JP3233851B2 - Electrochemical detection method of gene and its device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an apparatus and a method for detecting DNA.

[0002]

2. Description of the Related Art In gene analysis in the fields of biology and medicine, a hybridization method is used as a means for detecting DNA having a specific sequence.

[0003] Among them, particularly, so-called Southern hybridization is generally used. In general, this Southern hybridization is performed by first applying the test D
After fragmenting NA with one or more restriction enzymes,
Separate by size on gel electrophoresis. Next, the test DNA is denatured into single-stranded DNA, and then immobilized on a nylon filter or nitrocellulose paper. Then, the denatured single-stranded DNA is hybridized with a complementary single-stranded DNA (hereinafter, referred to as a DNA probe) forming a base pair labeled with a radioisotope (hereinafter, referred to as RI). Wash the filter (nitrocellulose paper). After washing, the filter (nitrocellulose paper) is subjected to autoradiography and developed, whereby DNA having a specific sequence that hybridizes with the probe DNA is detected.

[0004]

The conventional methods such as the above-mentioned southern hybridization use a radioisotope as a label, which requires not only a large expense and labor for a radioactive substance handling facility and its maintenance, but also for researchers. Health safety is not guaranteed. It takes more than 24 hours to detect a band by autoradiography. Further, when the amount of the test DNA is small, there is a problem that long-time exposure is required to expose the film, and the outline of the band becomes unclear.

[0005] In the Southern hybridization method, a method has been proposed in which a fluorescent substance is used as a probe label instead of RI. Although this method is superior to RI in terms of safety and speed, it causes discoloration due to excitation light,
The measurement has a drawback that a dedicated fluorescence measurement device is required, and it is difficult to introduce a certain amount or more of a fluorescent substance as a probe for internal quenching of the fluorescence. In recent years, a method of detecting DNA by luminescence has also been put to practical use, but a dedicated measuring device is required as in the case of the fluorescence method.

[0006] As described above, various detection methods have been studied. In order to detect the presence or absence of a specific gene sequence, in each case, DNA obtained from a sample is fragmented with a restriction enzyme and then subjected to electrolysis. The problem that the DNA must be fractionated according to size by electrophoresis or the like and the test DNA must be immobilized on nitrocellulose paper or the like, which is complicated,
Unresolved.

[0007]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and detects a DNA sequence having a specific sequence without fragmenting the DNA of a specimen. It is to provide a method and an apparatus.

According to the present invention, a single-stranded DNA probe is immobilized on an electrode surface, and a complementary double-stranded DN of a test DNA and a probe DNA in a solution system in the presence of an intercalator.
The present invention relates to an invention in which A is formed, and the detection of the formed double-stranded DNA is performed by detecting and / or measuring a change in current of an electrode as an electrochemical signal.

Accordingly, the present invention relates to a method for detecting DNA having a specific sequence, comprising the steps of: using an electrode on which a DNA probe modified with a conductive substance is immobilized, and a sample containing DNA in the presence of an intercalator. And the DNA
A method of forming a hybrid DNA of a probe and DNA; and a step of detecting and / or measuring a current of the electrode after the reaction.

[0010] In a preferred embodiment, the method includes a step of washing the electrode after the reaction.

In a preferred embodiment, the conductive substance is a compound having a redox activity. More preferably, said compound is ferrocene.

[0012] In a preferred embodiment, the intercalator is a ferrocene-modified sewn intercalator. What is a sewn intercalator?
An agent that forms a complex in which two substituents respectively protrude into the main groove upon intercalation into NA. Thus, one of the substituents acts as a stopper when the threaded intercalator comes off the DNA. Thereby, dissociation from double-stranded nucleic acids is extremely slow. Single strands dissociate quickly because this does not occur. Therefore, a sewn intercalator can be suitably used.

Further, the present invention relates to an apparatus for detecting DNA having a specific sequence, an electrode on which a DNA probe modified with a conductive substance is immobilized; a sample containing the DNA probe and DNA immobilized on the electrode. And a reaction in the presence of an intercalator to form a hybrid DNA; and detecting and / or detecting the current of the electrode after the reaction.
Or means for measuring;

In a preferred embodiment, there is provided a means for cleaning the electrode after the reaction.

In a preferred embodiment, the conductive substance is a compound having a redox activity.
A compound having redox activity is ferrocene.

In a preferred embodiment, the intercalator is a ferrocene-modified threaded intercalator.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION As an electrode used in the method or apparatus of the present invention, any electrode can be used as long as it can fix nucleic acid. Preferably, gold, glassy carbon, carbon and the like are used.

Examples of conductive substances that modify DNA include:
Any substance having conductivity can be used.
Particularly, a substance having an oxidation-reduction activity is preferable. Ferrocene, catecholamine, metal bipyridine complex, metal phenanthrin complex and viologen are preferred, and ferrocene is particularly preferred.

As the probe DNA, any of DNA extracted from a biological sample cut with a restriction enzyme and purified by electrophoretic separation or the like, or chemically synthesized DNA can be used. It is preferable that the sequence of the probe DNA is determined in advance. DNA sequencing is well known.

The probe DNA may be modified at the 5 'or 3' end with, for example, an amino group in order to bind to a conductive substance. A method known to those skilled in the art can be used to bind the conductive substance to DNA. Taking the DNA having an amino group introduced at the 5 ′ end and ferrocene as an example, the DNA is prepared using a suitable buffer solution (for example, sodium carbonate-
Dissolved in sodium bicarbonate buffer)
An organic solvent containing ferrocene carboxylic acid N-hydroxysuccinimide ester (for example, DMSO) is added and reacted, and purified using HPLC or the like, whereby a DNA having ferrocene bonded to the 5 ′ terminal side can be obtained.

The probe modified with the conductive substance is fixed to an electrode. As the immobilization method, a known method can be used. For example, if the electrode is gold, a thiol group can be introduced into the DNA and the DNA can be bound to the electrode via a gold-sulfur coordination bond between gold and sulfur. Methods for introducing thiol groups into DNA are described in Mizuo MAEDA, Koji NAKANO, Shinji U
CHIDA, and Makoto TAKAGI, Chemistry Letters, 1805-
1808 (1994) or BAConnolly, Nucleic Acids Re
s., 13 , 4484 (1985). DNA having a thiol group obtained by the above method is dropped on a gold electrode,
By leaving at low temperature (for example, 4 ° C) for several hours,
Ferrocene-modified DNA can be immobilized on a gold electrode.

As another method, carboxylic acid is introduced into the electrode surface by oxidizing glassy carbon with potassium permanganate, and immobilized by forming an amide bond with the amino group of the nucleic acid. Immobilization to glassy carbon, Kell
y M. Millan and Susan R. Mikkelsen, Analitical Che
mistry 65 , 2317-2323 (1993).

By introducing the above-obtained electrode to which a probe DNA modified with a conductive substance is bound into a sample solution containing a DNA sample, a DNA having a sequence complementary to the probe DNA is hybridized. , A hybrid DNA is formed. Methods for hybridizing DNA are well known.

The apparatus of the present invention preferably has a heating means for heating the reaction solution. If necessary, it may have a cooling means. This is because the double-stranded DNA is dissociated into a single strand by heating and hybridized with the probe.

This hybridization is performed in the presence of an intercalator. The coexistence of an intercalator can speed up the formation of hybridization and stabilize the resulting hybrid DNA.

The intercalator which can be used in the present invention includes a ferrocene compound, a catecholamine compound, a metal bipyridine complex, a metal phenanthrin complex, a viologen compound and the like. A threaded intercalator into which ferrocene, catecholamine, a metal bipyridine complex, a metal phenanthrin complex, viologen, or the like is introduced is preferred, and a ferrocene-modified threaded intercalator is particularly preferred.

The intercalator in the hybridization reaction solution can be used in a concentration range from several nM to several mM.
Preferably it is 0.1 mM to 5 mM, most preferably 0.5 mM.

The intercalator is a hybrid DNA
To form a kind of charge transfer complex. If no hybrids are formed (ie, single-stranded), no intercalation occurs. The amount of current flowing through the electrodes changes due to the intercalation. This current is a double-stranded DN (formed by hybridization)
This is due to oxidation and reduction of the intercalator intercalated into A. The degree of double-stranded DNA formation can be detected as the degree of intercalation. Therefore, by detecting and / or measuring the amount of the current, the presence of the hybrid DNA is detected or the amount of the hybrid DNA is measured. Therefore, the device of the present invention has means for detecting and / or measuring the amount of current. Examples of such means (apparatus) include a cyclic voltammogram, a differential pulse voltammogram, and a potentiostat.

Upon detection / measurement of the amount of current, after the hybridization reaction in the presence of the intercalator, the electrode is washed to remove free intercalator. Therefore, the device of the present invention may have means for cleaning the electrodes.

As described above, the electrode used for the apparatus of the present invention, on which the DNA probe modified with a conductive substance is fixed, and the DNA probe fixed on the electrode and D
A means for reacting a sample containing NA with an intercalator to form a hybrid DNA and a means for detecting and / or measuring the current of the electrode after the reaction are provided, and the device of the present invention can be produced. .

[0031] DNA having a specific sequence can be detected using the above apparatus.

A method for detecting DNA having a specific sequence includes, for example, an electrode on which a DNA probe modified with a conductive substance prepared by the above-described preparation method is immobilized,
Is reacted in the presence of an intercalator to form a hybrid DNA of the DNA probe and the DNA. Hybridization conditions are well known. When specifying more strictly, hybridization conditions, appropriate salt concentration and time, etc. can be set depending on the base composition or length of the probe used.

After the hybridization reaction, the electrode is washed, unnecessary intercalators are washed away, and the amount of electric current of the electrode is detected and / or measured, so that the hybridized DNA is detected and / or measured.

Hereinafter, the present invention will be described by way of examples, but it goes without saying that the present invention is not limited to only the examples.

[0035]

【Example】

(A: Preparation of electrode on which DNA probe modified with ferrocene was immobilized) (1) Synthesis of ferrocenecarboxylic acid N-hydroxysuccinimide ester Ferrocenecarboxylic acid 0.5 g (2.2 mM) and N-hydroxysuccinimide 0.29 g (2.5 mM) ) In a 20 ml dioxane solution,
0.5 g (2.5 mM) of dicyclohexylcarbodiimide under stirring
ml dioxane solution was added and stirred at room temperature for 24 hours.
Of the components precipitated in the reaction mixture, a dark yellow component was separated by silica gel chromatography. The physical properties of the dark yellow component are as follows.

[0036]

[Table 1]

(2) Synthesis of ferrocene-modified DNA probe As a DNA probe, a thymidine 20-mer (T ₂₀ A)

[0038]

Embedded image

Was synthesized. Synthesis was performed using an automated DNA synthesizer (Model 391 PCR-MAT manufactured by Applied Biosystems).
^ETM / PCR-MATE EP). T ₂₀ A26 nmol (E
₂₆₀ = 8800 M ^-1 cm ^-1 ) was dissolved in 20 μl of 1.3 mM sodium carbonate-sodium hydrogen carbonate buffer (pH 9.0). The ferrocenecarboxylic acid obtained in the above (1)
6m containing 1.3μmol of N-hydroxysuccinimide ester
l of DMSO solution was added and shaken at room temperature overnight. Transfer the reaction mixture to TEAA (triethylamine-acetic acid buffer)
(PH 7.0), and then diluted to 1 ml with a NAP-10 column (Pharma
Using cia Sephadex G-25), a portion of 2.5 ml from a retention volume of 1 ml was fractionated using a TEAA buffer as an eluent. This was concentrated under reduced pressure (40 ° C or less) and reverse phase HPLC (LiChrospher 100RP-18).
(e) / Cica-MERCK). Yield 7.4 nmol (29%)
Met.

(3) Thiolation of Ferrocene-Modified DNA Probe Thiolation of ferrocene-modified DNA can be carried out by a known method.
A. Connolly, NucleicAcids Res., 13 , 4484 (1985).

(4) Immobilization of ferrocene-modified probe DNA on the electrode surface dT _20- mer solution into which the thiol group was introduced and obtained in (3)
1 μl (3.4 × ^{10 −10} mol) was dropped on a gold electrode (0.79 mm ² ) and left at 4 ° C. for 1 hour. After that, wash the electrode with water,
A thiol group-introduced dT ₂₀ mer was recovered from the washing water. HPLC
At, a thiol group introduced dT ₂₀ mer which has not reacted were measured, calculating the dT ₂₀ mer amount that is fixed to the electrode 2.9X
It was 10 ^-11 mol (29 pmol). This is Tris-HCl-EDTA
(PH 7.0) Stored in buffer.

(B: Preparation of Ferrocene-Modified Threaded Intercalator) The ferrocene-modified threaded intercalator was prepared by mixing the ferrocenecarboxylic acid N-hydroxysuccinimide ester prepared in the above A (1) with an amine prepared by the following method. It was synthesized using the body.

(1) Synthesis of amine compound 2,4,5,8-naphthalenetetracarboxylic dianhydride 2 g (7.45
mol) and 1,4- (3-aminopropyl) piperazine 40m
l (0.194 mmol) was refluxed in 30 ml of THF for 8 hours. After cooling to room temperature, it was reprecipitated in hexane,
Crystals were collected by thickening. Further, it was dissolved in a minimum amount of chloroform and reprecipitated in a polyester. After removing the precipitate, the ether is removed under reduced pressure. After re-dissolving in chloroform and re-precipitating with hexane, the crystals were recovered. The yield was 330 mg and the yield was 52%.

[0044]

[Table 2]

(2) Synthesis of Ferrocene-Modified Threaded Intercalator The ferrocenecarboxylic acid N-hydroxysuccinimide ester prepared in the above A (1) is reacted with the amine (NaDI) prepared in the method of B (1). And synthesized.

Amine compound 300 mg (0.474 mol) and the above A
600 mg (1.8 mmole) of the ester prepared in (1) was dissolved in 30 ml of chloroform. This was stirred at room temperature for 50 hours. This was subjected to TLC on silica gel and developed using methanol as a solvent. At the origin, NaDI, a ferrocenecarboxylic acid N-hydroxyimide ester with an Rf value of 0.8, and a spot that was considered to be a broad target product with an Rf value of 0.2 were confirmed. After evaporating the reaction solution under reduced pressure, the residue was dissolved in methanol and developed with a silica gel column using methanol as a developing solvent to obtain a fraction considered to be the desired product. The methanol was distilled off under reduced pressure and dissolved in chloroform. After filtration, the filtrate was concentrated, reprecipitated with water, and recrystallized with acetone to collect crystals. TLC (MeOH) after recrystallization from acetone was only a spot having an Rf value of 0.2.

[0047]

[Table 3]

The yield of the recovered substance was 33 mg and the yield was 7%. The obtained intercalator had an absorption maximum at 382 nm.

[0049] (C: Evaluation of electrode) The DNA fragment of complementarily binding to the probe DNA with a synthetic oligonucleotide dA _20. dA ₂₀ 29 pmol and above
30% DM containing 0.5mM intercalator created in (B)
The electrode with the ferrocene-modified probe DNA (dT ₂₀ ) immobilized on the surface was immersed in SO-41 mM (AcOK-AcOH) buffer (pH 5.2). The reaction was performed at 40 ° C. for 10 minutes.

After the reaction is completed, the electrode is pulled up, and the electrode is held for 5 seconds.
Washing was performed with 30% DMSO-41 mM (AcOK-ACOH) buffer (pH 5.2) to remove free intercalator.

FIG. 1 shows the measurement of the cyclic voltammogram of this electrode. Measurements were performed using 41 mM potassium acetate-acetic acid buffer (pH 5.2), 30% DMSO, and a scanning speed of 100 m.
The test was performed under the condition of V / sec.

[0052] increasing current 619mV where dT ₂₀ of 29pmol of the present invention is derived from ferrocene intercalators bound electrodes was 0.15Myuei. Therefore, the binding of dA ₂₀ to dT ₂₀ , that is, the probe DN immobilized on the electrode
It turns out that a specific sequence can be detected by binding of DNA complementary to A.

Using this electrode, 30% containing various concentrations of dA ₂₀ and 0.5 mM of the intercalator prepared in (B) above.
The electrode on which the ferrocene-modified probe DNA (dT ₂₀ ) was immobilized was immersed in a DMSO-41 mM (AcOK-AcOH) buffer (pH 5.2). The reaction was performed at 40 ° C. for 10 minutes.

After completion of the reaction, the electrode is pulled up, and the electrode is held for 5 seconds.
Washing was performed with 30% DMSO-41 mM (AcOK-ACOH) buffer (pH 5.2) to remove free intercalator.

FIG. 2 shows the result of measuring the cyclic voltammogram of this electrode. From the detection limit of the increasing current,
The detection limit of DNA that can be detected by the above electrodes is several fmol
It was estimated. From this figure, it can be seen that DNA having a specific sequence
It can be seen that detection and quantification of are possible.

This DNA detection limit is equivalent to or higher in sensitivity than the current RI method, fluorescent label method, and luminescence method.

[0057]

According to the present invention, a probe DNA modified with a conductive substance is immobilized on an electrode, and in the presence of an intercalator,
After reacting with the DNA sample, the electrode current is detected and / or
Or, by measuring, DNA having a specific sequence
Is detected and / or measured. According to this method, detection can be performed in real time without requiring several days as in the conventional autoradiography using RI.
In addition, there is no need for development work, which is essential for autoradiography. Furthermore, in autoradiography, lack of photosensitivity is confirmed for the first time by development, and when the photosensitivity is insufficient, the film needs to be exposed again, resulting in a time loss. In the present invention, such a time loss is completely eliminated. Furthermore, since the test DNA does not need to be treated with a restriction enzyme or fractionation by electrophoresis, a DNA having a specific sequence can be easily detected and measured without using a fractionation device.

[Brief description of the drawings]

FIG. 1 is a view showing a measurement of a cyclic voltammogram of an electrode of the present invention.

FIG. 2 is a diagram showing the performance of the electrode of the present invention.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-5-199898 (JP, A) Kelly M. Millan and Susan R. See Mikkelsen, Anal. Chem. , 1993, Vol. 65, no. 17, p2317-2323 Keji Hashimoto and two others, Anal. Chim. Acta. , 1994, Vol. 286, no. 2, p219-224 Shigeori Takenaka, et al. Chem. Soc. , Chem. Commun. , 1990, Vol. 21, pp. 1485-1487 Shigeori Takenaka, et al., Anal. Biochem. , 1994, Vol. 218, p436-443 (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/327 G01N 27/06 BIOSIS (DIALOG) CAPLUS (STN) JICST file (JOIS)

Claims (16)

(57) [Claims] 1. A method for detecting a gene having a specific sequence, which comprises at least either an oxidizing activity or a reducing activity.
An electrode having a DNA probe modified with a compound immobilized thereon is reacted with a sample containing DNA in the presence of an intercalator, and a hybrid DNA of the DNA probe and DNA is reacted.
A method of electrochemically detecting a gene, comprising: a step of forming and a step of detecting and / or measuring a current of the electrode after the reaction. 2. The method according to claim 2, wherein said at least oxidizing activity or reducing activity is
The method for electrochemically detecting a gene according to claim 1 , wherein the compound having any of the above-mentioned properties is a compound having a redox activity. 3. The at least oxidizing or reducing activity.
The method for electrochemically detecting a gene according to claim 1 , wherein the compound having any of the above-mentioned properties is any of ferrocene, catecholamine, metal bipyridine complex, metal phenanthrin complex, and viologen. 4. A method for detecting a gene having a specific sequence, comprising reacting a DNA-immobilized electrode with a sample containing DNA in the presence of an intercalator to obtain a hybrid DNA of the DNA probe and DNA. And reacting an intercalator between the layers of the hybrid DNA so that the substituent of the intercalator has a main groove and a sub-groove.
A method for electrochemically detecting a gene, comprising: forming a complex protruding from a groove; and detecting and / or measuring a change in an amount of current flowing through an electrode due to the formation of the complex . 5. A method for detecting a gene having a specific sequence, comprising reacting a DNA-immobilized electrode with a sample containing DNA in the presence of an intercalator to obtain a hybrid DNA of the DNA probe and DNA. A method for electrochemically detecting a gene, comprising: a step of reacting a sewn intercalator with the hybrid DNA; and a step of detecting and / or measuring a current of the electrode after the reaction. 6. The sewn intercalator reacts with the hybrid DNA to form a complex in which the two substituents of the sewn intercalator protrude into the main groove and the minor groove of the double helix of the hybrid DNA, respectively. The method for electrochemically detecting a gene according to claim 5, wherein 7. The intercalator according to claim 1, wherein the intercalator is a ferrocene-modified threaded intercalator.
The method for electrochemically detecting a gene according to any one of the above. 8. The electrochemical electrochemical gene according to claim 1, wherein the intercalator is a threaded intercalator into which a catecholamine, a metal bipyridine, a metal phenanthrin complex, or a viologen compound is introduced. Detection method. 9. An apparatus for detecting a gene having a specific sequence, wherein the apparatus has at least either an oxidizing activity or a reducing activity.
An electrode on which a DNA probe modified with a compound is immobilized, a means for reacting the DNA probe immobilized on the electrode with a sample containing DNA in the presence of an intercalator to form hybrid DNA, and an electrode after the reaction. Means for detecting and / or measuring the current of the gene. 10. The at least oxidative activity or reduction
The apparatus for electrochemically detecting a gene according to claim 9, wherein the compound having any of the activities is a compound having a redox activity. 11. The at least oxidizing activity or the reducing
10. The apparatus for electrochemically detecting a gene according to claim 9, wherein the compound having any activity is any of ferrocene, catecholamine, metal bipyridine complex, metal phenanthrin complex, and viologen. 12. An apparatus for detecting a gene having a specific sequence, comprising: reacting a DNA probe-immobilized electrode with a DNA probe-immobilized sample in the presence of an intercalator. An apparatus for electrochemically detecting a gene, comprising: means for forming a hybrid DNA, thread-type intercalation into the hybrid DNA, and means for detecting and / or measuring the current of the electrode after the reaction. 13. The sewn intercalator forms a complex in which the two substituents of the sewn intercalator protrude into the main groove and the minor groove of the double helix of the hybrid DNA, respectively. And
An apparatus for electrochemically detecting a gene according to claim 12. 14. The intercalator according to claim 9, wherein said intercalator is a ferrocene-modified threaded intercalator.
4. The apparatus for electrochemically detecting a gene according to claim 3. 15. The method according to claim 15, wherein the intercalator is a catecholamine, a metal bipyridine, a metal phenanthrin complex,
The electrochemical detection device for a gene according to any one of claims 9 to 13, wherein the device is a threaded intercalator into which a viologen compound is introduced. 16. At least oxidizing activity or reducing activity
An electrode for detecting a specific DNA sequence, wherein a DNA probe modified with a compound having any one of the above is immobilized on the electrode.