same manner as when a single well having a diameter of 5 µm or more was formed (FIG. 4), and then on the substrate on which the electrode wiring 13 was formed using a spin coater, similarly to the process shown in FIG. 5A. The insulating resist 14 (ZEP520: Xeon Co., Ltd.) is apply | coated and baked at 90 degreeC for 2 minutes (FIG. 7A). Then, the electron beam 15 is irradiated at 75 kV (FIG. 7B), and the electrode surface is exposed by developing the resist 14, thereby forming an array of a plurality of wells 131 a on the biomolecule array region 131. (FIG. 7C). By baking this at 200 degreeC for 5 minutes, the resist 14 is fixed (FIG. 7D). 8 shows a schematic top view of a chip on which wells are formed.

By using a conventional semiconductor manufacturing technique, a well can be formed on a substrate, and thus, the well can be formed under other conditions that are well known to those skilled in the art, without being limited to the above conditions.

Next, using a method well known in the biochemistry art, a lipid double layer is formed on the electrode surface of the well bottom part. Specifically, the chip is first immersed in a piranha solution (H ₂ O ₂ : H ₂ SO ₄ = 1: 3 [v / v]) for 10 minutes to remove organic impurities on the electrode surface at the bottom of the well. Pure water, washed with ethanol (FIG. 9A), and then immersed in an ethanol solution of 1-octanedecanethiol (ODT) (3) to allow the ODT (3) to surface the electrode. (FIG. 9B).

Subsequently, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) (4) was dissolved in phosphoric acid buffer (PBS, pH7.4) (10 mM), and POPC (4) A liposome solution is prepared. This liposome solution is dropped on a chip and left for 1 hour in an environment of 100% humidity to form a lipid double layer of POPC 4 on the electrode surface (Fig. 9C).

Alternatively, after mixing 99.9 mol% POPC (4) and 0.1 mol% ODT (3) in chloroform well, the solvent is exchanged with PBS to prepare a mixed liposome solution, and the liposome solution is added dropwise to the chip. The lipid double layer of POPC 4 is formed on the surface (FIG. 9C).

In either case, the resist 14 needs to be a material to which the -SH group of the ODT is not bonded, so that the fat double layer is immobilized only on the electrode surface of the well bottom part.

Next, 1 mM (11-ferrocenyl) undecylpolyoxyethylene (Fe-PEG) (5) and 0.1 mM biotinylated 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine Phosphoric acid buffer mixed solution (pH7.4) of (Bio-PE) (21) was added dropwise to the chip on which the lipid double layer was formed, and left to stand for 1 hour to form Fe-PEG (5) and Bio-PE (21) on the lipid double layer. ) Is anchored (FIG. 9D). At this time, by appropriately adjusting the molar concentration ratio in the mixed solution of Fe-PEG (5) and Bio-PE (21), the Bio-PE (21) is dispersed and anchored on the lipid double layer at an appropriate density.

Since the lipid double layer is insulating, it is impossible to detect the phenomenon on the fat double layer as an electrical signal. Therefore, Fe-PEG (5) is used for the purpose of making the surface of a lipid double layer conductive.

A 0.2 mg / ml aqueous solution of streptavidin 22 was added dropwise to the chip on which the biotin-modified lipid double layer was formed, and left to stand for 1 hour in an environment of 100% humidity. By specific binding, streptavidin 22 is bound (FIG. 9E). By washing with PBS, unbound streptavidin that is not immobilized in the lipid double layer is removed.

Streptavidin is a protein that binds biotin most strongly and stably (K _d = ^10-15 M) and binds to four molecules of biotin.

Using specific binding between streptavidin-biotin, a method of combining a molecule modified with streptavidin and another molecule modified with biotin is generally used. Therefore, a technique for modifying biotin to various proteins has already been established, and kits for it are also commercially available. Biotin-modified DNA is also commercially available.

Bio-PE (21) is dispersed and anchored on the lipid double layer, and it is considered that only one biotin is bound to one streptavidin molecule.

Finally, the probe antibody 23 is immobilized at the bottom of the well through specific binding of streptavidin-biotin by immersing the streptavidin immobilization chip in a solution of the previously biotinylated probe antibody 23 (FIG. 9F). ).

When streptavidin is immobilized on a substrate, it is already known that the effective binding number of biotin is one. It is considered that the binding site of streptavidin to which biotin is accessible by immobilization is limited. In particular, in the case of the present invention, since a protein is bound to biotin, it is considered that the biotinylated protein cannot access streptavidin further due to steric hindrance after one biotinylated protein binds to streptavidin. Therefore, it is considered sufficient that one biotinylated probe antibody 23 is bound to one immobilized streptavidin 22.

To produce such a biochip, nanoimprinting lithography (NIL) technology may be used.

In recent years, nanoimprint lithography (NIL) technology is one of the most expected technologies for forming high-resolution, nanoscale patterns in various fields such as electronics, photonics, magnetic devices, and biology.

NIL technology is a technique of pattern-forming in a resin layer by pressing a mold (also called a stamp or a template) to a resin layer, and there exist a thermal NIL technique and an optical NIL technique. In the thermal NIL technique, the resin layer is patterned by pressing the hard mold with a high pressure at a temperature equal to or higher than the glass transition point of the thermoplastic resin, cooling the state, and then removing the mold. In the optical NIL technique, the resin layer is patterned by pressing the mold onto the layer of the photocurable resin, irradiating light such as UV in the state, and then removing the mold.

In addition, there is a step-and-flash imprint lithography (SFIL) as a NIL technique for forming a relief pattern in a large area at a high throughput and low cost. SFIL is UV-NIL performed under the conditions of low temperature and low pressure. In SFIL, since a resin layer is formed only by dripping a low-viscosity photocurable resin on a board | substrate, it is not necessary to perform spin coating. By this technique, the micro pattern of 100 nm or less size can be formed with respect to a large area [nonpatent literature 1-5].

Patent document 5 manufactures the biochip of the structure similar to what was described in FIGS. 1-4 using NIL technique.

10 is a schematic diagram showing a step of producing a biochip by the NIL technique.

(1) UV-NIL method

Using the UV curable resin, an insulating resist resin layer for forming a nano pattern on a SiO ₂ substrate is formed.

First, on the SiO ₂ substrate 16, a 30 wt% UV curable resin diluted with ethanol containing 1 wt% DMPA was spin-coated at 3000 rpm for 20 seconds to form a resin layer of 450 nm thick UV curable resin (17). ). Subsequently, the preheating process is performed for 3 minutes at 70 degreeC on a hotplate, and a solvent is removed (FIG. 10A).

The mold 18 is pressed against the resin layer 17 for 1 minute at a pressure of 0.2 MPa at reduced pressure and at room temperature (FIG. 10B). Since UV curable resin to be used is a liquid of low viscosity, it can imprint at low pressure at room temperature.

UV light of 365 nm to the resin layer 17 of UV curable resin in the state which the pressure was applied to the mold 18 using the nanoimprint apparatus (NM-401: Meishoko Co., Ltd.) equipped with the UV lamp. By irradiating (19) for 1 minute (ultraviolet dose 200mJ / cm <2>), the resin layer 17 of UV curable resin is hardened | cured and the cured film 17a is formed.

The mold 18 is removed from the substrate (FIG. 10C), and the cured film 17a of the UV curable resin in which the nanopattern is formed is heated at 120 DEG C for 1 hour to enhance mechanical strength and solvent resistance.

(2) thermal-NIL method

The thermosetting resin is used to form an insulating resist resin layer forming a nano pattern on a SiO ₂ substrate.

First, on the SiO ₂ substrate 16 cleaned using a UV-ozone cleaner, a thermosetting resin is spin coated at 3000 rpm for 20 seconds to form a resin layer 17 of a 120 nm thick thermosetting resin. Subsequently, preheating is performed for 10 minutes at 80 degreeC on a hotplate, and a solvent is removed (FIG. 10A).

Thereafter, instead of irradiating the UV light 9, the mold 18 is pressed against the resin layer 17 of the thermosetting resin at a relatively high temperature (150 ° C.) and pressure (10 MPa) for 5 minutes (FIG. 10B).

The resin layer 17 of the thermosetting resin is cured by cooling the resin layer 17 of the thermosetting resin in a state where a pressure is applied to the mold 18. Thereafter, the mold is removed from the substrate (Fig. 10C) to form a cured film 17a of a thermosetting resin.

Although only the method of forming a well using the NIL method is shown in this figure, after forming an electrode wiring on the board | substrate 16, a well can be formed on an electrode wiring. When the well is formed by the NIL method, a thin cured film having a thickness of about 10 to 15 nanometers remains on the electrode wiring surface. This unnecessary cured film is removed by performing dry etching for 30 seconds by O ₂ plasma (150 W ICP, RIE 30 W, 250 mTorr, 49 sccm O ₂ ), and etching inductively coupled plasma reactive ions (ICP-RIE), and exposing the electrode surface. In addition, a biochip having an array of a plurality of wells may be formed on a biomolecule array region.

If a biosensor disclosed or suggested in Patent Literatures 1 to 6 is used as a working electrode, and a biosensor including at least a counter electrode and a reference electrode is produced, the target molecules are mutually interacted with the probe material on the biochip. The change of the electrical signal at the time of acting can be detected with high sensitivity, the presence of the target molecule in a sample can be detected, and the amount of the presence can be measured further. Usually, a redox current is used as an electric signal.

Depending on the kind of target molecule, the probe substance can be measured in a wide range of fields using proteins such as DNA and antibodies, liposomes, β-amidoid oligomers, reducing sugars and the like.

[Patent Document 1] Japanese Patent No. 4324707

[Patent Document 2] Japanese Patent No. 4497903

[Patent Document 3] Japanese Patent Laid-Open No. 2008-032554

[Patent Document 4] Japanese Patent Laid-Open No. 2008-105978

[Patent Document 5] Japanese Patent Laid-Open No. 2010-239827

[Patent Document 6] Japanese Patent Laid-Open No. 2010-136157

[Non-Patent Document 1] SY Chou, PR Krauss, JP Renstrom, Appl . Phys . Lett. 1995 , 67 , 3114.

[Non-Patent Document 2] SY Chou, PR Krauss, JP Renstrom, Science 1996 , 272 , 85.

[Non-Patent Document 3] J. Haisma, M. Verheijen, K. Vandenheuvel, J. Vandenberg, J. Vac . Sci . Technol . B 1996 , 14 , 4124.

[Non-Patent Document 4] P. Ruchhoeft, M. Colburn, B. Choi, H. Nounu, S. Johnson, T. Bailey, S. Damle, M. Stewart, J. Ekerdt, SV Sreenivasan, JC Wolfe, CG Willson , J. Vac . Sci . Technol . B 1999 , 17, 2965.

[Non-Patent Document 5] M. Colburn, S. Johnson, M. Stewart, S. Damle, TC Bailey, B. Choi, M. Wedlake, T. Michaelson, SV Sreenivasan, J. Ekerdt, CG Willson, Proc . SPIE - Int . Soc . Opt . Eng . 1999 , 3676 , 379.

[Non-Patent Document 6] LJ Guo, PR Krauss, SY Chou, Appl . Phys . Lett . 1997 , 71 , 1881.

Although the biosensor using the biochip disclosed in patent documents 1-6 as a working electrode is very useful, the present inventors focused on the big fluctuation | variation whenever the redox potential obtained is measured. The present inventors found out that the change in the redox potential is due to the wetting of the insulating resist film of the biochip. That is, since the resist composition is used for the insulating film of the biochip, the resin composition wets with water when the biochip is immersed in the measurement sample, so that a change in the film thickness and the insulating property of the insulating resist film occurs, resulting in the redox potential obtained. I think it is affecting.

It is therefore an object of the present invention to provide a biochip which does not generate measurement fluctuations of the sensor by wetting the insulating resist film.

The present invention is a biochip in which an array of wells for immobilizing probe materials is formed, and electrode wirings on the biochip are exposed at the bottom of the well. An appropriate probe substance can be fixed to the bottom of the plurality of wells formed in the insulating film of the metal oxide by the detection target.

More specifically, the present invention is a biochip formed with one or a plurality of wells for arranging and immobilizing probe materials, wherein one or a plurality of electrode wirings are formed on a substrate, and the one or a plurality of electrode wirings, A biochip in which one or a plurality of wells is formed is formed by forming one or a plurality of holes that are covered with an insulating film of a metal oxide and reach an electrode surface in the insulating film on each region of the one or the plurality of electrode wirings.

In the biochip according to the present invention, a probe substance selected from the group consisting of nucleic acids, proteins, peptides, liposomes, β-amiroid oligomers and reducing sugars is formed on an electrode wiring surface exposed at the bottom of one or a plurality of wells. Can be immobilized

In the biochip of the present invention, the metal oxide is selected from the group consisting of TiO ₂ , SiO ₂ , and ZnO.

The present invention also provides a biosensor comprising at least a working electrode, an antipole, and a reference electrode, wherein the working electrode is immobilized by arranging the biochip of the present invention, that is, a probe material. A biochip in which one or a plurality of wells is formed, wherein one or a plurality of electrode wirings are formed on a substrate, and the one or a plurality of electrode wirings are covered with an insulating film of a metal oxide, and the one or the plurality of electrode wirings. A biochip in which one or a plurality of wells is formed by forming one or a plurality of holes reaching the electrode surface in the insulating film on each region of the substrate, on the electrode wiring surface exposed to the bottom of the one or the plurality of wells, A probe substance selected from the group consisting of nucleic acids, proteins, peptides, liposomes, β-amyloid oligomers and reducing sugars is immobilized and, prior to processing, the first electrical signal of the biochip Measure and process the biochip in a test solution and then measure a second electrical signal of the biochip; Then, from the difference between the intensities of the first electrical signal and the second electrical signal, an unlabeled target molecule that specifically binds to one or a plurality of probe substances present in the test solution is detected. Provide a biosensor.

As used herein, "treatment" refers to a process necessary for specific binding of a probe substance to a non-labeled target molecule. Therefore, in the biochip before the treatment, non-display target molecules are not bound to the one or the plurality of probe substances. If the non-labeled target molecule is present in the test solution, the non-labeled target molecule is specifically bound to the one or more probe substances by this treatment. Before and after performing the above-described processing on the biochip of the present invention, an electrical signal is measured, and if there is a change in the electrical signal, it can be confirmed that non-display target molecules existed in the test solution.

In the present invention, in particular, a redox current value is used as the electrical signal.

That is, according to the present invention, the presence or absence of specific binding of the non-labeled target molecule to the probe substance is detected from the change of the redox state on the biomolecule array of the biochip. Therefore, specific binding can be detected only by measuring the redox current value without displaying a fluorescent substance or the like on the target molecule, thereby making it possible to perform a quick and simple measurement.

In other words, if there is a statistically significant change between the electrical signal measured in a system in which the target molecule is not bound to the probe material and the electrical signal measured in a system in which the target molecule is bound to the probe material, the target change is targeted. Specific binding of molecules to probe materials.

The present invention also provides a method for manufacturing a biochip having one or a plurality of wells for arranging and immobilizing a probe material, wherein the one or the plurality of electrode wirings are formed on a substrate, and the one or the plurality of electrode wirings are formed of a metal oxide. Manufacturing a biochip in which one or a plurality of wells are formed by covering with a switching film and then forming one or a plurality of holes reaching the electrode surface in the insulating film on each region of the one or the plurality of electrode wirings. It also provides a method.

The biochip of the present invention may be produced by applying any conventional technique.

In particular, in the biochip manufacturing method of the present invention, a technique for forming a well in an insulating film of the metal oxide is selected from the group consisting of nanoimprinting technique, KrF stepper patterning, E-beam method, and convergent ion beam method.

Since the biochip of the present invention uses a metal oxide as an insulating film, compared to a conventional biochip using a resist as an insulating film, when immersed in a sample solution, there is no adverse effect on the electrical properties due to wetting, thereby enabling stable measurement. .

In addition, since the biochip of the present invention uses a metal oxide as an insulating film, the size of the well can be made smaller than that of a conventional biochip using a resist as an insulating film. Thus, when a probe material such as a protein is immobilized inside the well, Increasing the number of probe materials into the well is reduced, and the sensing capability of the probe material is improved, so that a higher precision measurement is possible.

1 is a top view of a conventional biochip.
2 is a top view of a biomolecule array region of a conventional biochip.
3 is a cross-sectional view of a biomolecule array region of a conventional example.
4 is a schematic view illustrating a manufacturing process of a conventional biochip by photofabrication technology.
5 is a schematic view illustrating a manufacturing process of a conventional biochip by photo fabrication technology.
6 is a schematic view illustrating a manufacturing process of a conventional biochip by photo fabrication technology.
7 is a schematic view illustrating a manufacturing process of a conventional biochip by photo fabrication technology.
8 is a schematic view illustrating a manufacturing process of a conventional biochip by photofabrication technology.
9 is a schematic view illustrating a manufacturing process of a conventional biochip.
10 is a schematic diagram illustrating a manufacturing process of a conventional biochip by nanoimprint lithography technique.
11 is a schematic diagram illustrating a manufacturing process of a biochip of the present invention by a photofabrication technique.
12 is an electron micrograph of the upper surface of the biomolecule array region of the biochip of the present invention.
13 is a result of measuring the voltametry of the biochip of the embodiment of the present invention.
14 is a result of measuring the voltametry of the biochip of the comparative example.

Example 1 [Biochip Using Metal Oxide Film as Insulation Film]

(1) manufacture of biochips

Biochips having nano-sized wells were manufactured using general electron beam (e-beam) lithography.

First, as shown in Figs. 4A to 4F, electrodes are formed on a substrate in the same manner as in the conventional method.

Next, by using a spin coating method (5000 rmp: 60 seconds), a 2% chlorobenzene solution of an e-beam resist (polymethyl methacrylate) was coated on the electrode to a thickness of 150 nm, and then cured through heat treatment. The film 61 was formed (FIG. 11A).

The cured resist film 61 was irradiated with an electron beam 62 in a predetermined pattern (Fig. 1 lb), and then the substrate was placed in a developing solution (methyl ethyl ketone: isopropyl alcohol = 1: 3) for 30 seconds. The resist film 61 was developed by immersion (FIG. 11C).

Thereafter, SiO ₂ was deposited by direct current magnetron sputtering (metal target, room temperature deposition, pressure: 5 mTorr, DC power: 60 W) using a common metal oxide deposition apparatus to form a metal oxide film 63 (FIG. 11D). ).

Further, the substrate was immersed in acetone for 30 minutes or more to remove the resist film 61 remaining after deposition and SiO ₂ deposited on the resist, thereby exposing the electrode surface to form an array of a plurality of wells 63a (Fig. 11e). Although the schematic top view of the obtained biochip is not shown, it is similar to FIG. 8 except that the insulating resist film 14 is an insulating film 63 of a metal oxide (here, SiO ₂ ).

In the above manner, an array of wells of very small size with a diameter of 60 nm and a depth of 40 nm was formed. An electron microscope image of the array portion of the well is shown in FIG. 12.

Next, as in the conventional example shown in FIGS. 9A to 9F, a biochip A having immobilized streptavidin on the exposed electrode wiring was fabricated.

(2) electrochemical measurement

The electrochemical measurement of the biochip was performed at room temperature using BAS100B / W potentiometer (Bioanalytical Systems, Inc.). In the measurement, a biochip was used as a working electrode, Ag / AgCl in 3M KCL was used as a reference electrode, and a platinum line was used as a counter electrode (1 mM).

EC before each measurement was carried out at the pre-treated for 1 min at + 1.7V for a bio-chip to form an array of wells of the nano-sized from H ₂ SO ₄ solution.

Cyclic voltametry (CV) was measured at a scanning speed of 200 mV / sec in a solution containing 100 mM phosphate buffered saline (pH 7.4). The setting of CV measurement is as follows.

Initial potential: -0.3 V, final potential: 0.6 V, amplitude: 25 mV, step potential: 4 mV, period: 50 Hz.

The result of having obtained the CV curve immediately after immersing the biochip (A) in the solution and from 30 minutes to 240 minutes after immersion is shown in FIG.

As can be seen from the results of FIG. 13, it is shown that a biochip using a metal oxide film as an insulating film can be measured very stably without fluctuation of the CV curve even when immersed in a solution for a long time.

The nano-structured biosensor made by the above method is easier to manufacture with a smaller size than the conventional polymer, and the durability and stability of the polymer is particularly good since the insulation of the metal oxide is very good. It was found to be good.

Comparative Example 1 [Biochip Using Resist Film as Insulating Film]

(1) manufacture of biochips

An electrode wiring was formed in the same manner as when a single well having a diameter of 5 µm or more was formed (FIG. 4), and then on the substrate on which the electrode wiring 13 was formed using a spin coater, similarly to the process shown in FIG. 5A. The insulating resist 14 (ZEP520: Xeon Co., Ltd.) is apply | coated and baked at 90 degreeC for 2 minutes (FIG. 7A). Subsequently, the electron surface 15 was irradiated at 75 kV (FIG. 7B), and the electrode surface was exposed by developing the resist 14 to form an array of a plurality of wells 131 a on the biomolecule array region 131. (FIG. 7C). The resist 14 was fixed by baking this at 200 degreeC for 5 minutes (FIG. 7D). The schematic top view of the chip | tip with which the well was formed is the same as that shown in FIG.

Next, similarly to Example 1, the biochip B by which streptavidin was immobilized on the exposed electrode wiring was produced.

(2) electrochemical measurement

Under the same conditions as in Example 1, electrochemical measurement of the biochip was performed by the cyclic voltammetry (CV) method.

The result of having obtained the CV curve immediately after immersing the biochip (B) in the solution and from 20 minutes to 120 minutes after immersion is shown in FIG. 14.

As can be seen from the result of FIG. 14, when the biochip using the resist as the insulating film was immersed in the solution, the CV curve greatly varied.

Compared with the biochip which uses a conventional resist film as an insulating film, since the measurement stability is high, it can apply to the field which requires high precision measurement.

1: biochip
10: substrate
11: photoresist
12a: UV light
12b: Photomask
13: electrode wiring
131: Biomolecule Array Region
131a, 131b: wells
132: Pad
14: insulating resist film
15: electron beam
16: substrate
17: resin layer
17a: cured film
18: Mold
19: UV light
2: probe
21: biotinylated 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Bio-PE)
22: streptavidin
23: biotinylated probe antibody
3: 1-octanedecanthiol (ODT)
4: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC)
5: (11-ferrocenyl) undecylpolyoxyethylene (Fe-PEG)
61: resist for electron beam lithography
62: electron beam
63: metal oxide film
63a: Well

Claims (7)

A biochip having one or a plurality of wells for arranging and immobilizing probe materials, wherein one or a plurality of electrode wirings are formed on a substrate, and the one or a plurality of electrode wirings are covered with an insulating film of a metal oxide, A biochip in which one or a plurality of wells is formed by forming one or a plurality of holes reaching an electrode surface in an insulating film on one region of one or a plurality of electrode wirings. The method of claim 1,
A biochip, wherein a probe substance selected from the group consisting of nucleic acids, proteins, peptides, liposomes, β-amidoid oligomers and reducing sugars is immobilized on an electrode wiring surface exposed to the bottom of one or a plurality of wells. The method of claim 1,
A biochip, wherein the metal oxide is selected from the group consisting of TiO ₂ , SiO ₂ , and ZnO. A biosensor comprising at least a working electrode, a counter electrode, and a reference electrode, wherein the working electrode is a biochip in which one or a plurality of wells are formed for arranging and immobilizing a probe material, wherein the working electrode is a biosensor formed on a substrate. Electrode wirings are formed, and the one or more electrode wirings are covered with an insulating film of metal oxide, and one or more holes reaching the electrode surface are formed in the insulating film on each region of the one or the plurality of electrode wirings. A biochip in which one or a plurality of wells is formed by forming,
A probe substance selected from the group consisting of nucleic acids, proteins, peptides, liposomes, β-amyloid oligomers and reducing sugars is immobilized on the electrode wiring surface exposed to the bottom of one or the plurality of wells,
Before processing, the first electrical signal of the biochip is measured,
After processing the biochip in a test solution, the second electrical signal of the biochip is measured; next,
And detecting the non-labeled target molecule specifically binding to the one or the plurality of probe substances present in the test solution from the difference in intensity between the first electrical signal and the second electrical signal. 5. The method of claim 4,
The biosensor whose electrical signal is a redox current value. A method of manufacturing a biochip having one or a plurality of wells for arranging and immobilizing probe materials, the method comprising:
Forming one or a plurality of electrode wirings on the substrate,
The one or more electrode wirings are covered with a metal oxide blocking film, and then
A method for manufacturing a biochip, wherein one or a plurality of wells are formed in an insulating film on each region of the one or a plurality of electrode wirings by forming one or a plurality of holes reaching the electrode surface. The manufacturing method of Claim 6 whose technique of forming a well in the insulating film of this metal oxide is selected from the group which consists of a nanoimprinting technique, KrF stepper patterning, an E-beam method, and a converging ion beam method.

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