JP2001013103A - Method for detecting and quantitatively determining sample nucleic acid fragment by scanning electrochemical microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which sensitively detects and quantitatively determines sample nucleic acid fragments which are respectively complementary to a DNA fragment and a PNA fragment fixed on a surface of an analysis element by using a DNA analysis element and a PNA analysis element. SOLUTION: By bringing a solution which contains sample nucleic acid fragments into contact with a DNA analysis element or a PNA analysis element in which DNA fragments or PNA fragments are respectively fixed in plural regions sectioned on a surface of a substrate, under the presence of a hybrid DNA-binding electrochemical active molecule or a hybrid PNA-binding electrochemical active molecule, the sample nucleic acid fragments which are complementary to the DNA fragments or the PNA fragments fixed in the analysis elements, and also the electrochemical active molecules are combined. This is a method for detecting and determining quantitatively the complementary sample nucleic acid fragments in which an electric current generated in the electrochemical active molecule-binding region on the surface of the analysis elements is measured by the application of an electric potential to the surface of the analysis elements by a scanning electrochemical microscope.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a large number of DNs which are very useful for simultaneous analysis of gene expression, mutation, polymorphism, etc.
The present invention relates to a method for electrochemically detecting a complementary sample nucleic acid fragment using a DNA analysis element in which A fragments are arranged on a substrate surface or a PNA analysis element in which many PNA fragments are arranged on a substrate surface.

[0002]

2. Description of the Related Art A target DNA fragment in a sample is detected by using a known DNA fragment having a complementarity to the base sequence of the DNA fragment and a known DNA fragment in the sample.
In general, the hybrid formed with the fragment A is detected by an electrochemical method, a fluorescence method, a radioisotope method, or the like. Complementary known D
Since the NA fragment is usually used by being immobilized on a solid support, it is called "probe" or "probe DNA". A method for detecting a sample DNA fragment using a probe DNA is widely used for detecting pathogenic bacteria and screening genes.

[0003] However, the DNA fragment is negatively charged, and the electric repulsion generated between the probe DNA and the sample DNA fragment hinders the hybridization and lowers the detection sensitivity. Hybridization is generally carried out in a buffer in which a certain concentration of salt is present, since such charging of the DNA fragment becomes significant when the salt concentration is low.

[0004] Japanese Patent Application Laid-Open No. 9-288080 discloses a method for detecting a sample DNA fragment using a probe DNA and an electrochemically active sewn intercalator. In this method, the current flowing between the conductive group of the intercalator inserted into the hybrid and the probe DNA is measured to obtain the sample DNA.
This method is extremely excellent in that the detection of fragments does not cause the discoloration seen in the fluorescence method, can solve the problem of safety in the radioisotope method, and does not require direct labeling of the sample DNA fragments.

However, this method cannot be said to be a method that can sufficiently satisfy a practical level in that the intercalator also binds to the probe DNA to some extent, thereby affecting the measurement background.

On the other hand, so-called antisense molecules, ie,
When a gene is expressed, it binds to a single-stranded DNA transcription region or RNA translation region with high selectivity, and a molecule that restricts the function of that region is known as an antisense drug in the field of gene therapy. I have. As this antisense molecule, PNA (peptide nucleic acid) is known as a substance that mimics DNA (or RNA). PNA has a nucleobase in its molecule like DNA and the like, and specifically hybridizes to DNA and the like having a complementary base sequence to form a double strand (PENiel
sen et al., Science, 254, 1497-1500 (1991)). PN
As described above, A has almost no functional difference from DNA, but has a completely different structure, and has a basic skeleton of polyamide having N- (2-aminoethyl) glycine as a unit. Contains no phosphoric acid.

[0007]

Embedded image

For this reason, it is electrically neutral and does not charge even under conditions where no salt is present. The nucleobase is
It is usually bound to the polyamide skeleton via a methylenecarbonyl group. Although PNA is functionally almost the same as DNA, it is also clear that compared to DNA, the formed double strand is more stable and strictly recognizes the complementary DNA base sequence. (Naomi Sugimoto, Abstracts of the 74th Annual Meeting of the Chemical Society of Japan, p. 1287). It is a molecule that is expected to be applied to various diagnostic agents other than antisense drugs (Japanese Patent Publication No. 6-509063).

PNA can be synthesized by a liquid phase method or a solid phase method as in the case of the peptide, and the synthesized one is commercially available. For the synthesis method of PNA, see Table 6
No. 509063; U.S. Pat. No. 2,758,988; PENielsen et al., Journal of American Ch.
emical Society, 114,1895-1987 (1992), PENielsenet
al., Journal of American Chemical Society, 114, 9677
-9678 (1992).

JP-A-11-332595 discloses a PNA probe in which a PNA fragment is immobilized on the surface of a solid support, and a method for detecting a DNA fragment using the PNA probe. This PNA probe is obtained by immobilizing a PNA fragment on a measurement chip for a surface plasmon resonance biosensor by the avidin-biotin method. Target DNA using the PNA probe
The detection of the fragments is performed by measuring the surface plasmon resonance signal.

Also, a PNA probe and a sample DNA
Using a nucleic acid fragment labeled with a radioisotope,
A method of detecting a sample DNA fragment having complementarity by measuring the labeled amount of a hybrid is also known (PENielsen et al., Science, 254, 1497-1500 (199).
1)).

[0012]

SUMMARY OF THE INVENTION The present invention relates to a method for analyzing a DNA element using a DNA analysis element, a nucleic acid fragment in a sample complementary to a DNA fragment immobilized on the surface of the analysis element, and a PNA analysis element. It is an object of the present invention to provide a method for detecting and quantifying a nucleic acid fragment in a sample having complementarity to a PNA fragment immobilized on a surface with high sensitivity.

[0013]

SUMMARY OF THE INVENTION The present invention relates to an aqueous liquid comprising a sample nucleic acid fragment dissolved or dispersed in a DNA analysis element in which a DNA fragment is fixed in each of a plurality of regions partitioned on a substrate surface. In the presence of a hybrid DNA-binding electrochemically active molecule to bind a sample nucleic acid fragment having complementarity with the DNA fragment fixed to the analytical element in the aqueous liquid, A sample having complementarity, wherein an active molecule is also bound, and then a current generated in the electrochemically active molecule binding region on the surface of the analytical element is measured while applying a potential by a scanning electrochemical microscope. Nucleic acid fragment detection method
A1.

[0014] The present invention also provides (1) a DNA analysis element having a DNA fragment fixed to each of a plurality of regions partitioned on the substrate surface, the DNA having an unknown concentration and being fixed to the analysis element. By contacting a sample aqueous liquid in which a sample nucleic acid fragment having complementarity with the fragment is dissolved or dispersed and a hybrid DNA-binding electrochemically active molecule, the DNA immobilized on the analytical element in the sample aqueous liquid Binding a sample nucleic acid fragment having complementarity with the fragment and also binding a hybrid DNA-binding electrochemically active molecule; and (2) applying a potential to the analytical element by a scanning electrochemical microscope to perform the analysis. The current generated in the hybrid DNA binding electrochemically active molecule binding region on the device surface is measured, and the value of the current is measured by the same sample nucleic acid fragment as described above. A step of determining the concentration of a sample nucleic acid fragment having complementarity in the sample aqueous liquid by collating with a calibration curve showing the relationship between the concentration of the solution or the dispersed sample aqueous liquid and the current. Quantitative method for sample nucleic acid fragments-A2.

In a preferred embodiment of the method A2 for quantifying a sample nucleic acid fragment of the present invention, the calibration curve used in the step (2) is
A DNA analysis element in which a DNA fragment is fixed to each of a plurality of regions partitioned on the substrate surface contains sample nucleic acid fragments complementary to the DNA fragment fixed to the analysis element at known concentrations different from each other. A sample nucleic acid fragment having complementarity with a DNA fragment immobilized on the analytical element in the sample aqueous liquid by contacting each of three or more sample aqueous liquids in which the sample nucleic acid fragment is dissolved or dispersed. And also bind the hybrid DNA-binding electrochemically active molecule, and then apply a potential with a scanning electrochemical microscope to generate a current generated in the hybrid DNA-binding electrochemically active molecule-binding region on the surface of the analytical element. It was created by measuring.

Preferred embodiments of the method for detecting a sample nucleic acid fragment having complementarity-A1 and the method for quantifying a sample nucleic acid fragment-A2 of the present invention are as follows. (A) The base sequence of the DNA fragment is known. (B) The hybrid DNA-binding electrochemically active molecule is
It is a ferrocene-modified electrochemically active threaded intercalator having redox activity.

According to the present invention, there is further provided a PNA obtained by fixing a PNA fragment to each of a plurality of regions partitioned on the substrate surface.
By contacting the NA analysis element with an aqueous liquid in which the sample nucleic acid fragment is dissolved or dispersed in the presence of the hybrid PNA-binding electrochemically active molecule, the PNA fixed to the analysis element in the aqueous liquid is contacted. Along with binding the sample nucleic acid fragment having complementarity with the fragment, the electrochemically active molecule is also bound, and then a potential is applied by a scanning electrochemical microscope to generate a potential in the electrochemically active molecule binding region on the surface of the analysis element. There is also a method B-1 for detecting a sample nucleic acid fragment having complementarity, characterized by measuring a current.

According to the present invention, (1) a PNA fragment having a PNA fragment fixed to each of a plurality of regions defined on the substrate surface is fixed to the PNA analytical element having an unknown concentration. By contacting a sample aqueous liquid in which a sample nucleic acid fragment having complementarity with a PNA fragment is dissolved or dispersed and a hybrid PNA-binding electrochemically active molecule, the sample is fixed to the analytical element in the sample aqueous liquid. Binding a sample nucleic acid fragment having complementarity with the PNA fragment and also binding a hybrid PNA-binding electrochemically active molecule; and (2) applying a potential to the analytical element by a scanning electrochemical microscope, The current generated in the hybrid PNA-binding electrochemically active molecule-binding region on the surface of the analysis element is measured, and the value of the current is determined using the same sample nucleic acid A step of determining the concentration of a complementary sample nucleic acid fragment in the sample aqueous liquid by comparing with a calibration curve indicating the relationship between the concentration of the sample aqueous liquid in which the piece is dissolved or dispersed and the current. Quantitative method for sample nucleic acid fragments-B2.

In a preferred embodiment of the method for quantifying a sample nucleic acid fragment-B1 of the present invention, the calibration curve used in the step (2) is
A PNA analysis element in which a PNA fragment is immobilized on each of a plurality of regions partitioned on the substrate surface contains sample nucleic acid fragments having complementarity with the PNA fragment immobilized on the analysis element at known concentrations different from each other. A sample nucleic acid fragment having complementarity with a PNA fragment immobilized on the analysis element in the sample aqueous liquid by contacting each of three or more sample aqueous liquids in which the sample nucleic acid fragment is dissolved or dispersed. And also bind the hybrid PNA-binding electrochemically active molecule, and then apply a potential with a scanning electrochemical microscope to generate a current generated in the hybrid PNA-binding electrochemically active molecule-binding region on the surface of the analytical element. It was created by measuring.

Sample nucleic acid fragment-B having complementation according to the present invention
Preferred embodiments of the detection method 1 and the method for quantifying a sample nucleic acid fragment-B2 are as follows. (A) The nucleotide sequence of the PNA fragment is known. (B) The hybrid PNA-binding electrochemically active molecule is
It is a ferrocene-modified electrochemically active threaded intercalator having redox activity.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows a typical sample nucleic acid fragment detection method A1 of the present invention. Each of the plurality of regions partitioned on the surface of the substrate (21) has a DNA fragment (11).
An aqueous solution in which the sample nucleic acid fragment (12) is dissolved or dispersed is brought into contact with the DNA analysis element (31) in which is immobilized in the presence of the hybrid DNA-binding electrochemically active molecule (41). DNA fragment (11) in the aqueous liquid
When the sample nucleic acid fragment (12a) having complementarity with the above is bound together with the electrochemically active molecule (41),
A complex (51) in which (41) is bound to a hybrid DNA formed by (11) and (12a) fixed to the substrate (21) can be obtained. A scanning electrochemical microscope (61) is provided on the surface of the analytical element on which this (51) is fixed.
Are brought close to each other, and after applying a potential, the response current is measured to obtain an image diagram (71). Circles in (71) indicate a plurality of regions partitioned on the surface of the substrate (21),
Among them, black circles indicate the binding region of the hybrid DNA-binding electrochemically active molecule (41) where the response current was detected. The PNA analysis element is an analysis element in which a PNA fragment instead of a DNA fragment is fixed to each of a plurality of regions partitioned on the surface of the substrate (21), and the sample nucleic acid of the present invention using the PNA analysis element Fragment detection method-B1
Is the same as when a DNA analysis element is used.

Hereinafter, the method for detecting a sample nucleic acid fragment of the present invention will be described.
Each component of A1 and its quantification method-A2 will be described. Unless otherwise specified, the method for detecting a sample nucleic acid fragment of the present invention—B1 and the method for quantifying the same—B2
And A2.

Hereinafter, terms used in the present specification are defined as follows. “Hybrid DNA” refers to a double-stranded fragment formed by a DNA fragment fixed on a DNA analysis element and a sample nucleic acid fragment. “Hybrid PNA” refers to a double-stranded fragment formed by a PNA fragment immobilized on a PNA analysis element and a sample nucleic acid fragment. "PNA
The term "fragment" does not include a fragment of PNA obtained by synthesis, which is partially fragmented by an operation such as cleavage. “Hybrid DNA-binding electrochemically active molecule” refers to a compound that binds to a formed hybrid and has electrochemical activity irrespective of the type of a sample nucleic acid fragment that hybridizes with a DNA fragment immobilized on a DNA analysis element. A thing. However, the hybrid DNA-binding electrochemically active molecule is a single-stranded DN in addition to the formed hybrid.
It may also temporarily bind to the A fragment or a single-stranded sample nucleic acid fragment. The same applies to the “hybrid PNA-binding electrochemically active molecule”. “Binding” refers to a state in which the hybrid DNA-binding electrochemically active molecule is inserted into the structure of the hybrid DNA, and a state in which the hybrid DNA is electrostatically interacting with the hybrid DNA outside the hybrid DNA structure. Although the detection method using a scanning electron microscope detects an electric signal,
The DNA analysis element is used for a sample nucleic acid fragment and a hybrid
Immersed in an electrolyte solution containing A-binding electrochemically active molecules,
Next, using a counter electrode, a potential is applied between the hybrid DNA-immobilized substrate and the hybrid DNA-binding electrochemically active molecule in the electrolyte solution between the substrate on which the hybrid DNA is immobilized and the counter electrode. This is different from a general “electrochemical detection method” for detecting a current flowing through a cell. In that sense, it should be noted that the detection method of the present invention is not so-called electrochemical.

[Substrate] The substrate is preferably an electrically insulating hydrophobic carrier or an electrically insulating low hydrophilic carrier. In addition, even if the surface has unevenness and low flatness, it can be preferably used. As the material of the substrate, glass, cement, ceramics such as ceramics or new ceramics, polyethylene terephthalate, cellulose acetate, polycarbonate such as bisphenol A, polystyrene, polymers such as polymethyl methacrylate, silicon, activated carbon, porous glass, porous ceramics, Porous silicon, porous activated carbon, woven or knitted fabric, nonwoven fabric, filter paper, short fibers, porous materials such as membrane filters and the like can be mentioned, but various polymers, glass or silicon are particularly preferable. This is due to the ease of surface treatment and the ease of analysis by electrochemical methods. The thickness of the substrate is not particularly limited.
It is preferably in the range of 00 μm.

As the substrate, electrodes, optical fibers, photodiodes, thermistors, ISFETs, MOSFEs
T, a piezo element, a surface acoustic wave element and the like can also be preferably used. For example, in the case of an electrode, it is preferable to use an electrode provided on a substrate having no conductivity as described above, and the electrodes are preferably arranged so as not to be in contact with each other and regularly. . Examples of the material of the electrode include a carbon electrode such as graphite and glassy carbon, a noble metal electrode such as platinum, gold, palladium, and rhodium; an oxide electrode such as titanium oxide, tin oxide, manganese oxide, and lead oxide; Si, Ge, and ZnO. And a semiconductor electrode such as CdS, and an electron conductor such as titanium. It is particularly preferable to use gold or glassy carbon. These electron conductors may be covered with a conductive polymer or may be covered with a monomolecular film. As a plurality of electrodes arranged on a non-conductive substrate, it is preferable to use a non-conductive substrate surface that has been treated with the above-described electronic conductor. It is particularly preferred to use. Before the surface treatment with the electron conductor, the substrate may be provided with a layer made of a charged hydrophilic polymer substance or a layer made of a crosslinking agent on the substrate. By providing such a layer, unevenness of the substrate can be reduced. Further, depending on the substrate, a hydrophilic polymer substance having an electric charge can be contained in the substrate, and a substrate subjected to such treatment can be preferably used.

[0026] As an example in which a plurality of electrodes are arranged on a substrate having no conductivity, a literature (Sosnowski, RG et al.,
Acad. Sci. USA, 94, 1119-1123 (1997)), and a silicon chip on which nucleic acids are not fixed can also be preferably used. Further, like a printed wiring board, may be printed on the board.

[DNA Fragment] The DNA fragment can be divided into two types depending on the purpose. In order to examine gene expression, it is preferable to use a polynucleotide such as cDNA, a part of cDNA, or EST. These polynucleotides may be of unknown function, but are generally based on sequences registered in a database.
It is prepared by amplifying by a PCR method using an NA library, a genomic library or a whole genome as a template. Those not amplified by the PCR method can also be preferably used. In addition, in order to examine gene mutations and polymorphisms, it is preferable to synthesize various oligonucleotides corresponding to the mutations and polymorphisms based on a known sequence as a standard, and use them. Furthermore, in the case of nucleotide sequence analysis, it is preferable to use 4 ⁿ (n is the length of a base) synthesized oligonucleotides. DN
The base sequence of the fragment A is preferably determined in advance by a general base sequence determination method, and its base type is also preferably known. The DNA fragment is preferably a 3 to 50 mer, particularly preferably a 10 to 25 mer. It is also preferable to use such a plurality of types of DNA fragments in order to fix DNA fragments having different base sequences to each of a plurality of regions partitioned on the substrate surface as the DNA fragments.

As a method for immobilizing a DNA fragment, a known method can be used. An appropriate method for fixing the DNA fragment to the substrate can be selected according to the type of the fragment and the type of the substrate (Protein / Nucleic Acid / Enzyme, Vol., 4
3, No. 13, 2004-2011 (1998)). For example, if the DNA fragment is c
In the case of DNA or PCR products, a method of utilizing the charge of DNA to electrostatically bond to a substrate surface-treated with a cation such as polylysine, polyethyleneimine, or polyalkylamine can be used. When immobilizing synthetic nucleotides, a method of directly synthesizing on a substrate, or a method of synthesizing an oligomer having a functional group for covalent bonding introduced at an end in advance and covalently bonding to a surface-treated substrate can be used. . For example, when the surface of the substrate is vapor-deposited with gold, a mercapto group is introduced into the 5 ′ or 3 ′ end of the DNA fragment, and the DNA fragment is fixed to the substrate via a coordination bond between gold and sulfur. I do. A method for introducing a mercapto group into the DNA fragment is described in the literature (M. Maeda et al., Ch.
em. Lett., 1805-1808 (1994) and BAConnolly, Nucle.
ic Acids Res., 13, 4484 (1985)).
When the substrate surface is coated with glassy carbon, the carboxylic acid group is introduced into the substrate surface by oxidizing the glassy carbon with potassium permanganate. Can be fixed to the surface. For the actual immobilization method,
References (KMMillan et al., AnalyticalChemistry, 65,
2317 to 2323 (1993)). D above
The NA fragment may be a double-stranded DNA fragment prepared in advance. When the double-stranded DNA fragment is immobilized on a substrate which has been subjected to a deposition process with gold, the 5 ′ of one strand of the double-stranded DNA fragment is
Alternatively, a mercapto group is introduced at the 3 'end (preferably, the 5' end). Examples of the functional group for covalent bonding include an amino group, an aldehyde group, a mercapto group, and biotin. When glass or silicon is used as the substrate, it is preferable to use a known silane coupling agent for the surface treatment.

The fixation of the DNA fragment is preferably carried out by dropping an aqueous solution in which the DNA fragment is dissolved or dispersed on a region on the substrate. The concentration of the DNA fragment to be spotted is preferably in the range of several pM to several mM, and particularly preferably in the range of several pM to several nM. The amount of spotting is
It is preferably in the range of 1 to 100 nL, particularly preferably in the range of 1 to 10 nL. The aqueous liquid containing the DNA fragment may contain an additive for increasing the viscosity of the aqueous liquid. Such additives include sucrose, polyethylene glycol, glycerol and the like. After spotting, the DNA fragments are fixed to the region on the substrate when left at a predetermined temperature for several hours. Conditions for spotting differ depending on the type and size of the substrate to be used. The spotting can be performed by a manual operation, but it is also preferable to perform the spotting using a spotter provided in a commonly used DNA chip manufacturing apparatus. After spotting, it is also preferable to carry out incubation. After the incubation, it is preferable to wash and remove unspotted DNA fragments. After immobilizing the DNA fragment on the substrate, the surface is treated with a compound having a hydrophilic group at one end of an alkylene group having 1 to 6 carbon atoms and a functional group at the other end having a functional group binding to the substrate. It is preferable to perform the coating treatment. As such a compound, in the case of a substrate on which gold is deposited, 2-mercaptoethanol or a derivative thereof can be used. The life of the DNA analysis device manufactured as described above ranges from several days to several weeks.

[PNA Fragment] The PNA fragment preferably used in the present invention is a compound represented by the following formula (I).

[0031]

Embedded image (I)

In the formula, B ¹¹ is a ligand and represents a natural nucleobase (A, T, C, G, I or U) or a base analog. B ^11, the position found in nature, i.e., adenine, the purine containing guanine or inosine, in position 9, for pyrimidines including thymine, uracil or cytosine, bonded at position 1. A base analog is an organic base similar to a non-naturally occurring nucleic acid base, and is a compound in which a purine ring or pyrimidine ring is partially substituted with C to N or N to C, or a purine ring or pyrimidine ring. Is a compound (Sulfhydryl group or halogen atom-introduced compound) partially modified. B ¹¹ may be an aromatic moiety not containing a nucleic acid base, an alkanoyl group having 1 to 4 carbon atoms, a hydroxyl group or a hydrogen atom. Base analogs include 7-deazaadenine, 6-azauracil and 5-azacytosine. Exemplary nucleobase ligands and exemplary synthetic ligands are illustrated in WO 92/20702, where 5-propylthymine and 3-deazauracil are known to increase binding affinity to DNA fragments ( JP-A-11-236396). Other useful non-natural nucleobases include 6-thioguanine and pyrazolo [4,
3d] -Pyrimidines are useful (International Application PCT / U
S92 / 04795). Further, B ¹¹ may be a DNA intercalator, a reporter ligand (for example, a fluorophore), a protein label of hapten or biotin, a spin label, or a radiolabel. B ¹¹ is particularly preferably a nucleobase (A, T, C, G or U).

R ¹¹ represents a hydrogen atom or a group representing a side chain of a natural α-amino acid. Examples of the group representing the side chain of the natural α-amino acid include an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and a carbon atom containing an alkyl group having 1 to 6 carbon atoms. Number 7 to 26
An aralkyl group, a heteroaryl group having 6 to 20 carbon atoms, a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an alkylthio group having 1 to 6 carbon atoms, -NR ¹³
It is preferably a group selected from the group consisting of an R ¹⁴ group, a —SH group, and an alkyl group having 1 to 6 carbon atoms. The alkyl group having 1 to 6 carbon atoms may be further substituted with a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms or an alkylthio group having 1 to 6 carbon atoms. R ¹³ and R ¹⁴ are each independently a hydrogen atom,
It represents an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, an atom or a hydroxyl group selected from the group consisting of an alkylthio group having 1 to 3 carbon atoms and a hydroxyl group. The group representing the side chain of the natural α-amino acid may form an alicyclic ring or a heterocyclic ring together with the hydrogen atom of the carbon atom to which R ¹¹ is bonded.

L ¹¹ represents a linking group, preferably a —CO— group or a —CO—NR ¹² — group,
Particularly preferred is a group. R ¹² represents an atom or a group selected from the group consisting of a hydrogen atom, an alkylene group having 1 to 4 carbon atoms, a hydroxyl group, an alkoxy group having 1 to 4 carbon atoms and an amino group. The alkylene group having 1 to 4 carbon atoms, the alkoxy group having 1 to 4 carbon atoms and the amino group are all an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms or It may be substituted with a hydroxyl group.

D represents an integer of 1 to 60. d is 1
It is preferably an integer of from 40 to 40. a, b and c
Represents an integer of 0 to 5 each independently. It is preferable that a, b and c are all 1.

The PNA fragment used in the present invention has the following general formula:
The compound represented by the formula (II) is particularly preferred. In the formula, B ¹¹ and d each represent B
¹¹ and d.

[0037]

Embedded image (II)

As a method for fixing the PNA fragment to the substrate,
Known methods can be used (Protein / Nucleic Acid / Enzyme, Vol. 43, No. 13, 2004-2011 (1998), and
288080). As an immobilization method, an appropriate method can be selected according to the type of the PNA fragment and the type of the substrate. For example, a method of directly synthesizing a PNA fragment on a substrate, or a method of spotting a previously synthesized PNA fragment on the substrate surface and immobilizing it at the N-terminus or C-terminus can be used. It is preferable that the surface of the substrate be treated in advance to fix the PNA fragment. When a separately synthesized PNA fragment is used, it is also preferable to introduce a reactive group for binding to a substrate at one end of the synthesized PNA fragment. Examples of the reactive group include an amino group, an aldehyde group, a mercapto group, and biotin. As the surface treatment of the substrate, for example, when the substrate is glassy carbon, the substrate is preferably treated with potassium permanganate. When a plurality of electrodes arranged on a substrate having no conductivity are used as a substrate, different types of PN
It is preferred to fix the A fragment.

The fixation of the PNA fragment is preferably carried out by dropping an aqueous liquid containing the PNA fragment on a substrate. After spotting,
When left at a predetermined temperature for several hours, PNA fragments are fixed on the substrate surface. Conditions for spotting differ depending on the type of substrate used, the size of the substrate, and the like. The spotting can be performed by a manual operation, but it is also preferable to perform the spotting using a spotter provided in a commonly used DNA chip manufacturing apparatus. After spotting, it is also preferable to carry out incubation. After the incubation, it is preferable to wash and remove unfixed PNA fragments. The life of the PNA analytical element manufactured as described above ranges from several days to several weeks.

[Hybridization] Hybridization is carried out in the presence of a hybrid DNA-binding electrochemically active molecule when DNA is used, and in the case of using a hybrid PNA-binding electrochemically active molecule when a PNA analytical element is used. It is performed by bringing the sample nucleic acid fragment into contact with a DNA analysis element or a PNA analysis element in the presence. The hybrid DNA-binding electrochemically active molecule may be brought into contact with the formed hybrid DNA after completion of the hybridization. In this case, after completion of hybridization, washing is performed using a mixed solution of a surfactant (preferably, sodium dodecyl sulfate) and a buffer (preferably, citrate buffer), and unreacted sample nucleic acid is removed. Preferably, the fragments have been removed. Alternatively, at the time of hybridization, the electrochemically active molecule may be included in an aqueous liquid containing a sample nucleic acid fragment, so as to be brought into contact with the formed hybrid DNA. The electrochemically active molecule is added to a (single-stranded) sample nucleic acid fragment or a double-stranded sample nucleic acid fragment contained in the sample,
In order to avoid non-specific binding of the electrochemically active molecule, it is desirable to remove unreacted sample nucleic acid fragments after hybridization and then contact them. The same applies to hybrid PNA-binding electrochemically active molecules. A hybrid DNA-binding electrochemically active molecule or a hybrid PNA-binding electrochemically active molecule is
It is preferable to use in a concentration range of 10 nM to 10 mM. Hybridization is preferably carried out at a temperature in the range of room temperature to 70 ° C. and for a time in the range of 0.5 to 20 hours.
It is desirable to set optimal hybridization conditions according to the type of the sample nucleic acid fragment and the like. For example, when the purpose is to analyze gene expression, it is preferable to perform hybridization for a long time so that low-expressed genes can be sufficiently detected, and when the purpose is to detect a single nucleotide mutation, It is preferable to perform hybridization for a short time. After hybridization, washing is performed using a mixed solution of a surfactant (preferably sodium dodecyl sulfate) and a buffer (preferably citrate buffer) to remove unreacted sample nucleic acid fragments. preferable.

[Sample nucleic acid fragment] As the sample nucleic acid fragment,
DNA fragments extracted from biological samples, DNA fragments produced by genetic manipulation, etc. are cut with restriction enzymes and the like, and then DNA fragments purified by electrophoretic separation or the like, or single-stranded DNA fragments obtained by chemical synthesis. Preferably, it is used. In the case of a DNA fragment obtained from a biological sample or the like, a single-stranded D
It is preferable to dissociate into NA fragments. DNA fragments cleaved with restriction enzymes generally contain a plurality of types of D
The NA fragment will be used. When this is used as a sample DNA fragment, the DNA fragment may be of one type or a plurality of types. The sample DNA fragment is preferably in the range of several pM to several mM, particularly preferably in the range of several pM to several nM.

[Hybrid DNA-Binding Electrochemically Active Molecule and Hybrid PNA-Binding Electrochemically Active Molecule] The hybrid DNA-binding electrochemically active molecule is any molecule that binds to hybrid DNA and has electroactive activity. Those can also be used. As shown in FIG. 1, the hybrid DNA is preferably obtained by bringing a sample nucleic acid fragment into contact with a DNA analysis element. However, even without such a method, a hybrid DNA not involving the DNA analysis element can be used. It may be prepared separately. The same applies to the case where the DNA analysis element is replaced with a PNA analysis element. Hybrid DNA-binding electrochemically active molecules (or hybrid PNA-binding electrochemically active molecules) can be prepared by immersing a DNA analysis element (PNA analysis element) in a solution containing the corresponding electrochemically active molecule, or by using a DNA analysis element ( Hybrid DNA (or hybrid P) can be obtained by dropping a solution containing the corresponding electrochemically active molecule onto a PNA analytical element.
NA). Hybrid DNA
Bonding to the hybrid may be any of an intercalating type, a groove (main groove or sub-groove) connecting type to the hybrid, and the like.
As the hybrid PNA-binding electrochemically active molecule, any molecule can be used as long as it binds to hybrid PNA and has an electroactive activity. The mode of binding to hybrid PNA is the same as that for hybrid DNA. It is preferable that both the hybrid DNA-binding electrochemically active molecule and the hybrid PNA-binding electrochemically active molecule are used in a concentration range of 10 nM to 10 mM.

As the hybrid DNA-binding electrochemically active molecule and the hybrid PNA-binding electrochemically active molecule, the same molecule can be used, and such a molecule is a sewn-type intercalator labeled with a conductive group. It is particularly preferred that it is a curator. The sewn intercalator labeled with a conductive group is preferably a substance having a redox activity. The redox-active moiety is preferably a ferrocene compound, a catecholamine compound, a metal bipyridine complex, a metal phenanthroline complex, a viologen compound, or the like, and particularly preferably a ferrocene compound. The intercalator portion is preferably naphthalenediimide, anthracene, anthraquinone, or the like, and particularly preferably naphthalenediimide. Therefore, the intercalator is a ferrocene-naphthalenediimide derivative represented by the following formula (S. Takenaka et al., J. Chem.) Synthesized by the reaction of N-hydroxysuccinimide of ferrocenecarboxylic acid with the corresponding amine compound. Soc., Commun., 1111
(1998)).

[0044]

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Further, a ferrocene naphthalenediimide derivative represented by the following formula is also preferably used.

[0046]

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Here, X is a ferrocene derivative represented by the following formula.

[0048]

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[0049]

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[0050]

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[0051]

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The sewn intercalator labeled with a conductive group has a linker portion connecting the redox active portion and the intercalator portion. 1, represented by the above equation
The 4-dipropylpiperazinyl group corresponds to this linker moiety. Instead of this piperazinyl group, a quaternized imino group can also be introduced. The intercalator represented by the following formula into which a quaternized imino group has been introduced becomes cationic regardless of the pH of the reaction system, so that the binding of the hybrid DNA or hybrid PNA becomes stronger. An intercalator in which a quaternized imino group is introduced is particularly effective when a PNA analytical element is used. The group corresponding to the linker moiety is not limited to those described above. For example, instead of a piperazinyl group,
N-alkyl group (an alkyl group having 1 carbon atom
And preferably an alkyl group of 6 to 6, a methyl group,
It is also preferable to introduce an ethyl group or an n-propyl group). The oxidation-reduction potential of the ferrocene molecule differs due to the difference in the structure of the group corresponding to the linker portion.

[0053]

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When the above-mentioned naphthalenediimide derivative is used as the sewn-in intercalator, the naphthalenediimide derivative binds to the hybrid DNA with high specificity, and is arranged every two bases to be saturated with the hybrid DNA. This means that two ferrocene molecules of the naphthalenediimide derivative are densely arranged in the main groove and the minor groove of the hybrid DNA, respectively. Therefore, the naphthalenediimide derivative is a hybrid DNA
From the hybrid DNA
And a naphthalenediimide derivative can form a stable complex. In addition, the binding of the naphthalenediimide derivative to the hybrid DNA in the intercalation mode is generally confirmed by a change in viscosity observed when the intercalator is brought into contact with the hybrid DNA. For example, the virus SV40
It is known that the viscosity of a closed circular plasmid represented by the formula (1) changes with the change in the supercoil when intercalation occurs. On the other hand, the intercalator does not bind to the single-stranded DNA fragment, or once bound, dissociates immediately to become a free intercalator.

[Detection of Response Current] The detection of the response current generated in the hybrid DNA binding electrochemically active molecule binding region on the surface of the DNA analysis element was performed by using a scanning electrochemical microscope (S
ECM). The current was detected by using the DNA analysis element after hybridization with the hybrid DN.
It may be carried out in the solution immersed in and contacted with the solution containing the A-binding electrochemically active molecule, or may be carried out by pulling up the DNA analysis element from the solution. The same applies to the case where a PNA analysis element is used.

A scanning electrochemical microscope (SECM) generally comprises a probe / sample fixture, a bipotentiostat, high-resolution data processing, and a three-dimensional micropositioner. A scanning electrochemical microscope (SECM) scans the xy plane of a small substrate using an equipped probe, and three-dimensional SECM images of chemical changes (redox reaction) near the substrate surface. This is a general term for systems having features given as figures. The term “microscope” generally indicates that a change on a minute substrate is output as an image diagram. Therefore, any system having such characteristics is included in the scanning electrochemical microscope (SECM). 3D SECM
The image is actually obtained by observing the probe current as a function of the probe position. As the probe, an insulating glass, a noble metal or a carbon fiber microelectrode embedded in a polymer is used.

Using an aqueous liquid in which a sample nucleic acid fragment is dissolved or dispersed at an unknown concentration, the DNA in the aqueous liquid is
The concentration of the sample nucleic acid fragment having complementarity with the DNA fragment immobilized on the analysis element can be determined. As a typical example, first, the concentration of an unknown
A sample aqueous liquid in which a sample nucleic acid fragment having complementarity to a DNA fragment fixed to the analysis element is dissolved or dispersed is brought into contact with a hybrid DNA-binding electrochemically active molecule. Next, a potential was applied to the analytical element by a scanning electrochemical microscope, and a hybrid D on the analytical element surface was applied.
The current generated in the NA binding electrochemically active molecule binding region is measured. Then, by comparing the current value with a calibration curve showing the relationship between the current and the concentration of the sample aqueous liquid in which the same sample nucleic acid fragment is dissolved or dispersed as described above,
The concentration of the complementary sample nucleic acid fragment in the sample aqueous liquid is determined. It is preferable to use an aqueous sample solution obtained by dissolving or dispersing a sample nucleic acid fragment in a concentration range of 1 to 100 nM. The calibration curve has three or more sample nucleic acid fragments each containing a sample nucleic acid fragment having complementarity with a DNA fragment fixed to the DNA analysis element at a known concentration different from each other, and Contacting each sample aqueous liquid, and then measuring a current generated in the hybrid DNA-binding electrochemically active molecule binding region on the surface of the analytical element while applying a potential to the analytical element by a scanning electrochemical microscope. Is preferably created in advance.

[0058]

[Example 1] Detection using DNA analysis element on which hybrid DNA was immobilized (1) Preparation of DNA analysis element A 5 'end was placed on a glass substrate having a side of 2 × 10 ³ (μm) on which gold was deposited. An aqueous solution (1 nL) of a hybrid DNA (HS-DNA: A ₂₀ T ₂₀ ) (50 nM) composed of a 20-mer of adenine having a mercaptohexyl group and a 20-mer of thymine was spotted using a spotter apparatus. And the same glass substrate at concentrations of 25 nM and 12.5 n
M, and 1 nL of an aqueous solution of HS-DNA was spotted on each, and the spotted aqueous solution was allowed to stand for 1 hour so as not to dry. Then, unfixed HS-DNA was washed with distilled water, and DNA analysis was performed. An element was manufactured. (2) Detection of Sample DNA Fragment
Ferrocenated naphthalenediimide represented by the following formula (50 μ) synthesized according to the method described in
M), and immersed in an electrolyte solution (a mixed solution of 0.1 M acetic acid / potassium acetate aqueous solution (pH 5.6) -0.1 M potassium chloride aqueous solution) containing M), and a model 900 scanning electrochemical microscope (CH Instruments) , A probe potential of 0.7 V was applied, and the surface was measured at a scanning speed of 0.002 seconds and a height of the probe from the substrate of about 20 μm. FIG. 2 is a three-dimensional SECM image obtained by imaging this measurement result. The response current value is 6.51 × 10 ⁻¹¹ (A) (corresponding to black in FIG. 2) to about 1.20 × 10 ^−8. (A) (corresponding to the gray in FIG. 2). FIG. 3 is a profile diagram of FIG. (1), (2) and (3) show the portions where the aqueous solution of HS-DNA was spotted on the substrate at a concentration of 50, 25, and 12.5 nM, respectively. (Dotted on each two rows) shows that a response current equivalent to 1.20 × 10 ⁻⁸ (A) was detected in any part. Therefore, 12.5 × 10 ^-18 to 50 × 1
Hybrid DNA in amounts ranging from ^0-18 moles can be detected.

[0059]

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Reference Example Hybrid DNA (HS-D
The same operation as in Example 1 was performed except that NA) was changed to a single-stranded DNA fragment (HS-dA ₂₀ ), and the substrate surface was measured. (1 ′), (2 ′) and (3 ′) in FIG.
An aqueous solution of -dA ₂₀ each 50,25,12.5nM
The part spotted with the density of is shown. For this reason, in some regions, a high response current was detected due to irregularities on the substrate surface, etc.
It can be seen that only about 1/1000 of the current amount in the case of (1) was detected.

[Example 2] Detection using hybrid PNA-immobilized PNA analytical element (1) Preparation of PNA analytical element A PNA fragment represented by the following formula: PNA-H ₂ N-Lys
-T ₁₀ -H (hereinafter referred to as "PNA-T _10".), Literature (PENielsen et al., Journal of AmericanChemical
Society, 114, 1895-1897 (1992) and 114, 9677-9678 (199
It was synthesized according to the method described in 2)). T represents thymine.

[0062]

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The area having a mercapto group on the surface is 2.2.
A phosphoric acid buffer of 1,2-bis (vinylsulfonylacetamide) ethane is dropped on a 5 mm ² gold electrode, and a 50 nM PNA fragment ( PNA-T ₁₀₎ and 50nM
Of was added dropwise a mixed solution (1 nL) of the 10-mer of adenine (DNA-A _10), and left at room temperature for 1 hour, the hybrids of PNA and PNA-T ₁₀ and DNA-A ₁₀ that have not been fixed to the electrode After washing with distilled water, a PNA analytical element was prepared. (2) Detection of Sample DNA Fragment The same operation as in (2) of Example 1 was performed using the PNA analysis element prepared in (1) above in place of the DNA analysis element of Example 1, and the response current was measured. As a result, substantially the same result as the result shown in the three-dimensional SECM image obtained in Example 1 was obtained.

Example 3 Single-stranded DNA Fragment was Immobilized
(1) Preparation of DNA analysis element A side on which gold was deposited was 2 × 10^Three(Μm) glass substrate
Adenine having a mercaptohexyl group at the 5 'end
20-mer (HS-DNA: A₂₀) (50 nM) in water
The liquid (1 nL) is spotted using a spotter, and spotted.
After leaving for 1 hour to prevent the aqueous solution from drying,
Undefined HS-DNA is washed with distilled water, and DN
A analytical element was prepared. (2) Detection of Sample DNA Fragment
20-mer (DNA: T ₂₀) (100nM)
Drop 10 μL of M Tris buffer (pH 7.5) solution
And at 25 ° C. while keeping the solution from drying out.
Incubated for hours. After incubation, the analytical element
Surface 0.1 M sodium dihydrogen phosphate-dihydrogen phosphate
After washing with an aqueous sodium solution (pH 7.0),
The thymine 20 mer was removed. Then, of Example 1
The response current was measured in the same manner as in (2).
HS of 50 nM in the three-dimensional SECM image indicated by 1
-The result was almost the same as the spotting spot of DNA.

Example 4 PN with PNA Fragment Immobilized
Detection using A analysis element (1) Preparation of PNA analysis element Phosphate buffer solution of 1,2-bis (vinylsulfonylacetamide) ethane was dropped on a gold electrode having an area of 2.25 mm ² having a mercapto group on the surface. 50 nM was applied to the gold electrode having a vinylsulfonyl group at one end free.
(For PNA-T _10, described in Example 2) PNA-T ₁₀ of the dropwise an aqueous solution (1 nL) of, for 1 hour at room temperature, washed with PNA-T ₁₀ that was not fixed to the electrode with distilled water As a result, a PNA analysis element was produced. (2) Detection of sample DNA fragment On the PNA analytical element prepared in the above (1), a 10-mer containing ademin 10-mer (DNA: A ₁₀ ) (100 nM)
10 μL of a mM Tris buffer solution (pH 7.5) was added dropwise at 25 ° C. while keeping the solution from drying.
Incubated for hours. After the incubation, the surface of the analysis element was washed with a 0.1 M aqueous solution of sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 7.0) to remove unreacted adenine decamer. Next, when the response current was measured in the same manner as in (2) of Example 1, 50 nM HS was measured in the three-dimensional SECM image shown in Example 1.
-The result was almost the same as the spotting spot of DNA.

[0066]

According to the detection method using the scanning electron microscope of the present invention, it is possible to detect an electric signal indicating the formation of hybrid DNA without using a so-called electrochemical means. That is, according to the present invention, a sample nucleic acid fragment having complementarity with a DNA fragment immobilized on a DNA analysis element can be easily detected, and a large number of sample nucleic acid fragments can be simultaneously detected. It is also possible to quantify the concentration of the sample nucleic acid fragment in the sample. In addition, the present invention provides
Output terminal normally provided in the A sensor (Japanese Patent Laid-Open No. 9-2
No. 88080) and no special labeling is required for the sample nucleic acid fragment to be detected. Furthermore, PN
Even in the case where the A analysis element is used, simple detection and quantification of the sample nucleic acid fragment can be performed similarly to the case of the above-mentioned DNA analysis element. In particular, in a method using a PNA analysis element,
Since the electrical repulsion between the PNA fragment and the sample nucleic acid fragment is suppressed, more efficient hybridization can be performed. Since it is possible to avoid the binding between the PNA fragment that does not form a hybrid and the hybrid PNA-binding electrochemically active molecule, more sensitive detection can be realized.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a typical method for detecting a sample nucleic acid fragment of the present invention.

FIG. 2: Hybrid D bound to hybrid DNA
4 is a three-dimensional SECM image by a scanning electrochemical microscope showing detection of a current flowing between an NA-binding electrochemically active molecule and the surface of a DNA analysis element.

FIG. 3 is a profile diagram of a three-dimensional SECM image.

[Explanation of symbols]

11 DNA Fragment 12a Sample Nucleic Acid Fragment Complementary to DNA Fragment 21 Sample Nucleic Acid Fragment 31 DNA Analysis Element 41 Hybrid DNA-binding Electrochemically Active Molecule 51 Hybrid DNA-Binding Electrochemically Active Molecule Bonds to Hybrid DNA Immobilized on Substrate Complex 61 formed by scanning electrochemical microscope 71 3D image diagram

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 G01N 33/58 A 33/58 C12N 15/00 A G01N 27/46 336B (72) Inventor Masashi Ogawa 2-30-30 Nishiazabu, Minato-ku, Tokyo Inside Fujisha Shin Film Co., Ltd. (72) Inventor Shigeori Takenaka 4-23-21 Mainosato, Koga City, Fukuoka Prefecture (72) Kenichi Yamashita Jonan, Fukuoka City, Fukuoka Prefecture Tsutsumi-ku, Ward 17-104 (72) Inventor Makoto Takagi 3-4-29 Shonan-cho, Hakata-ku, Fukuoka City, Fukuoka Prefecture

Claims (10)

[Claims] 1. A DNA analysis device comprising a plurality of regions partitioned on a substrate surface and a DNA fragment fixed to each of the plurality of regions.
By contacting the aqueous liquid formed by dissolving or dispersing the sample nucleic acid fragment in the presence of the hybrid DNA-binding electrochemically active molecule, complementarity with the DNA fragment immobilized on the analysis element in the aqueous liquid is obtained. Along with binding the sample nucleic acid fragments having the same, the electrochemically active molecules are also bound, and then a current generated in the electrochemically active molecule binding region on the surface of the analysis element is measured by applying a potential by a scanning electrochemical microscope. A method for detecting a sample nucleic acid fragment having complementarity. 2. A DNA analysis element having a DNA fragment fixed to each of a plurality of regions partitioned on a substrate surface, a DN having an unknown concentration and fixed to the analysis element.
The sample nucleic acid fragment having complementarity with the A fragment is fixed to the analytical element in the sample aqueous liquid by contacting the sample aqueous liquid in which the sample nucleic acid fragment is dissolved or dispersed and the hybrid DNA-binding electrochemically active molecule. Binding a sample nucleic acid fragment having complementarity with the DNA fragment and also binding a hybrid DNA-binding electrochemically active molecule; and (2) applying a potential to the analytical element by a scanning electrochemical microscope, The current generated in the hybrid DNA-binding electrochemically active molecule binding region on the surface of the analysis element is measured, and the value of the current is determined by comparing the current and the concentration of the sample aqueous liquid in which the same sample nucleic acid fragment is dissolved or dispersed as described above. A step of determining the concentration of a complementary sample nucleic acid fragment in the sample aqueous solution by comparing with a calibration curve indicating the relationship That method of quantifying the sample nucleic acid fragment. 3. The calibration curve used in the step (2) is applied to a DNA analysis element in which a DNA fragment is fixed to each of a plurality of regions partitioned on the substrate surface, and the DNA fixed to the analysis element. The sample nucleic acid fragments containing the sample nucleic acid fragments having complementarity with the fragments at different known concentrations are brought into contact with each of the three or more sample aqueous liquids obtained by dissolving or dispersing the sample nucleic acid fragments. Along with binding the sample nucleic acid fragment having complementarity to the DNA fragment fixed to the analytical element, and also binding the hybrid DNA-binding electrochemically active molecule, and then applying a potential by a scanning electrochemical microscope, the analytical element The method according to claim 1, wherein the current is generated by measuring a current generated in a hybrid DNA binding electrochemically active molecule binding region on the surface. 3. The quantification method according to 2. 4. The method according to claim 1, wherein the base sequence of the DNA fragment is known. 5. The method according to claim 1, wherein the hybrid DNA-binding electrochemically active molecule is a ferrocene-modified electrochemically active embroidery intercalator having a redox activity. The described method. 6. A PNA analysis element in which a PNA fragment is fixed to each of a plurality of regions partitioned on a substrate surface,
By contacting the aqueous liquid formed by dissolving or dispersing the sample nucleic acid fragment in the presence of the hybrid PNA-binding electrochemically active molecule, complementarity with the PNA fragment immobilized on the analytical element in the aqueous liquid is obtained. Along with binding the sample nucleic acid fragment having the same, the electrochemically active molecule is also bound, and then a potential is applied by a scanning electrochemical microscope to measure a current generated in the electrochemically active molecule binding region on the surface of the analysis element. A method for detecting a sample nucleic acid fragment having complementarity, characterized in that: 7. A PNA analysis element having a PNA fragment immobilized in each of a plurality of regions partitioned on a substrate surface, and a PN immobilized on the analysis element having an unknown concentration.
The sample nucleic acid fragment having complementarity with the A fragment is fixed to the analytical element in the sample aqueous liquid by contacting the sample aqueous liquid in which the sample nucleic acid fragment is dissolved or dispersed and the hybrid PNA-binding electrochemically active molecule. Binding a sample nucleic acid fragment having complementarity with the PNA fragment and also binding a hybrid PNA-binding electrochemically active molecule; and (2) applying a potential to the analytical element by a scanning electrochemical microscope, The current generated in the hybrid PNA binding electrochemically active molecule binding region on the surface of the analysis element is measured, and the value of the current is compared with the concentration of the sample aqueous liquid in which the same sample nucleic acid fragment is dissolved or dispersed and the current. A step of determining the concentration of a complementary sample nucleic acid fragment in the sample aqueous solution by comparing with a calibration curve indicating the relationship That method of quantifying the sample nucleic acid fragment. 8. A calibration curve used in the above step (2) is obtained by using a PNA analysis element in which a PNA fragment is fixed to each of a plurality of regions partitioned on the substrate surface, and a PNA fixed to the analysis element. The sample nucleic acid fragments containing the sample nucleic acid fragments having complementarity with the fragments at different known concentrations are brought into contact with each of the three or more sample aqueous liquids obtained by dissolving or dispersing the sample nucleic acid fragments. The sample nucleic acid fragment having complementarity with the PNA fragment immobilized on the analytical element is combined with the hybrid PNA-binding electrochemically active molecule, and then a potential is applied by a scanning electrochemical microscope. The method is characterized in that it is created by measuring a current generated in a hybrid PNA binding electrochemically active molecule binding region on the surface. 3. The quantification method according to 2. 9. The method according to claim 6, wherein the base sequence of the PNA fragment is known. 10. The method according to claim 6, wherein the hybrid PNA-binding electrochemically active molecule is a ferrocene-modified electrochemically active stitching intercalator having redox activity. The described method.