JP2005164388A - Protein chip and biosensor using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a small-sized biosensor of high sensitivity, capable of rapidly and simply detecting specific proteins without performing labelling with a fluorescent substance or the like. <P>SOLUTION: One or a plurality of microwells are arranged on the surface of an electrode by a microprocessing technique and a lipid double layer is formed on the surface of the electrode to produce a biomolecule array chip which keeps probe protein fixed thereon. A change in an oxidation-reduction current value at the interaction of a target molecule with this probe protein is detected with high sensitivity. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a biochip having a protein microarray as a probe and a biosensor using the same. More specifically, the present invention relates to a highly sensitive biosensor capable of detecting specific binding of a target molecule to a protein microarray by electric signal detection.

In recent years, biochips have attracted attention in fields such as new drug development processes and medical diagnosis, and research and development have been actively promoted.
A biochip is a biomolecule array such as a nucleic acid such as DNA, a protein such as an enzyme or an antibody, a peptide, etc. A specific reaction that occurs when a specific target substance is bound to a probe substance such as an immobilized biomolecule array by immobilizing cells or the like is detected. In particular, because high-throughput detection and analysis can be performed in large quantities using a small amount of sample, biochip-related technologies are used in the functional analysis technology of biomolecular arrays in the post-genome era that requires large-scale and parallel processing. It is essential.

Typical examples of biochips include DNA chips (DNA microarrays) that detect the presence of complementary sequences by immobilizing DNA on a substrate at high density, and proteins that detect and interact with proteins. There are chips (protein chips).
Also, devices and instruments (lab-on-chip, μTAS () that form channels and circuits on the substrate surface of silicon, glass, etc. using microfabrication technology and perform reaction, separation, detection, etc. in a micro space. micro-total analysis system), bio-MEMS (micro-electro-mechanical systems, etc.) have been put into practical use, and they are sometimes referred to as biochips.

In the case of a protein chip, it may be considered that the probe of the DNA chip is basically changed from DNA to protein, but in the case of a protein, in order to express a function that interacts with a target molecule, unlike DNA. Higher order structure is required. Therefore, when a protein is immobilized on a solid support, it is necessary not to destroy its higher order structure. Further, depending on the relative positional relationship between the interaction site and the solid phase carrier, the interaction may be sterically hindered. Therefore, a protein chip cannot be produced simply by replacing the DNA of a conventional DNA chip with a protein.
In protein chips, in order to control non-specific adsorption (when two existing substances tend to bind to each other, they bind to other unrelated substances) Immobilization techniques are difficult due to the need to keep it right and stability in the atmosphere. For this reason, protein chips are currently not as popular as DNA chips.

  Patent Document 1 discloses a method for detecting a large amount of protein at a time using a protein chip in which antigens or antibodies are arranged one-dimensionally or two-dimensionally on a planar substrate. According to this method, the antigen or antibody is fluorescently labeled, the protein is bound by an antigen-antibody reaction, washed to remove the unbound antigen or antibody, and finally the bound protein is detected by fluorescence observation. doing.

  In recent years, the importance of point-of-care testing (POCT) has increased as a clinical test in the medical field, and as one of the POCT devices, it has become smaller as a portable biosensor. The automated analysis system will be effective.

However, in the method disclosed in Patent Document 1, as described above, since protein detection is performed by fluorescence observation, pretreatment of a fluorescent label and a fluorescent device are required.
It is difficult to meet the demands of POCT.

JP 2001-004630 A

In order to solve the above problems, the object of the present invention is to develop an innovative method for immobilizing a protein on a solid phase carrier without causing steric hindrance or inactivation regardless of the type of protein, The purpose is to develop a general-purpose technology that can produce high-density arrays that are controlled and addressed.
Another object of the present invention is to solve the problems of conventional fluorescence detection protein chips and to develop a microelectrode array protein chip that is excellent in terms of simplicity, portability, and development cost. Therefore, the present inventors examined a method for immobilizing a probe protein on an electrode, produced a microelectrode array type protein chip, and simultaneously applied multiple types of target molecules without any labeling molecule modification. A new “next generation protein chip electrode array system” for chemical detection was constructed.
And finally, this invention aims at applying the protein chip by this invention to a quick and simple clinical test.

  In the present invention, one or a plurality of microelectrodes are arranged on a plane by a microfabrication technique, and a biomolecule array chip is prepared by immobilizing a probe protein on each of the one or a plurality of microelectrodes. Changes in electrical signals when molecules interact are detected with high sensitivity.

  In the biochip of the present invention, the protein to be a probe can be selected from antibodies and other proteins depending on the type of target molecule. For example, when a protein is selected as the target molecule, an antibody against the selected target protein may be used as the probe as the probe protein.

  More specifically, the present invention is a biochip in which one or more probe proteins are arrayed and immobilized, wherein one or more electrode wires are formed on a substrate, and the one or more electrode wires are One or a plurality of wells are formed by providing one or a plurality of holes that reach the electrode surface in the insulating film on each region of the one or a plurality of electrode wirings that are covered with the insulating film. A biochip having a probe protein immobilized on the bottom of each well is provided.

  In the biochip according to the present invention, a first molecule is bound to the probe protein, a second molecule that specifically binds to the first molecule is immobilized at the bottom of the well, and the first molecule and the The probe protein is immobilized on the bottom of the one or more wells by specific binding with a second molecule.

  In the biochip according to one aspect of the present invention, a lipid bilayer is formed at the bottom of the well, and an anchor compound to which a third molecule is bound is anchored to the lipid bilayer and bound to the anchor compound. The probe protein is immobilized on the bottom of the one or more wells by specific binding between the third molecule and the second molecule.

  The cell membrane is mainly composed of non-covalently bound lipids and protein molecules, and has a structure in which protein molecules are dissolved in a lipid bilayer composed of phospholipids. For the purpose of immobilizing the probe protein on the electrode wiring without affecting the higher-order structure of the probe protein, the present inventors did not immobilize the protein directly on the electrode surface, but imitated a lipid membrane simulating the cell membrane structure. A multilayer was formed on the electrode surface, and the probe protein was arranged on it.

  In the present invention, the first molecule is preferably biotin, the second molecule is preferably streptavidin, and the third molecule is preferably biotin.

  In one aspect of the present invention, the one or more probe proteins are antibodies.

  The biochip according to the present invention has one or more electrode wirings formed on a substrate, the one or more electrode wirings are covered with an insulating film, and an insulating film on one region of each of the one or more electrode wirings One or more wells are formed by providing one or more holes reaching the electrode surface, and then the probe protein is immobilized on the bottom of the one or more wells.

Furthermore, the present invention is a biosensor comprising at least a working electrode, a counter electrode and a reference electrode,
Here, the working electrode is a biochip in which one or more probe proteins are arrayed and immobilized, and one or more electrode wirings are formed on a substrate, and the one or more electrode wirings are One or a plurality of wells are formed by providing one or a plurality of holes that reach the electrode surface in the insulating film on each region of the one or a plurality of electrode wirings that are covered with the insulating film. Is a biochip in which the probe protein is immobilized on the bottom of the well,
Measuring a first electrical signal of the biochip prior to processing;
After treating the biochip in a test solution, measuring a second electrical signal of the biochip;
An unlabeled target molecule that specifically binds to the one or more probe proteins is detected from a difference in intensity between the first electrical signal and the second electrical signal. Providing biosensors.

  Here, “treatment” refers to a process necessary for specifically binding a probe protein and an unlabeled target molecule. Therefore, before the treatment, unlabeled target molecules are not bound to the one or more probe proteins in the biochip. If the unlabeled target molecule is present in the test solution, the unlabeled target molecule is specifically bound to the one or more probe proteins by this treatment. Before and after performing the above treatment on the biochip of the present invention, an electrical signal is measured, and if there is a change in the electrical signal, it is confirmed that an unlabeled target molecule was present in the test solution. Can do.

In the present invention, in particular, an oxidation-reduction current value is used as an electrical signal.
That is, according to the present invention, the presence or absence of specific binding of unlabeled target molecules to the probe protein is detected from the change in the redox state on the biomolecule array of the biochip. Therefore, since specific binding can be detected only by measuring the oxidation-reduction current value without labeling the target molecule with a fluorescent substance or the like, rapid and simple measurement is possible.
That is, when there is a statistically significant change between the electrical signal measured in the system where the target molecule is not bound to the probe protein and the electrical signal measured in the system where the target molecule is bound to the probe protein The significant change can be related to the specific binding between the target molecule and the probe protein.

  In one aspect of the present invention, the unlabeled target molecule is an antigen, and one or more probe proteins immobilized on the biochip are antibodies to the target molecule. For example, the unlabeled target molecule is human serum albumin (HSA), and one or more probe proteins immobilized on the biochip are anti-HSA antibodies.

When measuring an electrical signal using the biosensor of the present invention, in a preferred embodiment, an unlabeled target molecule having Au particles attached to its surface is used.
Thereby, even if the target molecule is a protein that is an insulator, the surface thereof becomes conductive, so that it becomes easy to detect a change in the electrical signal due to specific binding.

By using the biochip according to the present invention, it is possible to recognize the presence of a target molecule without using a fluorescent label, and thus it is possible to construct a miniaturized biosensor that can be measured quickly and easily. .
Further, according to the present invention, the lipid bilayer is immobilized on the electrode and the probe protein is immobilized thereon, so that the specificity of the probe protein is impaired without destroying the higher order structure of the probe protein. There is nothing.
Furthermore, since different probe proteins can be immobilized on a plurality of electrodes in the biochip according to the present invention, a plurality of types of target molecules can be detected simultaneously.

Example 1 Production of Protein Chip A top view of a protein chip according to the present invention is shown in FIG. In this example, eight electrode wirings 13 were formed on the glass substrate 10, and the protein chip 1 capable of measuring eight points at a time was formed.
A biomolecule array region 131 is formed at one end of the electrode wiring 13, and a pad 132 for taking out an electrical signal detected in the biomolecule array region is formed at the other end.
FIG. 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 more wells 131a for immobilizing biomolecules are formed by providing one or more holes in the insulating film. .
When the diameter of the well 131a is 5 μm or more in the biomolecule array region 131, a single well can be formed as shown in FIG. 2a. A plurality of wells can be arranged as shown in 2b.

  A cross-sectional view of the biomolecule array region 131 is shown in FIG. In this example, first, a Ti thin film 13a was formed on a glass substrate 10, and an Au thin film 13b was formed thereon, thereby forming an electrode wiring 13 made of an Au / Ti laminated thin film. Although an Au thin film can be directly formed on a glass substrate, since the adhesive strength of the Au thin film is weak, a Ti thin film was used to prevent peeling of the Au thin film and improve the reliability of the biochip. In addition to Ti, Cr can also be used.

As shown in this figure, the hole formed in the insulating resist film reaches the surface of the electrode wiring in the lower layer. That is, the side surface of the well formed in the biomolecule array region is defined by the insulating resist film 14, and the bottom of the well is defined by the surface of the electrode wiring.
With this configuration, as shown in FIG. 3b, biomolecules that become probes are immobilized only on the exposed electrode surface at the bottom of the well 131a.

  Human serum albumin (HSA) is well known for its properties and structure, is readily available commercially, and antibodies against HSA can also be readily generated, so in this example the target protein HSA was used as 6 (not shown), and goat anti-HSA antibody was used as probe 2.

In order to produce the protein chip of the present invention, photofabrication technology, which is one of semiconductor manufacturing technologies, was used. Here, the term “photofabrication technique” means a technique that combines a photolithography technique, a vapor deposition technique, an etching technique, and the like.
Since the photofabrication technology has already been established as one of IC and LSI manufacturing technologies, it can be miniaturized and the manufacturing process can be automated like a conventional DNA chip, leading to mass production of chips and cost reduction.

4 to 9 are schematic views showing a process for producing the DNA chip of the present invention. First, a process for producing a single well (first specific example) having a diameter of 5 μm or more will be described. Photoresist 11, which is a photosensitive material, is applied onto the substrate 10 using a spin coater and baked at 90 ° C. for 2 minutes (FIGS. 4a and 4b). In this embodiment, a glass substrate is used, but any material such as an alumina substrate, a silicon substrate, or a resin substrate such as a silicone resin can be used.
Next, using a photomask, exposure is performed with ultraviolet rays 12a for 20 seconds (FIG. 4c), and the photoresist 11 is developed to form a wiring pattern for the electrode wiring 13 (FIG. 4d). There are two types of photoresists, one that dissolves in a developing solution due to decomposition of the bond upon exposure (positive type) and the other that does not dissolve due to polymerization (negative type). AZ1500: Clariant Japan Co., Ltd.) was used.

Thereafter, a metal thin film serving as an electrode material is formed on the substrate by an evaporation technique such as vacuum evaporation or RF sputtering (FIG. 4e).
Furthermore, the electrode wiring 13 was formed on the glass substrate 10 by immersing this in an organic solvent such as acetone and peeling the resist 11 (FIG. 4f). FIG. 6A shows a schematic top view of the chip on which the electrode wiring 13 is formed. In FIG. 6, an electrode wiring in which a circular biomolecule array region 131 is formed at one end and a square pad 132 for taking out an electrical signal is formed at the other end is shown. This is an example of electrode wiring, and is not limited to this shape. A person skilled in the art can transform into any shape that can be used as a biochip mounted on a biosensor.
Further, the number of electrode wirings can be appropriately increased or decreased according to the usage form.

  Next, an insulating resist 14 (AZ1500) is applied on the substrate on which the electrode wiring 13 is formed using a spin coater, and baked at 90 ° C. for 2 minutes (FIG. 5a). Next, the film was exposed to ultraviolet rays 12a for 20 seconds using a photomask (FIG. 5b), the resist 14 was developed to expose the electrode surface, and a well 131a was formed on the biomolecule array region 131 (FIG. 5c). This was baked at 150 ° C. for 5 minutes to fix the resist 14 (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 having a diameter of submicron or less (second specific example) will be described. An electrode wiring is formed in the same manner as when a single well having a diameter of 5 μm or more is fabricated (FIG. 4), and then on the substrate on which the electrode wiring 13 is formed using a spin coater, as in the step shown in FIG. 5a. An insulating resist 14 (ZEP520: Nippon Zeon Co., Ltd.) is applied to the substrate and baked at 90 ° C. for 2 minutes (FIG. 7a). Next, the electron beam 15 was irradiated at 75 kV (FIG. 7b), the resist 14 was developed to expose the electrode surface, and an array of a plurality of wells 131a was formed on the biomolecule array region 131 (FIG. 7c). . This was baked at 200 ° C. for 5 minutes to fix the resist 14 (FIG. 7d). A schematic top view of the chip in which the well is formed is shown in FIG.

  If a conventional semiconductor manufacturing technique is used, a well can be formed on a substrate. Therefore, the well is appropriately formed under other conditions well known to those skilled in the art without being limited to the above conditions. it can.

Next, a lipid bilayer was formed on the electrode surface at the bottom of the well using methods well known in the field of biochemistry. Specifically, first, the chip is 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, After pure and thorough washing and washing with ethanol (Fig. 9a), it was immersed in an ethanol solution of 1-octadecanthiol (ODT) 3 to bind ODT3 to the electrode surface via its -SH group (Fig. 9b). ).
Next, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) 4 is dissolved in phosphate buffer (PBS, pH 7.4) (10 mM) to prepare a liposome solution of POPC4. This liposome solution was dropped on the chip and allowed to stand in an environment of 100% humidity for 1 hour to form a lipid bilayer of POPC4 on the electrode surface (FIG. 9c).
Alternatively, after 99.9 mol% POPC4 and 0.1 mol% ODT3 are mixed well in chloroform, the solvent is exchanged with PBS to prepare a mixed liposome solution, and this liposome solution is dropped onto the chip, A lipid bilayer of POPC4 is formed on the electrode surface (FIG. 9c).
In either case, the resist 14 needs to be made of a material that does not bind to the —SH group of ODT so that the lipid bilayer is immobilized only on the electrode surface at the bottom of the well.

  When the surface of the electrode on which the lipid bilayer was formed was observed using an atomic force microscope (AFM; Seiko Instruments Inc. SPI3800), it was confirmed that the lipid bilayer was uniformly formed on the entire electrode surface ( Not shown).

  As another method for immobilizing the lipid bilayer on the electrode surface, an amino coupling method can also be used. Specifically, a chip washed with a piranha solution is immersed in an ethanol solution of 3,3′-dithiodipropionic acid (DTDP), and DTDP is bonded to the electrode surface via its —S—S— group. The chip was then placed in a mixed solution of N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) -3-carbodiimide hydrochloride (EDC) in dioxane and water (9: 1 [v / v]). Immerse to activate the carboxyl group of DTDP. Here, the liposome solution of POPC4 is added, and the lipid bilayer of POPC4 is immobilized on the electrode surface by binding the amine group of POPC4 and the carboxyl group of DTDP.

Next, 1 mM (11-ferrocenyl) undecyl polyoxyethylene (Fe-PEG) 5 and 0.1 mM biotinylated 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Bio-PE) 21 The phosphate buffer mixed solution (pH 7.4) was dropped onto the chip on which the lipid bilayer was formed, and allowed to stand for 1 hour to anchor Fe-PEG5 and Bio-PE21 onto the lipid bilayer (FIG. 9d). At this time, by appropriately adjusting the molar concentration ratio in the mixed solution of Fe-PEG5 and Bio-PE21, Bio-PE21 is dispersed and anchored on the lipid bilayer with an appropriate density.
Since the lipid bilayer is insulating, it is impossible to detect a phenomenon on the lipid bilayer as an electrical signal. Therefore, Fe-PEG5 is used for the purpose of making the lipid bilayer surface conductive.

  Further, 0.2 mg / ml of an aqueous solution of streptavidin 22 was dropped onto the chip on which the biotin-modified lipid bilayer was formed, and left to stand in an environment of 100% humidity for 1 hour, so that Bio-PE21 and biotin specific Streptavidin 22 was bound by mechanical binding (FIG. 9e). Unbound streptavidin not immobilized on the lipid bilayer was removed by washing with PBS.

Streptavidin is a protein that binds to biotin most strongly and stably (K _d = 10 ⁻¹⁵ M) and binds to four molecules of biotin.
A technique of binding a molecule modified with streptavidin and another molecule modified with biotin using a specific binding between streptavidin and biotin is generally used. Therefore, techniques for modifying biotin on various proteins have already been established, and kits for that purpose are also commercially available. Biotin-modified DNA is also commercially available.

  Bio-PE21 is dispersed and anchored on the lipid bilayer, and it is considered that only one biotin is bound to one streptavidin molecule.

  Finally, the streptavidin-immobilized chip was immersed in a pre-biotinylated probe antibody 23 solution to immobilize the probe antibody 23 on the bottom of the well through specific binding of streptavidin-biotin (FIG. 9f).

  It is already known that the effective binding number of biotin is 1 when streptavidin is immobilized on a substrate. This is probably because the binding site of streptavidin accessible to biotin is limited by immobilization. In particular, in the case of the present invention, since a protein is bound to biotin, it is considered that after one biotinylated protein is bound to streptavidin, no further biotinylated protein is accessible to streptavidin due to steric hindrance. Therefore, it is considered that one immobilized streptavidin 22 has one biotinylated probe antibody 23 bound thereto.

Example 2: Evaluation of HSA binding ability of protein chip 27 MHz crystal for measuring quartz crystal microbalance (QCM; Initiative AFFINIX Q) under the same conditions as when protein chip 1 produced in Example 1 was produced. Probe antibody 23 was immobilized on the surface of the vibrator, and the binding ability of target protein 6 (HSA) was evaluated.

  Specifically, first, as shown in FIG. 7e, 5 μl of a 1 mg / ml probe antibody 23 (biotinylated goat anti-HSA antibody) solution was injected using a sample at the stage of binding to streptavidin 22 ( The injection time is indicated by an arrow), the oscillation frequency decreased with time, and it was confirmed that the probe antibody 23 was bound to streptavidin 22 (FIG. 10a). Furthermore, when 5 μl of a 100 μg / ml HSA solution was injected using the sample to which the probe antibody 23 was bound, it was confirmed that the oscillation frequency decreased and HSA was bound (FIG. 10 b).

  For comparison, as shown in FIG. 7d, 5 μl of a 1 mg / ml solution of probe antibody 23 (biotinylated goat anti-HSA antibody) was injected using a sample at the stage where streptavidin 22 was not bound (injection time). As shown by the arrows, the oscillation frequency decreased with time, and it was confirmed that the probe antibody 23 was bound on the lipid bilayer (FIG. 11a), however, the sample was injected with 100 μg / ml HSA solution. However, although the oscillation frequency increased due to the injection shock, the oscillation frequency did not decrease, so it was confirmed that HSA does not bind unless streptavidin is present (FIG. 11b).

From the above results, it was found that by arranging the probe antibody on the lipid bilayer via streptavidin, the antigen can be efficiently bound to the target molecule.
That is, according to the present invention, the orientation of the probe protein can be controlled so that the probe protein and the target molecule can be bound efficiently.

Example 3: Evaluation of detection performance of protein chip Using the protein chip 1 (single well having a diameter of 200 μm) of the first specific example prepared in Example 1, a biosensor was constructed and a detection experiment of the target protein 6 was performed. Went.
Specifically, an electrochemical cell is constructed in a mixed aqueous solution of 5 mM K ₃ [Fe (CN) ₆ ] and 100 mM KCl, using protein chip 1 as a working electrode, platinum as a counter electrode, and silver / silver chloride as a reference electrode. Then, the oxidation-reduction potential of the protein chip 1 was measured by cyclic voltammetry. The measurement was performed at 25 ° C., and the potential sweep rate was 50 V / sec.

First, in order to confirm the function of the biosensor of the present invention, a current-potential curve was obtained when the target protein 6 was bound to each stage of the production process of the protein chip 1 and the probe antibody 23.
Specifically, as shown in FIG. 9e, the state in which streptavidin 22 is bound to Bio-PE21 (indicated by A in the figure); the state in which biotinylated probe antibody 23 is bound to streptavidin 22 (in the figure) , B); and current potential curves were obtained in three states (indicated by C in the figure) in which target protein 6 (HSA) was bound to biotinylated probe antibody 23 (FIG. 12).
In the current-potential curve shown in FIG. 12, the peak current value indicates the redox current value.

As can be seen from FIG. 12, when the biotinylated probe antibody 23 is bound to the streptavidin 22 (change from state A to state B), the target protein 6 is bound to the probe antibody 23 (from state B to state). Also, the absolute value of the oxidation-reduction current value decreased.
That is, even if the redox current value is measured under this condition, the change in the value cannot be associated with the specific binding of the target protein 6 to the probe antibody 23.

The present inventors have found that this phenomenon is caused by the fact that the surface of the lipid bilayer is not sufficiently conductive and the target protein is also insulative. Even if the state changed, the electrical signal could not be detected by the electrodes.
Therefore, the present inventors decided to improve the electrical signal transmission in this system by making the surface of the target protein conductive.

In the present invention, in order to make the target protein HSA conductive, Au particles are attached to the surface by electrostatic interaction.
Since the Au particles are negatively charged in the aqueous solution, the Au particles can be attached to the surface of the HSA if the surface charge of the HSA becomes positive. The isoelectric point pI of HSA is 4.9, and HSA is positively charged when the pH is below 4.9.

Therefore, a 5 nm diameter Au particle (BB International) was added to a 1 mg / ml HSA aqueous solution to prepare a mixed solution having a particle concentration of 5 × 10 ¹³ particles / l. Next, the pH of this mixed solution was adjusted to 4.6 and reacted at 37 ° C. for 10 hours to attach Au particles to the surface of HSA.

Using the target protein 6 ′ (Au—HSA) to which the Au particles were attached, a current-potential curve was obtained at each stage in the same manner as when the current-potential curve shown in FIG. 12 was obtained (FIG. 13).
In this case, when the biotinylated probe antibody 23 was bound to the streptavidin 22 (change from state A to state B), the absolute value of the oxidation-reduction current value decreased as described above. When Au-HSA was bound (change from state B to state C), the absolute value of the redox current value increased.
That is, by attaching Au particles to the surface of HSA, the change in redox current value could be associated with specific binding of the target protein 6 to the probe antibody 23.

Example 4: Production of a protein chip having an array of nano-sized wells A DNA chip of a second specific example in which wells having a diameter of 100 nm were formed in an array of 200 × 200 on the biomolecule array region was produced.

  An array of 100 nm diameter wells is formed in the biomolecule array region, and the probe protein 2 is immobilized on the electrode surface at the bottom of a plurality of wells by the method described in Example 1 (FIG. 9). A DNA chip was prepared.

The surface of the biomolecule array region 113 of the second specific example was observed in a tapping mode using an atomic force microscope (AFM; Seiko Instruments Inc. SPI3800) (FIG. 14). A silicon cantilever (SI-DF-20, Seiko Instruments Inc.) having a resonance frequency of 125 kHz and a spring constant of 14 N / m was used.
From this AFM image, it was confirmed that streptavidin (the substance shown in white in the well) was immobilized in each well (area shown in a black circle). In addition, the presence of streptavidin was confirmed in the well uniformly throughout the observation field.

  Thus, a protein chip was obtained in which the probe protein was uniformly dispersed and immobilized in a plurality of wells formed in the biomolecule array region 113.

  From the above results, according to the present invention, it is not necessary to label the target protein with a fluorescent substance, it is possible to measure in an electrolyte solution, and a plurality of electrodes are produced using the microfabrication method. Therefore, it is expected as a technology that enables a quick and simple analysis and provides a next-generation DNA array system with excellent cost performance.

The top view of the protein chip of this invention. The top view of the biomolecule array area | region of the protein chip of this invention. Sectional drawing of the biomolecule array area | region of this invention. Schematic explaining the manufacturing process of the protein chip of the 1st specific example of this invention. Schematic explaining the manufacturing process of the protein chip of the 1st specific example of this invention. Schematic explaining the manufacturing process of the protein chip of the 1st specific example of this invention. Schematic explaining the manufacturing process of the protein chip of the 2nd specific example of this invention. Schematic explaining the manufacturing process of the protein chip of the 2nd specific example of this invention. Schematic explaining the manufacturing process of the protein chip of this invention. The graph which shows the crystal oscillator microbalance measured by the structure similar to the protein chip of this invention. The graph which shows the crystal oscillator microbalance of the comparative example measured with the structure similar to a protein chip. The redox current potential curve measured using the protein chip and HSA of the present invention. The redox current potential curve measured using the protein chip of the present invention and Au particle-attached HSA. The AFM image of the DNA chip of the 2nd example of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Biochip (protein chip), 10 ... Substrate, 11 ... Photoresist, 12a ... Ultraviolet, 12b ... Photomask, 13 ... Electrode wiring, 131 ... Biomolecule Array region, 131a ... well, 132 ... pad, 14 ... insulating resist film, 15 ... electron beam, 2 ... probe, 21 ... biotinylated 1,2-dioleoyl-sn -Glycero-3-phosphoethanolamine (Bio-PE), 22 ... streptavidin, 23 ... biotinylated probe antibody, 3 ... 1-octadecanethiol (ODT), 4 ... 1-palmitoyl- 2-oleoyl-sn-glycero-3-phosphocholine (POPC), 5 (11-ferrocenyl) undecyl polyoxyethylene (Fe-PEG), 6 ... Target protein.

Claims (11)

  A biochip in which one or more probe proteins are arrayed and immobilized, wherein one or more electrode wires are formed on a substrate, and the one or more electrode wires are covered with an insulating film, Alternatively, one or a plurality of wells are formed by providing one or a plurality of holes reaching the electrode surface in the insulating film on each region of the plurality of electrode wirings, and the probe protein is formed at the bottom of the one or the plurality of wells. The biochip immobilized.   A first molecule is bound to the probe protein, and a second molecule that specifically binds to the first molecule is immobilized at the bottom of the well, and between the first molecule and the second molecule The biochip according to claim 1, wherein the probe protein is immobilized on the bottom of the one or more wells by specific binding.   A lipid bilayer is formed at the bottom of the well, and an anchor compound to which a third molecule is bound is anchored to the lipid bilayer, and the third molecule bound to the anchor compound and the second molecule The biochip according to claim 2, wherein the probe protein is immobilized on the bottom of the one or more wells by specific binding between them.   The biochip according to claim 2 or 3, wherein the first molecule is biotin and the second molecule is streptavidin.   The biochip according to claim 3, wherein the third molecule is biotin.   The biochip according to claim 1, wherein the one or more probe proteins are antibodies. A biosensor comprising at least a working electrode, a counter electrode and a reference electrode,
Here, the working electrode is a biochip in which one or more probe proteins are arrayed and immobilized, and one or more electrode wirings are formed on a substrate, and the one or more electrode wirings are One or a plurality of wells are formed by providing one or a plurality of holes that reach the electrode surface in the insulating film on each region of the one or a plurality of electrode wirings that are covered with the insulating film. Is a biochip in which the probe protein is immobilized on the bottom of the well,
Measuring a first electrical signal of the biochip prior to processing;
After treating the biochip in a test solution, measuring a second electrical signal of the biochip;
An unlabeled target molecule that specifically binds to the one or more probe proteins is detected from a difference in intensity between the first electrical signal and the second electrical signal. Biosensor.   The biosensor according to claim 7, wherein the electrical signal is a redox current value.   The biosensor according to claim 7, wherein the unlabeled target molecule is an antigen, and one or more probe proteins immobilized on the biochip are antibodies to the target molecule.   The biosensor according to claim 9, wherein the unlabeled target molecule is human serum albumin (HSA), and the one or more probe proteins immobilized on the biochip are anti-HSA antibodies. The biosensor according to claim 9, wherein an unlabeled target molecule having Au particles attached to the surface thereof is used for measuring an electrical signal.

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