JP4941994B2 - Gene detection method - Google Patents

Description

  The present invention relates to a gene detection method for specifically detecting a specific gene present in a sample.

A technique for immobilizing a single-stranded nucleic acid probe having a base sequence complementary to a target gene to be detected on a substrate and confirming the hybridization between the single-stranded nucleic acid probe and the target gene by some method is today It has become an important basic technology for gene detection.
For example, in a DNA chip, multiple types of nucleic acid probes are fixed and aligned as spots on a substrate such as silicon or glass, and hybridization occurs when the gene in the sample and the base sequence of the nucleic acid probe are complementary. It is possible to simultaneously confirm the presence of many types of genes using. A systematic and comprehensive analysis of gene expression and functions using DNA chips reveals in detail the mechanism that causes the disease at the molecular level, and a revolutionary new diagnostic method targeting the cause of the disease as a target molecule And the development of therapeutic methods and drugs, the mechanism of causing the disease, the creation of customized prevention and treatment methods that take into account individual differences in susceptibility to the disease, and the establishment of diagnostic technology therefor. ing.

As existing techniques for detecting hybridization between a nucleic acid probe immobilized on a substrate and a target gene, a fluorescent labeling method, an enzyme labeling method, and an electrochemical detection method using an intercalator are the main ones.
In the fluorescent labeling method, first, an operation of introducing a fluorescent dye into a gene in a sample is performed, and the fluorescence measurement detects that the fluorescently labeled gene has hybridized with the nucleic acid probe. Although the fluorescent labeling method is the most widely used method, the operation of introducing a fluorescent dye into a sample gene is complicated and complicated, and thus has a drawback that it takes a long time of about 2 to 3 days to detect the gene. ing. Further, since the fluorescence detection is used as a measurement principle, the apparatus is unavoidable to be large and expensive.

  The enzyme labeling method is a technique for introducing an enzyme into a gene in a sample, and utilizes the fact that an enzyme reaction is performed on a nucleic acid probe spot by hybridizing an enzyme-labeled gene with a nucleic acid probe. By detecting enzyme reaction products by electrochemical methods, the equipment can be simplified and miniaturized. However, the labeling operation is complicated and complicated, and it takes a long time to detect genes. It is the same as the labeling method.

In recent years, an electrochemical detection method using an intercalator has been devised (see, for example, Patent Document 1 and Non-Patent Document 1). Intercalator is a general term for substances consisting of organic substances, metal complexes and the like that specifically bind to double-stranded nucleic acids. For the purpose of hybridization detection, an intercalator having electrochemical activity is used. When the nucleic acid probe and the gene are hybridized, the intercalator added to the sample solution is incorporated into the double strand formed from the nucleic acid probe and the gene, so the redox current of the incorporated intercalator is used as the nucleic acid probe spot. By measuring at, hybridization can be detected.
Japanese Patent No. 2573433 N. Komatsu, T. Nojima, S. Takenaka, “Analysis of Electrochemical Reaction of Ferrocenylnaphthalene Diimide Captured by Double-Stranded DNA during the Electrochemical Detection of DNA Hybridization”, Electrochemistry, 74, 2006, 65-67.

This detection method is simple because it does not require a chemical modification operation in which a gene in a sample is labeled with a fluorescent dye or an enzyme in advance, and the time required for gene detection can be shortened to about 1 hour. In addition, since the electrochemical method is used as a detection principle, the apparatus can be simplified and downsized. However, if an intercalator that has not been incorporated into the double-stranded nucleic acid coexists in the sample solution, it causes a measurement error. Therefore, there is a problem that an operation of removing this by washing or the like is indispensable.
In addition, intercalators are known not only to bind to double-stranded nucleic acids, but also to cause nonspecific physical adsorption on the substrate, leading to a decrease in sensitivity, thus preventing nonspecific physical adsorption. Chemical processing of the substrate is also required. Furthermore, because of the nature of binding to double-stranded nucleic acids, many intercalators are harmful to the human body, such as having mutagenic properties, and there are problems with easy use of intercalators.

  The present invention has been made in view of the above circumstances, and the problem is that it is more convenient and safer than the electrochemical method using an intercalator, and the hybridization between the target gene and nucleic acid probe can be performed in a short time. An object of the present invention is to provide a method for detecting an electrochemical gene that can be detected.

As a result of various studies on a method capable of detecting hybridization by electrochemical measurement without using an intercalator, the present inventors have obtained a gold electrode having a nucleic acid probe immobilized on its surface. When copper ions were electrodeposited using this, it was found that the appearance of electrodeposition changes before and after hybridization, and the gene detection method of the present invention has been completed using this phenomenon.
More specifically, the present invention utilizes the underpotential precipitation phenomenon of copper ions on the gold electrode surface. When depositing copper onto a gold electrode, it is known that, under certain conditions, deposition of copper atoms begins at a potential on the anode side of the equilibrium electrode potential of copper prior to bulk copper deposition. Yes. This is underpotential precipitation, which results from the deposition of about one layer of copper atoms on the (111) plane of the gold crystal (see, for example, Non-Patent Document 2).
The Chemical Society of Japan, “5th edition, Experimental Chemistry Course 24, Surface / Interface”, Maruzen Co., Ltd., published on January 30, 2007, pages 393-402

  The present inventors have found that underpotential precipitation occurs in a gold electrode on which a nucleic acid probe is immobilized. As an example, in Example 1 to be described later, the underpotential deposition potential was about 0.18 V (a value based on a silver-silver chloride electrode, the same applies hereinafter). On the other hand, as described later in Example 1, after immobilizing the nucleic acid probe, the gold electrode hybridized with the target DNA had an underpotential deposition potential of approximately 0.0 V, and was a copper balanced electrode. It was greatly shifted to the cathode side near the potential. By utilizing the cathode shift of this underpotential precipitation potential due to hybridization, it is possible to detect the presence of a specific gene in a sample.

That is, the following configurations 1 to 6 are employed in the present invention.
1. A gold electrode having a single-stranded nucleic acid probe having a base sequence complementary to the target gene is inserted into the electrolyte solution containing the target gene, and after adding copper ions, the gold electrode is used as a working electrode. A method for detecting a gene, wherein the target gene is detected by performing electrodeposition of copper ions and confirming a change in the electrodeposition phenomenon due to hybridization between the nucleic acid probe and the target gene.
2. 2. The gene detection method according to 1, wherein a shift of the underpotential deposition potential of copper ions to the cathode side in the gold electrode is confirmed as the electrodeposition phenomenon.
3. 2. The gene detection method according to 1, wherein, as the electrodeposition phenomenon, constant potential electrodeposition of copper ions on the gold electrode is performed in the vicinity of an underpotential deposition potential, and a decrease in the amount of copper deposition is confirmed.
4). 4. The gene detection method according to any one of 1 to 3, wherein a gold electrode having a (111) plane is used as the gold electrode.
5. Any one of 1 to 4, wherein a nucleic acid probe having an SH group is used as the single-stranded nucleic acid probe, and a gold electrode having a nucleic acid probe fixed on the surface by covalent bonding of the SH group and the gold electrode is used. A method for detecting the gene according to claim 1.
6). The method for detecting a gene according to any one of 1 to 5, wherein a water-soluble copper salt is used as the copper ion added to the electrolyte solution.

  According to the present invention, since it is not necessary to chemically modify a gene in a sample with a fluorescent dye or an enzyme, the detection operation is simple, and hybridization can be detected in a short time. Further, since it is not necessary to use an intercalator, the simplicity and safety of the detection operation are improved. Furthermore, since the detection method of the present invention is based on the electrochemical method, hybridization can be detected with a simple and small device.

In the method for detecting hybridization according to the present invention, a single-stranded nucleic acid probe having a base sequence complementary to a target gene to be detected is immobilized on the gold electrode surface. The shape and manufacturing method of the gold electrode used in the present invention are not particularly limited, and a gold thin film prepared on a substrate by gold single crystal, gold polycrystal, vapor deposition method, sputtering method, electroless plating, etc. Is used. Since underpotential precipitation of copper ions occurs only on the (111) plane of the gold single crystal, in the case of the gold single crystal, only the (111) plane is brought into contact with the electrolyte solution and used as a working electrode. The gold (111) surface having a suitable shape and smoothness as an electrode can be obtained, for example, by melting the tip of a gold wire with a hydrogen-oxygen flame and solidifying it into a spherical single crystal, and cutting the (111) surface from itself. It can be produced (see, for example, Non-Patent Document 3).
Electrochemical Society Measurement Method Subcommittee, `` Electrochemistry-I want to know and discuss it, '' Electrochemistry, 70, 2002, 51-58.

A gold thin film produced on a substrate by gold polycrystal, sputtering, electroless plating, or the like is used as an electrode after the surface is annealed and quenched. By annealing / quenching, most of the surface of the polycrystalline gold film or the thin gold film can have a (111) plane structure (see, for example, Non-Patent Document 4).
As the nucleic acid probe, both a gene extracted from a biological sample and a chemically synthesized DNA are used. The nucleic acid probe can be immobilized on the surface of the gold electrode by covalent bond, ionic bond, physical adsorption, etc. Preferably, a thiol group (SH group) is introduced into the nucleic acid probe in advance to form a strong covalent bond with gold. Let it form.
The Chemical Society of Japan, “5th edition, Experimental Chemistry Course 24, Surface / Interface”, Maruzen Co., Ltd., published on January 30, 2007, pages 283-301

By inserting the gold electrode on which the nucleic acid probe obtained above is immobilized into an appropriate electrolyte solution containing the target gene to be detected, hybridization occurs between the nucleic acid probe and the gene having a complementary base sequence. A double-stranded nucleic acid is formed. Next, copper ions are added to the sample solution. Copper ions are added by dissolving a water-soluble copper salt in the sample solution. The chemical form of the copper salt used in this case is not particularly limited, but preferably does not contain ions that are easily oxidized or reduced in addition to copper ions, for example, copper (II) sulfate, copper nitrate ( II), copper (II) perchlorate, copper (II) acetate and the like are used.
Further, after inserting a counter electrode and a reference electrode into the electrolyte solution, an underpotential deposition potential of copper ions is measured using the gold electrode as a working electrode (UPD potential A). For the measurement of the underpotential deposition potential, a widely used electrochemical measurement method such as cyclic voltammetry or differential pulse voltammetry can be used.

On the other hand, for comparison, the electrodeposition potential when no hybridization occurs is measured. That is, the gold electrode on which the nucleic acid probe obtained above is immobilized is inserted into an electrolyte solution that does not contain the target gene to be detected, and then copper ions are added. Further, after inserting a counter electrode and a reference electrode into the electrolyte solution, an underpotential deposition potential of copper ions is measured using the gold electrode as a working electrode (UPD potential B).
The composition of the electrolyte solution used at this time, the concentration of copper ions to be added, and the type of counter electrode and reference electrode to be used are the same as those at the time of measuring the UPD potential A. Since the UPD potential A measured as described above is largely shifted to the cathode side as compared with the UPD potential B, it can be confirmed that hybridization between the nucleic acid probe and the target gene has occurred.

  Further, in the method for confirming hybridization by the amount of copper deposited by constant potential electrodeposition, on the gold electrode on which the nucleic acid probe is immobilized by constant potential electrolysis with the electrode potential fixed in the vicinity of the UPD potential B. Metal copper is deposited on the substrate. At this time, when the target gene is contained in the electrolyte solution, the amount of metal copper deposited is significantly smaller than when the target gene is not contained in the electrolyte solution. It is possible to detect that hybridization has occurred.

The amount of metallic copper deposited on the gold electrode on which the nucleic acid probe is immobilized can be measured by an electrochemical stripping method, a surface plasmon resonance (SPR) method, a quartz crystal microbalance (QCM) method, or the like. As a preferred method, an electrochemical stripping method is used because measurement can be performed with the same apparatus configuration as the above-described constant potential electrolysis.
That is, the electrodeposited metal potential is shifted from the potential at which constant potential electrodeposition is performed to the potential on the anode side, whereby the metal copper once electrodeposited is redissolved. The amount of deposited metal copper is determined by measuring the amount of electricity flowing at this time (see, for example, Non-Patent Document 5).
Akira Fujishima, Masuo Aizawa, Toru Inoue, "Electrochemical Measurement Method", Gihodo Publishing, 1984, pp. 206-208

In the present invention, various genes can be detected by changing the nucleic acid probe to be used. Examples of the nucleic acid probe that can be used include a probe having a sequence complementary to the causative gene of a genetic disease, the gene of a pathogenic microorganism, or the whole or a partial base sequence of each virus.
When a probe having a sequence complementary to the whole or a part of the base sequence of a causative gene of a genetic disease is used as the nucleic acid probe, the genetic disease can be determined. Examples of genes causing such genetic diseases include hemochromatosis and genes responsible for mitochondrial cardiomyopathy.

When a probe having a sequence complementary to the whole or a part of the base sequence of a gene of a pathogenic microorganism is used, an infectious disease caused by the microorganism can be diagnosed. For example, Salmonella, enterohemorrhagic Escherichia coli, and Chlamydia can be mentioned as such microorganisms that infect the human body and cause infection.
When a probe having a sequence complementary to the whole or a part of the nucleotide sequence of a virus is used, it becomes possible to diagnose an infection caused by the virus. For example, herpes virus and influenza virus can be mentioned as such viruses that infect the human body and cause infection.
The length of the nucleic acid probe used in the present invention is not particularly limited, and a single stranded nucleic acid of several mer to several hundred mer can be used. In order to obtain a highly accurate detection result, several mer It is preferable to use one having a length of several tens of mer.

EXAMPLES Next, the present invention will be further described with reference to examples, but the following specific examples are not intended to limit the present invention.
(Example 1: Detection of hybridization by measuring the electrodeposition potential of copper; when a gold single crystal is used as a gold electrode)
(1) Immobilization of nucleic acid probe to gold electrode A gold electrode was prepared by the method described in Non-Patent Document 3 as follows. That is, the tip of a gold wire with a diameter of 0.8 mm was melted with a hydrogen-oxygen flame and solidified, and a spherical gold single crystal with a diameter of 2 mm was provided at the tip of the gold wire. The spherical portion was polished and processed into a hemispherical shape so that the gold (111) surface was exposed at the planar cross-sectional portion of the hemisphere. The area of the gold (111) surface thus prepared was 3.1 mm ² . At the time of electrochemical measurement, the gold wire portion of this gold electrode is used to hang it on the electrolyte solution surface, and the gold (111) surface is brought into contact with the electrolyte solution by the hanging meniscus method. Only the surface operates as a working electrode (for the hanging meniscus method, see, for example, Non-Patent Documents 2 and 3).

As the nucleic acid probe, a synthetic nucleic acid (SEQ ID NO: 1) having the structural formula 5 ′ HS (CH ₂ ) ₆ -GACTTTAAGCAAGAAAC 3 ′ consisting of nucleic acid residue 17 and having an SH group at the 5 ′ end was used. 140 μg of the nucleic acid probe was dissolved in 50 mM NaClO ₄ to make a total volume of 50 ml to obtain a solution having a nucleic acid probe concentration of 5 μM. The gold electrode was immersed in the nucleic acid probe solution for 5 minutes to immobilize the nucleic acid probe on the gold electrode. The gold electrode was removed from the nucleic acid probe solution, sufficiently washed with water, and dried. In this case, the nucleic acid probe is bound on the gold electrode via Au-S bond, that is, immobilized in the form of Au-5 ′ S (CH ₂ ) ₆ -GACTTTAAGCAAGAAAC 3 ′ (SEQ ID NO: 2). Is known. When the immobilization density of the nucleic acid probe to the gold electrode was estimated by electrochemical reductive desorption method, it was 2.3 μg / cm ² , that is, 4.2 × 10 ⁻¹⁰ mol / cm ² .

(2) Hybridization of nucleic acid probe and target DNA The target DNA is 5 ′ GTTTCTTGCTTAAAGTC 3 ′ (SEQ ID NO: 3) having a base sequence complementary to the nucleic acid probe. First, the target DNA was dissolved in 4 × SSC (0.6 M NaCl, 0.06 M sodium citrate) buffer solution as a sample solution to obtain a solution having a concentration of 50 μM (258 μg / ml). The gold electrode on which the nucleic acid probe prepared in (1) is immobilized is immersed in this solution containing the target DNA for 60 minutes to allow hybridization, and then the gold electrode is removed from the solution, washed with water, and then dried. I let you.

(3) Measurement of current-potential curve for gold electrode with nucleic acid probe immobilized
Using the (111) plane of the gold electrode on which the nucleic acid probe prepared in (1) was immobilized, the current-potential curve was measured by cyclic voltammetry. A 0.1 M NaClO ₄ containing 1 mM Cu (ClO ₄ ) ₂ is used as an electrolytic solution, and a platinum electrode as a counter electrode and a silver / silver chloride electrode (saturated KCl) as a reference electrode are inserted in the electrolytic solution in advance. Further, the (111) surface of the gold electrode was further brought into contact with the electrolytic solution, allowed to stand for 1 minute, and then a current-potential curve was measured at a potential scanning rate of 20 mV per second. The current-potential curve obtained at this time is shown as curve 1 in FIG. It can be seen from curve 1 that a cathode peak corresponding to UPD deposition of copper ions appears at an electrode potential of 0.18 V (UPD potential B).

(4) Measurement of the current-potential curve for the gold electrode that was hybridized with the target DNA after immobilizing the nucleic acid probe
The current-potential curve was measured by cyclic voltammetry using the (111) surface of the gold electrode on which the nucleic acid probe prepared and hybridized was prepared as the working electrode. A 0.1 M NaClO ₄ containing 1 mM Cu (ClO ₄ ) ₂ is used as an electrolytic solution, and a platinum electrode as a counter electrode and a silver / silver chloride electrode as a reference electrode are inserted in the electrolytic solution in advance. Further, the (111) surface of the gold electrode was brought into contact with the electrolytic solution, allowed to stand for 1 minute, and then a current-potential curve was measured at a potential scanning rate of 20 mV per second. The current-potential curve obtained at this time is shown as curve 2 in FIG. From curve 2, it can be seen that a cathode peak corresponding to UPD deposition of copper ions appears at an electrode potential of 0.0 V (UPD potential A). Since UPD potential A is largely shifted to the cathode side as compared with UPD potential B in (3), it is confirmed that hybridization has occurred.

(Example 2: Detection of hybridization by measuring the amount of deposited copper using an electrochemical stripping method: When a gold single crystal is used as a gold electrode)
(1) Measurement of copper stripping electricity quantity at the gold electrode to which the nucleic acid probe was fixed. The (111) surface of the gold electrode to which the nucleic acid probe prepared in Example 1 (1) was fixed was coated with 1 mM Cu (ClO ₄ ) ₂ . A 0.1 M NaClO ₄ solution is brought into contact, and a platinum electrode as a counter electrode and a silver / silver chloride electrode as a reference electrode are inserted into the solution to form a three-electrode arrangement. First, using a gold electrode as a working electrode, the electrode was held at 0.12 V for 1 minute, and copper ions were electrodeposited at a constant potential on the gold electrode. As this holding potential, a potential slightly on the cathode side from the UPD potential B was selected. After that, when the potential was swept to the anode side at a rate of 20 mV per second, a stripping current due to remelting of copper was observed. The stripping current-potential curve obtained at this time is shown in FIG. The shaded area in FIG. 2 gives the amount of electricity for copper stripping, and the value was 4.0 × 10 ⁻⁴ C / cm ² . From this amount of electricity, it can be seen that 2.1 × 10 ⁻⁹ mol / cm ² of copper was deposited.

(2) Measurement of copper stripping electricity quantity for gold electrode hybridized with target DNA after immobilization of nucleic acid probe. Immobilization of nucleic acid probe prepared in Example 1 (2) and hybridization. The (111) surface of the gold electrode was brought into contact with a 0.1 M NaClO ₄ solution containing 1 mM Cu (ClO ₄ ) _2, and a platinum electrode as a counter electrode and a silver / silver chloride electrode as a reference electrode were inserted into the solution. 3 electrode arrangement. First, using a gold electrode as a working electrode, the electrode was held at 0.12 V for 1 minute, and copper ions were electrodeposited at a constant potential on the gold electrode. As the holding potential, a potential slightly on the cathode side from the electrodeposition potential B was selected. After that, when the potential was swept to the anode side at a rate of 20 mV per second, a stripping current due to remelting of copper was observed. The stripping current-potential curve obtained at this time is shown in FIG. The shaded area in FIG. 3 gives the amount of electricity for stripping copper, and the value was 0.50 × 10 ⁻⁴ C / cm ² . From this amount of electricity, it can be seen that 0.26 × 10 ^-9 mol / cm ² of copper was deposited, and the amount of deposited copper was much smaller than that measured in (1), so hybridization occurred. It is confirmed that At this time, the amount of copper deposited on the gold electrode on which the nucleic acid probe is immobilized can be reduced to half or less as a guideline for confirmation of hybridization.

(Example 3: Detection of hybridization by measuring the electrodeposition potential of copper; when a gold wire is used as a gold electrode)
(1) Fixing of nucleic acid probe to gold electrode A gold electrode was prepared by processing a 0.8 mm diameter gold wire as follows. That is, the gold wire was cut and its end face was mirror-polished with an alumina bubbling. Next, the end surface of the mirror-finished gold wire was annealed with a hydrogen-oxygen flame, immediately immersed in pure water and rapidly cooled, and used as a gold electrode. The portion other than the end face of the gold wire (the side face of the gold wire) was electrically insulated by coating with an epoxy resin so that only the end face acts as an electrode.
As the nucleic acid probe, a synthetic nucleic acid (SEQ ID NO: 1) having the structural formula 5 ′ HS (CH ₂ ) ₆ -GACTTTAAGCAAGAAAC 3 ′ consisting of nucleic acid residue 17 and having an SH group at the 5 ′ end was used as in Example 1. . 140 μg of the nucleic acid probe was dissolved in 50 mM phosphate buffer at pH 7.0 to make a total volume of 50 ml, and a solution with a nucleic acid probe concentration of 5 μM was obtained. The gold electrode was immersed in the nucleic acid probe solution for 5 minutes to immobilize the nucleic acid probe on the gold electrode. The gold electrode was removed from the nucleic acid probe solution, sufficiently washed with water, and dried. In this case, the nucleic acid probe is bound on the gold electrode via Au—S bond, that is, immobilized in the form of Au-5 ′ S (CH ₂ ) ₆ -GACTTTAAGCAAGAAAC 3 ′ (SEQ ID NO: 2). Is known. The immobilization density of the nucleic acid probe on the surface of the gold electrode was estimated by an electrochemical reductive desorption method. As a result, it was 1.7 μg / cm ² , that is, 3.2 × 10 ⁻¹⁰ mol / cm ² .

(2) Hybridization of nucleic acid probe and target DNA As in Example 1, the target DNA is 5 ′ GTTTCTTGCTTAAAGTC 3 ′ (SEQ ID NO: 3) having a base sequence complementary to the nucleic acid probe. First, the target DNA was dissolved in 4 × SSC (0.6 M NaCl, 0.06 M sodium citrate) buffer solution as a sample solution to obtain a solution having a concentration of 50 μM (258 μg / ml). The gold electrode on which the nucleic acid probe prepared in (1) is immobilized is immersed in this solution containing the target DNA for 60 minutes to allow hybridization, and then the gold electrode is removed from the solution, washed with water, and then dried. I let you.

(3) Measurement of current-potential curve for gold electrode with nucleic acid probe immobilized
Using the gold electrode on which the nucleic acid probe prepared in (1) was fixed as a working electrode, a current-potential curve was measured by a cyclic voltammetry method. A 0.1 M NaClO ₄ containing 1 mM Cu (NO ₃ ) ₂ is used as an electrolytic solution, and a platinum electrode as a counter electrode and a silver / silver chloride electrode (saturated KCl) as a reference electrode are inserted in the electrolytic solution in advance. The gold electrode was further inserted here, allowed to stand for 1 minute, and then a current-potential curve was measured at a potential scanning speed of 20 mV per second. The measurement result in Example 1 (3) is shown in FIG. A current-potential curve having the same shape as curve 1 was obtained. From the position of the cathode peak appearing in this current-potential curve, the value of the UPD potential B was determined to be 0.14 V in this example.

(4) Measurement of the current-potential curve for the gold electrode that was hybridized with the target DNA after immobilizing the nucleic acid probe
The current-potential curve was measured by the cyclic voltammetry method using the gold electrode prepared in (2) to which the nucleic acid probe was immobilized and hybridization was performed. A 0.1 M NaClO ₄ containing 1 mM Cu (NO ₃ ) ₂ is used as an electrolytic solution, and a platinum electrode as a counter electrode and a silver / silver chloride electrode as a reference electrode are inserted in the electrolytic solution in advance. The gold electrode was further inserted here, and after standing for 1 minute, a current-potential curve was measured at a potential scanning speed of 20 mV per second. The measurement result in Example 1 (4) is shown in FIG. A current-potential curve having the same shape as curve 2 was obtained. From the position of the cathode peak appearing in the current-potential curve, it was determined that the value of the UPD potential A was 0.0 V in this example. Since UPD potential A is largely shifted to the cathode side as compared with UPD potential B in (3), it is confirmed that hybridization has occurred.

(Example 4: Detection of hybridization by measuring the amount of deposited copper using an electrochemical stripping method: When a gold wire is used as a gold electrode)
(1) Measurement of copper stripping electricity quantity at a gold electrode to which a nucleic acid probe is immobilized A gold electrode to which a nucleic acid probe prepared in Example 3 (1) is immobilized is 0.1 M NaClO containing 1 mM Cu (NO ₃ ) _2. Insert into ₄ and insert a platinum electrode as a counter electrode and a silver / silver chloride electrode as a reference electrode to form a three-electrode arrangement. First, using a gold electrode as a working electrode, the electrode was held at 0.12 V for 1 minute, and copper ions were electrodeposited at a constant potential on the gold electrode. As this holding potential, a potential slightly on the cathode side from the UPD potential B was selected. After that, when the potential was swept to the anode side at a rate of 20 mV per second, a stripping current due to remelting of copper was observed. The stripping current-potential curve obtained at this time showed the same shape as in FIG. 2, which was the measurement result in Example 2 (1). In the same manner as in Example 2 (1), the value of the amount of electricity for stripping of copper in this example was determined from the stripping current-potential curve and found to be 5.6 × 10 ⁻⁴ C / cm ² . From this amount of electricity, it can be seen that 2.9 × 10 ⁻⁹ mol / cm ² of copper was deposited.

(2) Measurement of copper stripping electric quantity for gold electrode hybridized with target DNA after immobilization of nucleic acid probe. Immobilization of nucleic acid probe prepared in Example 3 (2) and hybridization. The performed gold electrode is inserted into 0.1 M NaClO ₄ containing 1 mM Cu (NO ₃ ) _2, and a platinum electrode as a counter electrode and a silver / silver chloride electrode as a reference electrode are inserted to form a three-electrode arrangement. First, a gold electrode was used as a working electrode and held at 0.1 V for 1 minute. As the holding potential, a potential slightly on the cathode side from the electrodeposition potential B was selected. After that, when the potential was swept to the anode side at a rate of 20 mV per second, a stripping current due to remelting of copper was observed. The stripping current-potential curve obtained at this time showed the same shape as in FIG. 3, which is the measurement result in Example 2 (2). In the same manner as in Example 2 (2), the value of the amount of electricity for stripping of copper in this example was determined from the stripping current-potential curve and found to be 2.3 × 10 ⁻⁴ C / cm ² . From this amount of electricity, it can be seen that 1.2 × 10 ^-9 mol / cm ² of copper was deposited, and the amount of deposited copper was reduced to half or less compared to the measurement in (1). It is confirmed that hybridization has occurred.

It is a figure which shows the shape of the electric current-potential curve obtained in Example 1, and the position of UPD electric potential A and UPD electric potential B. FIG. It is a figure which shows the magnitude | size of the stripping electric current-potential curve obtained in Example 2 (1), and the magnitude | size of stripping electricity. It is a figure which shows the magnitude | size of the stripping electric current-potential curve obtained in Example 2 (2), and the magnitude | size of stripping electricity.

Claims (6)

  A gold electrode having a single-stranded nucleic acid probe having a base sequence complementary to the target gene is inserted into the electrolyte solution containing the target gene, and after adding copper ions, the gold electrode is used as a working electrode. A method for detecting a gene, wherein the target gene is detected by performing electrodeposition of copper ions and confirming a change in the electrodeposition phenomenon due to hybridization between the nucleic acid probe and the target gene.   The method for detecting a gene according to claim 1, wherein as the electrodeposition phenomenon, a shift of the underpotential deposition potential of copper ions on the gold electrode to the cathode side is confirmed.   The gene detection according to claim 1, wherein as the electrodeposition phenomenon, constant potential electrodeposition of copper ions on the gold electrode is performed in the vicinity of an underpotential deposition potential, and a decrease in the amount of copper deposition is confirmed. Method.   The method for detecting a gene according to any one of claims 1 to 3, wherein a gold electrode having a (111) plane is used as the gold electrode.   5. A nucleic acid probe having an SH group is used as the single-stranded nucleic acid probe, and a gold electrode having a nucleic acid probe immobilized on the surface by covalent bonding of the SH group and a gold electrode is used. The method for detecting a gene according to any one of the above.   The method for detecting a gene according to any one of claims 1 to 5, wherein a water-soluble copper salt is used as the copper ion added to the electrolyte solution.

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