JP4324707B2 - DNA chip and biosensor using the same - Google Patents

Description

  The present invention relates to a biochip for detecting unlabeled target DNA and a biosensor using the biochip. More specifically, the present invention relates to a highly sensitive biosensor capable of recognizing unlabeled single nucleotide polymorphic DNA by electric signal detection.

In recent years, biochips have attracted attention in fields such as gene research, new drug development processes, and medical diagnosis, and research and development have been actively promoted.
A biochip is a nucleic acid such as DNA, a biomolecule such as a protein such as an enzyme or an antibody, a peptide such as a peptide, or a cell on a solid surface (such as a silicon substrate, glass substrate, polymer, or gold substrate as a solid support) And the like, and a specific reaction that occurs when a specific target substance is bound to a probe substance such as an immobilized biomolecule is detected. In particular, biochip-related technologies are indispensable for functional analysis technologies for biomolecules in the post-genomic era that require large-scale and parallel processing because they can detect and analyze a large amount of samples with a small amount of samples. It has become.

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.

  Genome projects have accumulated gene sequence information for various organisms including humans. In today's post-genomic era, DNA chips have been studied among biochips as the most effective and widely used technology for effectively using such gene sequence information. The DNA chip is attracting attention as a technique for examining many kinds of genes with high throughput, and is considered to be a basic technology in a wide field (Non-Patent Document 1).

  The use of DNA chips makes it possible to analyze the difference in gene expression levels between certain conditions and target conditions, and it has become widely used in the field of gene function analysis. Applications of biochips in the field are being studied. In particular, analysis of genes involved in diseases is very important for early detection and early treatment of diseases, and expectations for gene diagnosis will increase further in the future.

  Conventionally, in the microarray technology in which DNA is arrayed and immobilized on a microchip, first, various types of probe single-stranded DNAs are prepared by pins (Patent Document 1), inkjet (Patent Documents 2 and 3), and photolithography. Immobilization on a glass substrate at a high density (production of a DNA array); fluorescence-labeled target single-stranded DNA is hybridized on the DNA array; and then the DNA array is washed and dried. Then, the interaction between the probe and the target is confirmed by imaging and analyzing the fluorescence-labeled target DNA bound on the substrate with a fluorescence microscope or a fluorescence scanner.

  In addition, focusing on the fact that DNA is negatively charged, an electronic addressing technique has been developed that achieves DNA immobilization by applying a positive potential to a specific position of probe DNA, and hybridization between probe DNA and target DNA. The time has been shortened. However, this technique also uses a fluorescent material for detection of hybridization.

  Thus, the conventional microarray technology has detected the interaction between the probe and the target by, for example, labeling the target with fluorescence and optically detecting the presence or absence of fluorescence emission. However, the conventional technology requires (1) a pretreatment step for fluorescently labeling the target DNA, and (2) a laser device for exciting the fluorescent label and a device for imaging and analyzing the emitted fluorescence. From the viewpoint of workability and economy, it was difficult to put it into practical use.

  In addition, in order to analyze the expression of a target gene using a conventional fluorescence detection type DNA chip, it is necessary to prepare tens of thousands of gene fragments and arrange them at high density on a substrate. It is used only in some research institutions and hospitals because of its cost.

Currently, single nucleotide polymorphism (SNP) analysis is rapidly progressing because single nucleotide level mutations in gene DNA are involved in specific diseases, and based on individual SNP information The development of new drugs and custom-made medical care that provides medical care tailored to individual differences are the most important application fields.
For the analysis of SNP, it is necessary to simultaneously measure many kinds of DNA duplexes having slightly different stability, and it is necessary to measure in a specific solution under a predetermined temperature condition. This measurement is not possible with the conventional DNA microarray technology that measures under dry conditions.

  Patent Document 4 describes a method for analyzing a polymorphism and / or mutation of a target nucleic acid from the amount of change in fluorescence intensity when a fluorescent dye is labeled on a nucleic acid probe and the target nucleic acid is hybridized to the probe. Yes.

In recent years, electrochemical detection of DNA hybridization has been conducted (Non-patent Documents 2 and 3).
The electrochemical technique has the advantage that the DNA analysis can be detected without labeling without labeling with a fluorescent label or the like, thereby speeding up the gene analysis, simplifying the analysis apparatus, and reducing the cost. Yes.

  For this reason, research on electrochemical DNA chips has increased rapidly in recent years (Patent Documents 5, 6 and 7), and it has been studied not only as a mere DNA sensing but also as a means of analyzing molecular mechanisms of cancer development. (Non-Patent Documents 4, 5 and 6). Mikkelsen et al. Also showed the characteristics of electrochemical DNA chips reported in recent years in comparison with other gene detection methods (Non-Patent Document 7).

  For example, Patent Document 5 discloses a DNA chip having an electrode on which a DNA probe to which an intercalator is bound is fixed. After contacting the DNA chip with a target substance that is a nucleic acid or a protein, a cyclic vol The input / output of an electrical signal generated by electron transfer between a DNA base pair and an intercalator is detected using tomography or the like.

These studies mainly modify the redox substance in probe DNA or target DNA, introduce an intercalator that specifically binds to DNA (Patent Documents 4, 5 and 6), or indicator-free ( Hybridization is detected by an indicator-free method.
Therefore, in each of these methods, the redox substance is modified, which increases the cost, requires the selection of an intercalator that specifically binds to DNA, or inserts an intercalator into the base pair and doubles it. Since it affects the helical structure, there is a problem that it is not suitable for the detection of SNPs. In addition, since the indicator-free method requires the presence of guanine (G) in the base sequence, there is a problem that the measurable range is limited.

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.
In addition, for the purpose of electrochemical gene expression analysis, it is necessary to have a method and apparatus for simultaneously measuring thousands to tens of thousands of microelectrode arrays. K. Cammann et al. By applying the reported technique (Non-Patent Documents 8 and 10), it is considered possible to develop a highly integrated microelectrode array type DNA chip.

  In addition, surface plasmon resonance sensor, method of measuring weight change due to hybridization using a quartz crystal microbalance (Patent Document 7) that detects interaction as a change in vibration frequency on a micro crystal resonator, surface polarization, etc. There are methods based on various principles such as polarization analysis that measures interaction as an increase in thin film thickness from analysis of reflected light, and immunochemical methods using antibodies that recognize the interaction, but it is small for portable biosensors. Such a device has not been put into practical use yet.

JP 2003-279576 A JP 2001-186880 A JP 2001-343386 A JP 2002-191372 A JP 2003-83968 A JP 2003-161730 A JP 2003-287538 A Nature Genetics, Vol. 21, No. 1 supplement January 1999 S. Takenaka, K. Yamashita, M. Takagi, Y Uto, and H. Kondo, "DNA Sensing on a DNA Probe-Modified Electrode Using Ferrocenylnaphthalene Diimide as the Electrochemically Active Ligand", Anal. Chem, 72, 1334-1341 ( 2000) M.I.Pividori, A. Merkoci, S. Alegret, "Electrochemical genosensor design: immobilisation of oligonucleotides onto transducer surfaces and detection methods", Biosensors & Bioelectronics, 15, 291-303 (2000) E. Palecek, M. Fojta, M. Tomschik, J. Wang, "Electrochemical biosensors for DNA hybridization and DNA damage", Biosensors & Bioelectronics, 13, 621-628 (1998) D. H. Johnson, K. C. Glasgow, and H. H. Thorp, "Electrochemical Measurement of the Solvent Accessibility of Nucleobases Using Electron Transfer between DNA and Metal Complexes", J. Am. Chem. Soc., 117, 8933-8938 (1995) P. A Ropp and H. H. Thorp, "Site-selective electron transfer from purines to electrocatalysts: voltammetric detection of a biologically relevant deletion in hybridized DNA duplexes", Chem Biol., 6, 599-605 (1999) S. R. Mikkelsen, "Electrochemical Biosensors for DNA Sequence Detection", Electroanalysis, 8, 15-19 (1996) B. Ross, K. Cammann, W. Mokwa, and M. Rospert, "Ultramicroelectrode arrays as transducers for new amperometric oxygen sensors", Sensors and Actuators B, 7, 758-762 (1992) H. Meyer, H. Drewer, J. Krause, K. Cammann, R. Kakerow, Y. Manoli, W. Mokwa, and M. Rospert, "Chemical and biochemical sensor array for two-dimensional imaging of analyte distributions", Sensors and Actuators B, 18-19, 229-234 (1994)

An object of the present invention is to solve the problems of the conventional fluorescence detection type DNA chip and to develop a microelectrode array type DNA chip which is excellent in terms of simplicity, portability and development cost.
In the examples of gene detection using integrated electrode chips reported so far, an electrochemical method is used as a method for immobilizing probe DNA on a microelectrode array. It is carried out.

In order to detect double-stranded DNA after hybridization of the DNA, the present inventors have made use of the fact that the DNA has electric activity and complementary single-stranded DNAs specifically bind to each other. I thought it was important to establish a DNA hybrid detection method using DNA.
Therefore, the present inventors examined a method for immobilizing probe DNA on an electrode, produced a microelectrode array-type DNA chip, and simultaneously performed a plurality of target DNAs electrochemically without any labeling molecule modification. A new “next-generation DNA chip electrode array system” was constructed.
The final object of the present invention is to apply the DNA chip according to the present invention to a rapid and simple clinical genetic test.

  In the present invention, one or a plurality of microwells are arranged on the electrode surface by a microfabrication technique, and a biomolecule array chip is prepared by immobilizing a probe biomolecule in each of the one or a plurality of microwells. Changes in redox current values when target molecules interact with each other are detected with high sensitivity.

  More specifically, the present invention is a biochip in which one or a plurality of probe single-stranded DNAs are arrayed and immobilized, wherein one or a plurality of electrode wirings are formed on a substrate, The electrode wiring is covered with an insulating film, and 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 one region of each of the one or the plurality of electrode wirings, The biochip has a probe single-stranded DNA immobilized on the bottom of one or more wells.

  In the present invention, “DNA” is intended to include not only biological nucleic acids but also derivatives and mimetics thereof.

  In the biochip according to the present invention, a first molecule is bound to one end of the probe single-stranded DNA, and a second molecule that specifically binds to the first molecule is immobilized to the bottom of the well. The probe single-stranded DNA is immobilized on the bottom of the one or more wells by specific binding between the first molecule and the second molecule.

In particular, in the biochip according to the present invention, the first molecule is biotin and the second molecule is streptavidin.
The single-stranded DNA is a fragment of DNA encoding leptin.

  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 a plurality of wells are formed by providing one or a plurality of holes reaching the electrode surface, and then the probe single-stranded DNA is immobilized on the bottom of the one or the plurality of 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 a plurality of probe single-stranded DNAs are arrayed and immobilized, wherein one or a plurality of electrode wirings are formed on a substrate, The electrode wiring is covered with an insulating film, and 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 one region of each of the one or the plurality of electrode wirings, A biochip in which probe single-stranded DNA is immobilized at the bottom of one or more wells;
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 DNA that hybridizes with the one or more probe single-stranded DNAs present in the test solution is detected from the intensity difference between the first electrical signal and the second electrical signal. A biosensor is provided.

Here, “treatment” refers to a process necessary for hybridizing a probe single-stranded DNA and an unlabeled target DNA. Therefore, before the treatment, in the biochip, unlabeled target DNA is not hybridized to the one or more probe single-stranded DNAs. If the unlabeled target DNA is present in the test solution, the unlabeled target DNA hybridizes to the one or more probe single-stranded DNAs by this treatment. The electrical signal is measured before and after performing the above treatment on the biochip of the present invention, and if there is a change in the electrical signal, it can be confirmed that unlabeled target DNA was present in the test solution. it can.
That is, there is a statistically significant change between the electrical signal measured in the system in which the target DNA is not hybridized to the probe DNA and the electrical signal measured in the system in which the target DNA is hybridized to the probe DNA. The significant change can be associated with the hybridization of the target DNA and the probe DNA.

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 hybridization of the unlabeled target DNA to the probe DNA is detected from the change in the redox state on the biomolecule array of the biochip. Therefore, hybridization can be detected only by measuring the oxidation-reduction current value without labeling the target DNA with a fluorescent substance or the like, so that rapid and simple measurement is possible.

By using the biochip according to the present invention, a target biomolecule can be detected without using a labeling molecule such as a fluorescent substance, and thus a biosensor that can be measured quickly and easily and is miniaturized is constructed. be able to.
In addition, since different probe DNAs can be immobilized on a plurality of electrodes in the biochip according to the present invention, a plurality of genes can be detected simultaneously.
Furthermore, since the biosensor according to the present invention is very sensitive and can detect SNPs, it must conventionally be cultured for a long time from the detection of SNP genetic mutations related to various diseases. It has various possibilities such as detection of pathogenic bacteria.

Example 1 Production of DNA Chip FIG. 1 shows a top view of a DNA chip according to the present invention. In this example, eight electrode wirings 13 were formed on the glass substrate 10, and the DNA 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.
Using this configuration, as shown in FIG. 3b, the biomolecules that become the probe 2 are immobilized only on the exposed electrode surface at the bottom of the well 131a.

In order to produce the DNA chip of the present invention, a photofabrication technique, which is one of semiconductor manufacturing techniques, 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, streptavidin was immobilized on the electrode surface at the bottom of the well using a method 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), DTDP was immersed in an ethanol solution of 3,3'-dithiodipropionic acid (DTDP) and the electrode surface via its -S-S- group Of N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) -3-carbodiimide hydrochloride (EDC) in dioxane and water (9: 1 [v / v]) mixed solution by immersing the chips was activated carboxyl group of DTDP. here, by adding a streptavidin solution, streptavidin to the activation DTDP via its NH ₂ group By washing with ligated to down (Figure 9b). Phosphate buffer solution (PBS), to remove unbound streptavidin that is not immobilized to the well bottom.
Therefore, the resist 14 needs to be made of a material that does not bind to the DTDP -SS group so that streptavidin is immobilized only on the electrode surface at the bottom of the well.
Finally, the streptavidin-immobilized chip was immersed in a pre-biotinylated probe DNA 22 in PBS to immobilize the probe DNA on the bottom of the well through specific binding of streptavidin-biotin (FIG. 9c).

  In this example, the probe DNA is SEQ ID NO: 1:

      5'-gag gag ttg ggg gag cac att-3 '

The artificial nucleic acid of 21 bases shown by these was used. In the field of biochemistry, it is known that if single-stranded DNAs of about 18 to 22 bases are used, hybridization will surely occur.
Biotinylated probe DNA 22 was prepared by binding biotin to the 5 ′ end of this artificial nucleic acid by a well-known method.

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.

  It is already known that when streptavidin is immobilized on a substrate, the effective binding number of biotinylated DNA is 1. The binding site of streptavidin accessible to biotin is limited by immobilization. Furthermore, after one biotinylated DNA binds to streptavidin, further biotinylated DNA cannot access streptavidin due to electrostatic repulsion between DNAs This is probably because of this. In particular, in the case of the present invention, since streptavidin is immobilized on the bottom of the well surrounded by a wall, it is sufficient that one biotinylated probe DNA 22 is bound to one immobilized streptavidin 21. Can be considered.

FIG. 10 shows a scanning near-field optical microscope (SNOM) image using fluorescence.
FIG. 10a is a SNOM image when streptavidin was immobilized on a SNICA observation mica substrate by the method described in Example 1, and biotinylated probe DNA was bound to the streptavidin. This probe DNA is labeled with fluorescein isothiocyanate (FITC). As can be seen from this figure, fluorescence emission was observed from the entire observation field of 5 × 5 μm square. That is, it was confirmed that streptavidin was uniformly arranged.
FIG. 10b is a SNOM image when probe DNA not labeled with FITC is bound to streptavidin and FITC-labeled target DNA is hybridized. As can be seen from this figure, fluorescence emission was observed from the entire observation field, and it was confirmed that hybridization occurred almost uniformly.
FIG. 10c is a SNOM image when a probe DNA not labeled with FITC is bound to streptavidin and a DNA having the same sequence as the probe DNA labeled with FITC is used as a target. As can be seen from this figure, no fluorescence was observed from the entire observation field. This is because the target DNA has the same sequence as the probe DNA, and thus hybridization does not occur at all due to electrostatic repulsion. Furthermore, the target DNA does not bind nonspecifically to the resist film other than the well. Show.
Thus, according to the method of the present invention, it was confirmed that the probe could be uniformly immobilized on the electrode surface.

Example 2: Evaluation of detection performance of DNA chip Biosensor using DNA chip 1 of the first specific example prepared by the method described in Example 1 (single well having a diameter of 200 μm on the biomolecule array region) And a detection experiment for target DNA 3 was performed.
Specifically, an electrochemical cell is constructed in a mixed aqueous solution of 5 mM K ₃ [Fe (CN) ₆ ] and 100 mM KCl, using DNA 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 DNA chip 1 was measured by cyclic voltammetry. The measurement was performed at 25 ° C., and the potential sweep rate was 50 V / sec.

First, as a control, a current-potential curve A was obtained using a DNA chip in which the target DNA 3 was not hybridized to the probe DNA 22 (FIG. 11).
Next, the non-hybridized DNA chip is immersed in a target DNA 3 solution (DNA concentration: 1 μM) in PBS (pH 7.4) at 30 ° C. for 24 hours, and then washed several times with PBS. Unbound target DNA 3 was sufficiently removed to prepare a DNA chip in which target DNA 3 was hybridized to probe DNA 22. Using this DNA chip, a current-potential curve B was obtained (FIG. 11).
In the current-potential curve shown in FIG. 11, the peak current value indicates the redox current value.

  In this example, the target DNA 3 is SEQ ID NO: 2:

      3'-ctc ctc aac ccc ctc gtg taa-5 '

The artificial nucleic acid of 21 bases shown by these was used. This artificial nucleic acid is completely complementary to the probe DNA (SEQ ID NO: 1).

  On average, the redox current value measured using the DNA chip hybridized with the target DNA 3 was reduced by 40% from the value of the control DNA chip. That is, the average rate of change in redox current due to the hybridization of the target DNA 3 with the probe DNA is −40%. Here, the rate of change (ΔI) of the oxidation-reduction current is given by the following formula:

_{ΔI = (I T -I P)} / I P

(Wherein I _P represents the redox current measured with the target DNA non-hybridized DNA chip; I _T represents the redox current measured with the target DNA hybridized DNA chip). did.

  Next, the same measurement was performed by changing the DNA3 concentration of the solution used when the target DNA3 was hybridized to the probe DNA22. The measurement results of the rate of change of the redox current are shown in Table 1 and FIG.

  From the above results, it was shown that the presence or absence of hybridization between the probe DNA and the target DNA in each well was successfully detected with the sensitivity of sub-femtomole using the DNA chip of the present invention.

Example 3 Detection of Single Nucleotide Polymorphic DNA (SNP) (1) Performance Evaluation Using Artificial Nucleic Acid Next, an artificial SNP-DNA that differs from the target DNA shown in SEQ ID NO: 2 by only one base is used. Then, a DNA chip of the first specific example (a single well having a diameter of 200 μm on the biomolecule array region) was prepared, and the detection performance of the DNA chip of the present invention was evaluated.
This SNP-DNA is SEQ ID NO: 3:

      3'-ctc ctc aac cct ctc gtg taa-5 '

Is a 21-base artificial nucleic acid. Using a target DNA (SEQ ID NO: 2) and SNP-DNA (SEQ ID NO: 3) that are completely complementary to the probe DNA (SEQ ID NO: 1), a redox current was obtained in the same manner as in Example 2. The value was measured.
An example of the result of comparing the rate of change of the oxidation-reduction current value is shown in FIG. Since the DNA chip of the present invention has 8 electrodes, it can measure 8 points at a time. FIG. 13 shows five of the eight measurement values obtained by one measurement.
The average rate of change in redox current with a target DNA perfectly complementary to the probe DNA was -40%, whereas it was -7% with SNP-DNA. That is, when the biochip of the present invention is used, only a single base in the DNA sequence is different, and a significant difference is shown in the redox current value, and the DNA chip of the present invention is very useful for SNP detection. Indicated.

  Further, in this example, one type of probe DNA was immobilized on a plurality of electrodes, and the measurement was performed. However, as shown in FIG. Since a significant difference is observed between target DNA complementary to SNP and SNP-DNA, the possibility of simultaneously detecting a plurality of SNP-DNAs by immobilizing different types of probe DNA on each of a plurality of electrodes Became clear.

(2) Performance Evaluation Using Leptin Leptin is known as a peptide hormone that is biosynthesized and secreted by adipocytes and brings about suppression of feeding and increased energy consumption. It is known that when this leptin is abnormal, the suppression of feeding does not function normally and causes overeating and obesity. Therefore, if a means capable of easily detecting the SNP of DNA encoding leptin is established, it is considered useful for elucidating the cause of obesity.
Therefore, the same oxidation-reduction measurement was performed using human leptin DNA as the biological DNA. Human leptin DNA is a gene that spans about 20 kb in the long arm of chromosome 7. In this experiment, SEQ ID NO: 4:

      5'-aga aga agt gca cga ccg gaa-3 '

A 21-base leptin DNA fragment represented by Biotinylated probe DNA 22 was prepared by binding biotin to the 5 'end of this leptin DNA fragment by a well-known method. As target DNA, SEQ ID NO: 5:

      3'-tct tct tca cgt gct ggc ctt-5 '

A 21-base leptin DNA fragment represented by This DNA is completely complementary to the probe DNA (SEQ ID NO: 4). Further, as SNP-DNA, SEQ ID NO: 6:

      3'-tct tct tca cat gct ggc ctt-5 '

The artificial nucleic acid of 21 bases shown by these was used.
In this experiment, the average rate of change in redox current value for the completely complementary target leptin DNA (SEQ ID NO: 5) was 40%, whereas the average value of redox current value for the SNP-DNA (SEQ ID NO: 6). The rate of change was -9%. From this measurement result, it was shown that the DNA chip of the present invention is also effective for SNP detection in actual biological DNA.

Example 4: Relationship between well diameter and detection sensitivity A DNA chip of a second specific example in which wells having a diameter of 100 nm were formed in a 200 × 200 array on a biomolecule array region was prepared, and Example 2 (first The same measurement as that of the specific example DNA chip) was performed to examine the influence of the well diameter on the detection sensitivity of the DNA chip.

  An array of 100 nm diameter wells was formed in the biomolecule array region, and the probe DNA 22 (SEQ ID NO: 1) was immobilized on the electrode surface at the bottom of a plurality of wells by the method described in Example 1 (FIG. 9). Two specific examples of DNA chips were 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 DNA chip was obtained in which the probe DNA was uniformly dispersed and immobilized in a plurality of wells formed in the biomolecule array region 113.

An electrochemical cell was constructed in the same manner as in Example 2 except that this DNA chip was used.
When the DNA chip of the first specific example in which a single well having a diameter of 200 μm is formed is used, the redox current before and after the target DNA (SEQ ID NO: 2) hybridizes to the probe DNA 22 (SEQ ID NO: 1). When the DNA chip of the second specific example in which a 100 nm diameter well array (200 × 200) was formed was used, the oxidation-reduction before and after hybridization was -40%. The average change rate of the current value was -60%, and the detection sensitivity was improved.

Streptavidin has a diameter of about 10 nm, and only a single streptavidin is immobilized on a 10 nm diameter well. In this example, 40,000 streptavidin are considered to exist in one biomolecule array region. It is done.
On the other hand, when the well diameter is 200 μm, it is considered that approximately 10 ¹⁸ streptavidin is present in one well in calculation.

The number of streptavidin immobilized on the array of diameter 10nm well, the detection sensitivity in the order of 1 in 10 ¹⁴ minutes of the number of streptavidin immobilized on computationally 200μm single well is improved, the diameter 200μm In the case of a single well, a large number of streptavidins are densely immobilized in the well, so that streptavidin is associated with each other and the orientation of the binding site is not controlled. In the case of an array of 100 nm wells, the hybridization efficiency between the target DNA and the probe DNA is reduced because the activated probe DNA is not bound effectively or the probe DNAs are close to each other. Streptavidin is immobilized on each well individually, and highly efficient Hybridization with Tsu door DNA and the probe DNA is considered to be because that happened.

  Examples 1-4 were performed using the DNA chip of the first specific example in which a single well having a diameter of 200 μm was formed, and the DNA chip of the present invention has sufficiently high SNP detection performance. However, from the results of this example, it was found that the detection sensitivity could be further increased by reducing the well diameter and controlling the streptavidin sequence.

  From the above results, according to the present invention, it is not necessary to label the target DNA with a fluorescent substance or the like, measurement in an electrolyte solution is possible, and a plurality of electrodes are produced using a 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 DNA chip of this invention. The top view of the biomolecule array area | region of the DNA chip of this invention. Sectional drawing of the biomolecule array area | region of this invention. Schematic explaining the manufacturing process of the DNA chip of the 1st specific example of this invention. Schematic explaining the manufacturing process of the DNA chip of the 1st specific example of this invention. Schematic explaining the manufacturing process of the DNA chip of the 1st specific example of this invention. Schematic explaining the manufacturing process of the DNA chip of the 2nd example of this invention. Schematic explaining the manufacturing process of the DNA chip of the 2nd example of this invention. Schematic explaining the manufacturing process of the DNA chip of this invention. DNA chip of the present invention SNOM image of a DNA array produced on a mica substrate with the same structure as the protein chip of the present invention. The current-potential curve measured using the DNA chip of the 1st specific example of this invention. The graph explaining the relationship between the change rate of the oxidation-reduction current value measured using the DNA chip of the 1st specific example of this invention, and the target DNA density | concentration in hybridization. The SNP-DNA detection result using the DNA chip of the 1st specific example of this invention. The AFM image of the DNA chip of the 2nd example of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Biochip (DNA chip), 10 ... Substrate, 11 ... Photoresist, 12a ... Ultraviolet, 12b ... Photomask, 13 ... Electrode wiring, 131 ... Biomolecule Array region, 131a ... well, 132 ... pad, 14 ... insulating resist, 15 ... electron beam, 2 ... probe, 21 ... streptavidin, 22 ... probe DNA, 3 Target DNA.

Claims (5)

One or more electrode wirings are formed on the substrate, the one or more electrode wirings are covered with an insulating film, and one or more wells are formed in the insulating film on each region of the one or more electrode wirings. Each of the one or more wells is formed with a side surface defined by the insulating film and a bottom portion defined by the surface of the electrode wiring;
One or more probe single-stranded DNAs are arrayed and immobilized on the bottom of each of the one or more wells, and a first molecule binds to one end of the probe single-stranded DNA. And a second molecule that specifically binds to the first molecule is immobilized on the surface of the electrode wiring defining the bottom of the well, and the specific binding between the first molecule and the second molecule The biochip , wherein the single-stranded DNA of the probe is immobilized on the bottom of the one or more wells, and the diameter of the one or more wells is 20 nm or less up to 20 nm . The biochip according to claim 1 , wherein the diameter of the one or more wells is 100 nm or less and 20 nm or less . A molecule of the first biotin, according to claim 1 biochip molecules the second is streptavidin.   The biochip according to claim 1, wherein the single-stranded DNA is a fragment of DNA encoding leptin. A biosensor comprising at least a working electrode, a counter electrode and a reference electrode,
Here, said working electrode, one or more electrode wires on the base plate is formed, the one or more electrode wires is coated with an insulating film, each of the one or more electrode wires on one region One or more wells are formed in the insulating film , wherein each of the one or more wells has a side surface defined by the insulating film and a bottom portion defined by the surface of the electrode wiring,
One or more probe single-stranded DNAs are arrayed and immobilized on the bottom of each of the one or more wells, and a first molecule binds to one end of the probe single-stranded DNA. And a second molecule that specifically binds to the first molecule is immobilized on the surface of the electrode wiring defining the bottom of the well, and the specific binding between the first molecule and the second molecule A biosensor, wherein the probe single-stranded DNA is immobilized on the bottom of the one or more wells, and the diameter of the one or more wells is submicron or less to 20 nm .

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