JP2020051889A - Electrochemical detection method of nucleic acid molecule - Google Patents

Abstract

To provide an electrochemical nucleic acid detection method having a low detection lower limit concentration and a wide dynamic range.SOLUTION: In an electrochemical detection method of a target nucleic acid in a subject sample, a poly-nucleobase modified electrode that is produced by binding a nucleobase probe made of a nucleobase of a nucleic acid binding oxidation-reduction marker low-binding property to a conductor is brought into contact with a liquid composition containing a subject sample. The nucleic acid binding oxidation-reduction marker is brought into contact with a poly-nucleobase modified electrode. The providing/receiving of an electron for the poly-nucleobase modified electrode that is caused by an oxidation-reduction reaction of the nucleic acid binding oxidation-reduction marker on the poly-nucleobase modified electrode is detected.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a method for electrochemically detecting nucleic acid molecules.

  In recent years, attention has been paid to small noncoding RNAs (ncRNAs) including microRNAs (miRNAs) of about 20 bases because they have various functions. In particular, when these ncRNA levels correlate with a disease, it can be used as a disease marker. Techniques such as fluorescence detection and electrochemical measurement are used for the detection of ncRNA.

  In the detection of ncRNA, it is common to capture by hybridization with a probe using a nucleic acid. For capture probes, not only natural nucleic acids such as DNA and RNA, but also nucleotide analogs such as peptide nucleic acids (PNA) may be used. PNA has a pseudo-peptide skeleton in which N- (2-aminoethyl) glycine is bonded by an amide bond instead of a sugar chain in DNA or RNA. PNA is used as a new probe because it can form a double strand with DNA or RNA (Non-patent Document 1 and Non-Patent Document 2).

PNA has no charge unlike DNA or RNA. An electrochemical measurement method applied to a probe utilizing this property is known. For example, a measurement method using a PNA probe and [Fe (CN) ₆ ] ^{4- / 3-} has been reported. In this method, the negatively charged [Fe (CN) ₆ ] ^{4- / 3-} in the solution easily reaches the electrode when DNA or RNA is not bound, but when the negatively charged DNA or RNA binds to PNA, The target DNA or RNA is detected by utilizing the fact that [Fe (CN) ₆ ] ^{4- / 3-} does not reach the electrode due to the repulsion between the charges.

Further, a method has been reported in which 8-amino-1-octanethiol is bonded to the surface of a gold electrode together with a PNA probe to give a positive charge to the surface of the electrode. In this method, a cation marker [Ru (NH ₃ ) ₆ ] ³⁺ is used. In the unbound state of DNA or RNA, [Ru (NH ₃ ) ₆ ] ³⁺ cannot reach the electrode due to the repulsive force with the positive charge due to 8-amino-1-octanethiol, but DNA or RNA having a negative charge is not deposited on the surface of the electrode. When the charge is canceled out by binding to PNA and lost, [Ru (NH ₃ ) ₆ ] ³⁺ reaches the electrode surface and causes a reduction reaction to detect DNA or RNA (Patent Document 1). Non-Patent Documents 3 and 4).

In addition, it has been reported that by binding PNA complementary to a target sequence to a gold electrode, nonspecific binding of cations to an electrode-bound probe was suppressed, and detection sensitivity was improved. For example, in Non-Patent Document 5, a PNA probe is bound to a gold electrode, and the PNA probe is densely packed near the surface of the gold electrode by electrostatic interaction with an anion of a phosphate group of DNA bound to the probe [Ru (NH ₃ )]. ₆ ] An electrical signal due to ³⁺ is detected.

As redox species to be used for electrochemical detection of nucleic acid, the above-mentioned ^{_{[Fe (CN) 6] 4-}} / 3-, [Ru (NH 3) 6] 3+ other, methylene blue (non-patent with nucleic acid binding properties Literatures 6 and 7) and methods using the difference in binding between nucleic acid single-stranded and double-stranded nucleic acids in Oracet Blue (Non-Patent Document 8) are used.

International Publication WO2002 / 073183

Nielsen, P .; E. FIG. Et al., Science; 254: 1497-1500 (1991). Michael Eholm et al., Nature; 365: 566-568 (1993). Hiroshi Aoki et al., Electroanalysis; 12 (16): 1272-1276 (2000). Hiroshi Aoki et al., Electroanalysis; 14 (19-20): 1405-1410 (2002). M. Steichen et al., Biosensors and Bioelectronics; 22: 2237-2243 (2007). Hossain-Ali Rafiee-Pour et al., Biosensors and Bioelectronics; 77: 202-207 (2016). Cheneng HE et al., Analytical Science; 33: 1155-1160 (2017). Mostafa Azimzadeh et al., Biosensors and Bioelectronics; 77: 99-106 (2016).

  The conventional electrochemical nucleic acid detection method has a feature that the lower detection limit concentration is lower than that of a biochemical method using enzymes or the like, but the lower detection limit concentration is in the range of 0.1 to 500 pM. It was 84.3 fM (Non-Patent Document 6 above). In addition, since the conventional method has a high background before hybridization, the method of measuring the binding by comparing before and after hybridization with the target nucleic acid, or the high current value shown before hybridization, It is a method of measuring by utilizing the fact that it becomes lower. In such a conventional method, two measurements must be performed before and after hybridization.

  The present inventors have conducted various studies on a detection method having a lower detection lower limit concentration and a wider dynamic range. As a result, by using a non-charged nucleic acid substance such as PNA as a probe and using a compound capable of binding to a nucleic acid having a phosphate skeleton as a redox marker, the lower detection limit concentration is low and the dynamic range is wide. It has been found that detection is possible. In particular, in the method according to one embodiment of the present invention, the use of a non-charged nucleic acid substance as the probe causes the redox marker to bind only slightly to the probe, so that the redox current before hybridization is significantly reduced, Measurement was unnecessary. As a result, since the target nucleic acid can be detected by one measurement after hybridization with the target nucleic acid, a simpler detection method was successfully provided.

That is, one embodiment of the present invention is a method for electrochemically detecting a target nucleic acid in a test sample,
A nucleic acid-binding redox marker, a polynucleobase-modified electrode formed by bonding a polynucleobase probe comprising a low-binding nucleobase to a conductor, and a liquid composition containing a test sample, and
The nucleic acid binding redox marker is brought into contact with the polynucleobase-modified electrode,
The present invention relates to a method for electrochemically detecting a target nucleic acid, which detects the transfer of electrons to the polynucleobase-modified electrode by a redox reaction of the nucleic acid-binding redox marker on the polynucleobase-modified electrode.

Another aspect of the present invention is a method for electrochemical quantification of a target nucleic acid in a test sample,
A nucleic acid-binding redox marker, a polynucleobase-modified electrode formed by bonding a polynucleobase probe comprising a low-binding nucleobase to a conductor, and a liquid composition containing a test sample, and
The nucleic acid binding redox marker is brought into contact with the polynucleobase-modified electrode,
Measuring the level of electron transfer to the polynucleobase-modified electrode by a redox reaction of the nucleic acid binding redox marker on the polynucleobase-modified electrode,
A method for electrochemically quantifying a target nucleic acid, comprising calculating a level of the target nucleic acid in the test sample from a level of electron transfer to the polynucleobase-modified electrode detected. About.

  By using methylene blue as a redox marker together with a PNA probe, the background in electrochemical detection of nucleic acids by hybridization can be reduced. This has made it possible to provide an electrochemical nucleic acid detection method having a low detection lower limit concentration and a wide dynamic range. For example, the lower limit of detection is about one digit lower than that of potassium ferrocyanide, which is a negatively charged redox marker, and the dynamic range has a width of two digits or more. In addition, since the background is low, it is not necessary to compare before and after hybridization between the probe and the target nucleic acid, and the nucleic acid can be detected only once after hybridization without performing measurement before hybridization. Became possible.

It is a graph which shows the oxidation-reduction peak of methylene blue, the upper figure shows the graph of the electric potential-current at the time of measuring the cyclic voltammetry (CV), and the lower figure shows the electric potential-current at the time of measuring the square wave voltammetry (SWV). Is shown. (A) shows a schematic diagram of a state before hybridization in an example of the present invention. (B) shows a schematic view of a state after hybridization in an example of the present invention. (C) A schematic diagram of a state before hybridization in a measurement using potassium ferrocyanide is shown. (D) shows a schematic view of the state after hybridization in the measurement using potassium ferrocyanide. It is a graph of the potential-current at the time of CV measurement before and after hybridization with potassium ferrocyanide. The upper figure shows the case where the target nucleic acid does not exist, and the lower figure shows the case where the target nucleic acid exists. It is a graph which shows the background current value in SWV measurement of a PNA probe and a DNA probe, the upper figure shows the graph of the potential-current when measuring the background current of a PNA probe, and the lower figure shows the background current of a DNA probe. 4 shows a graph of the potential-current when the value was measured. 4 is a graph showing a relationship between a target nucleic acid concentration and a peak current value ratio when potassium ferrocyanide is used as a redox marker. It is a graph which shows the target nucleic acid concentration and peak current value of SWV measurement when methylene blue is used as a redox marker.

  In the present application, “nucleobase” includes nucleotide analogs in addition to naturally occurring nucleotides. Naturally occurring nucleotides are deoxyribonucleotides or ribonucleotides having the bases adenine (A), guanine (G), cytosine (C), thymine (T), and / or uracil (U). The nucleotide analog means an artificial nucleotide having the same base as the above-described naturally occurring deoxyribonucleotide or ribonucleotide, but having an artificially modified chemical structure of ribose and / or a phosphodiester bond. For example, a glycol nucleic acid (Glycol nucleic acid: GNA), a bridged nucleic acid (Bridged Nucleic Acid: BNA), a 2 ′, 4′-crosslinked nucleic acid (Locked nucleic acid: LNA), a peptide nucleic acid (Peptide Nucleic Acid, PN: PN) Examples include threose nucleic acid (TNA) and morpholino nucleic acid (MNA). In the present application, GNA, BNA, LNA, PNA, TNA, and MNA may be interpreted as monomers or polymers depending on the context.

  In the present application, “polynucleobase” means a polymer compound in which the above-mentioned nucleobase is polymerized in a linear or branched manner. The polynucleobase may be a homopolymer composed of only one type of nucleobase (eg, only a naturally occurring polynucleotide or only a PNA constituent unit), or may be a homopolymer composed of two or more types of nucleobases (naturally occurring polynucleotides). A copolymer of nucleotide and PNA or BNA and LNA). Therefore, polynucleotides such as DNA and RNA, and polymers of GNA, BNA, LNA, PNA, TNA, and MNA are also included in the polynucleobase. In the present application, “nucleic acid” and “nucleic acid molecule” are synonymous and represent a general term for DNA and RNA.

  In the present application, the term "probe" or "polynucleobase probe" is synonymous, unless otherwise specifically interpreted, and is used to form a duplex by hybridization with a target sequence. Means the polynucleobase used. The probe includes a sequence that is at least partially complementary to the target sequence and is capable of binding to the target sequence. The probe may contain a sequence that is not complementary to the target sequence for binding to a labeling substance or for other purposes.

  In particular, when a DNA probe is used, the background is increased by the binding of methylene blue or the like to the probe. In the present application, a nucleic acid-binding redox marker low-binding nucleobase is used as a probe (nucleic acid binding). By using a redox marker low-binding probe, the background in the absence of the target nucleic acid is reduced. Since the nucleic acid-binding redox marker binds only to the hybridized nucleic acid, it is possible to detect even a low concentration of the target nucleic acid. Therefore, the polynucleobase constituting the probe is a polynucleobase having a low binding property to a nucleic acid binding redox marker such as methylene blue, and is, for example, PNA or MNA. It is preferable that the polynucleobase portion of the probe be composed of only the nucleic acid-binding redox marker low-binding polynucleobase, but if necessary, a low ratio (for example, 10% or less, 5% or less, 3% or less, 1% or less). (Hereinafter, 0.5% or less) nucleic acid-binding redox marker low-binding polynucleobase. Whether a probe or nucleobase has a low binding property to a nucleic acid-binding redox marker is determined by contacting a gold electrode to which the probe or nucleobase is bound with a nucleic acid-binding redox marker and applying a specific potential. Can be determined by measuring whether or not a change in current occurs due to the transfer of electrons to the electrode. If the current does not change or is negligibly low (for example, the same level as PNA), the lobe or nucleobase is determined to have low nucleic acid binding redox marker binding.

  The “nucleic acid binding redox marker” is a redox marker that binds to a nucleic acid molecule having a phosphate skeleton such as DNA and RNA but does not bind to an uncharged nucleic acid molecule or has low binding. In the present application, the nucleic acid-binding redox marker is preferably a compound that can bind to a single-stranded nucleic acid, and the compound may also bind to a double-stranded nucleic acid. Further, the nucleic acid-binding redox marker preferably does not include an intercalator that binds only to a nucleic acid duplex. The “redox marker” refers to a compound that has an oxidized state and a reduced state and that can transfer electrons by a redox reaction. Examples of such a nucleic acid binding redox marker include methylene blue, viologen analogs, ruthenium complexes and cobalt complexes.

  Methylene blue is a positively charged nucleic acid binding redox marker and has a redox potential near -250 mV (FIG. 1). There are several reports on the binding mechanism of methylene blue to nucleic acids, such as binding via guanine bases of single-stranded DNA (Yang, W et al., Electroanalysis; 14: 1299-1302 (2002)) and negatively. Binding by electrostatic interaction with charged phosphate groups (Zhao, G et al., Anal. Chim. Acta; 394: 337-344 (1999)), and directly to guanine-cytosine pairs in DNA duplexes. Incalating (Arias, P et al., J. Electronal. Chem; 634: 123-126 (2009)) has been reported. In addition, the redox reaction of methylene blue generates a large current value due to the transfer of two electrons, so that a small amount of nucleic acid can be detected as compared with a single-electron transfer type redox species. Difference is easy to detect. In particular, when performing electrochemical detection of a nucleic acid by hybridization with methylene blue, the background was suppressed by using a non-charged nucleic acid probe such as PNA instead of a negatively charged nucleic acid probe such as DNA, so that hybridization was performed with a small background. Only nucleic acids can be detected, and the detection sensitivity is further improved. This is because the background is increased by the binding of methylene blue to nucleic acid probes such as commonly used DNA. Since PNA connects bases by peptide bonds, it is considered that methylene blue bonds are small.

  In the present application, "target nucleic acid" means a nucleic acid of interest whose presence is to be detected or quantified by the method of the present invention. The target nucleic acid does not need to be derived from a natural product, and includes, for example, an artificial nucleic acid. For example, when the method of the present invention is used for the purpose of diagnosing a disease or disorder, the target nucleic acid means DNA or RNA derived from a living body. The length of the target nucleic acid is not particularly limited, but a target nucleic acid of 50 mer or less, 45 mer or less, 40 mer or less, 35 mer or less, 30 mer or less, 29 mer or less, 28 mer or less, 27 mer or less, 26 mer or less, or 25 mer or less is preferable. . For example, the length of the target nucleic acid can be 10 mer or more, 11 mer or more, 12 mer or more, 13 mer or more, 14 mer or more, 15 mer or more, 16 mer or more, 17 mer or more, or 18 mer or more. As an example, the chain length of the target nucleic acid is 10 to 50 mer, 10 to 40 mer, 13 to 30 mer, 15 to 28 mer, or 18 to 25 mer. In addition, a typical example of the target nucleic acid includes miRNA.

  In the present application, the “target sequence” is a sequence contained in a target nucleic acid and is a sequence to be detected. In another expression, the target sequence means a sequence complementary to or bindable to the probe used. The target sequence may be a full-length sequence or a partial sequence of the target nucleic acid. The length of the target sequence is 10 to 50 mer. For example, the chain length of the target sequence can be at least 10 mer, at least 11 mer, at least 12 mer, at least 13 mer, at least 14 mer, at least 15 mer, at least 16 mer, at least 17 mer, or at least 18 mer. In addition, the chain length of the target sequence can be 50 mer or less, 45 mer or less, 40 mer or less, 35 mer or less, 30 mer or less, 29 mer or less, 28 mer or less, 27 mer or less, 26 mer or less, or 25 mer or less. The chain length of the target sequence can be 10 to 40 mer, 13 to 30 mer, 15 to 28 mer, or 18 to 25 mer.

  In the present application, a “non-target nucleic acid” is a nucleic acid that is not intended to be detected by the present method. The “non-target sequence” means a sequence of a non-target nucleic acid.

  The `` electrode '' is not particularly limited as long as it can bind a polynucleobase probe and can detect a redox reaction by a redox marker, but, for example, is preferably a noble metal electrode, More preferably, it is a gold electrode. Gold is suitable for detecting a redox reaction with a redox probe. Further, the electrode may have a structure in which gold is attached to the surface of a carbon electrode such as glassy carbon or graphene oxide. Further, the polynucleobase probe does not need to be directly bonded to gold. For example, both acrylic fine particles and gold fine particles having a polynucleobase bonded thereto may be attached to the carbon electrode surface.

  The “non-nucleic acid-binding substance” is a substance that does not bind to the nucleic acid to be detected and plays a role of physically supporting the structure of the probe. The nucleic acid non-binding substance may be, for example, a short-chain alkanethiol or disulfide, and more specifically, 6-mercapto-1-hexanol, 6-hydroxy-1-hexanethiol (HHT). Can be.

  FIG. 2A shows a schematic diagram of an electrode used in the method of the present invention in a state where nucleic acids have not been hybridized. 1 denotes a capture PNA probe modified on a gold electrode, and 2 denotes 6-hydroxy-1-hexanethiol as a nucleic acid non-binding substance. Since no charge is generated on the surface of the gold electrode when DNA or RNA is not hybridized, a nucleic acid-binding redox marker such as methylene blue does not bind to the electrode surface. FIG. 2 (B) is a schematic view of an electrode used in the method of the present invention in a state where the target nucleic acid of the present invention has been hybridized. Reference numeral 1 denotes a capture PNA probe modified on a gold electrode, 2 denotes 6-hydroxy-1-hexanethiol as a nucleic acid non-binding substance, 3 denotes a target nucleic acid, and 4 denotes methylene blue. The binding of DNA or RNA generates an electric charge, and a nucleic acid-binding redox marker such as methylene blue binds to the electrode surface, and exchanges electrons with the electrode. FIG. 2C is a schematic diagram showing a state where DNA or RNA has not been hybridized in the measurement using potassium ferrocyanide. Reference numeral 1 denotes a captured PNA probe modified on a gold electrode, 2 denotes 6-hydroxy-1-hexanethiol as a nucleic acid non-binding substance, and 3 denotes potassium ferrocyanide. In the state where DNA or RNA is not hybridized, electric repulsion does not work, and potassium ferrocyanide can approach the electrode surface, so that electrons can be exchanged with the electrode. FIG. 2D is a schematic diagram showing a state where DNA or RNA has hybridized in the measurement using potassium ferrocyanide. Reference numeral 1 denotes a captured PNA probe modified on a gold electrode, 2 denotes 6-hydroxy-1-hexanethiol as a nucleic acid non-binding substance, 3 denotes potassium ferrocyanide, and 4 denotes a target nucleic acid. The binding of DNA or RNA causes an electric repulsion, and the potassium ferrocyanide cannot exchange electrons with the electrode.

1. Production of Polynucleobase Probe The polynucleobase probe used in the embodiment of the present invention can be produced by a method well-known in the art, based on a probe sequence designed to be complementary to a target sequence. In particular, a method is well known for chemical synthesis in which polynucleobases such as DNA and RNA, PNA, LNA and the like are linked one by one, and such a method can be employed. Further, if necessary, the synthesized polynucleobase probe can be bound to a modifying substance such as a solid phase or a label.

2. Production of Modified Electrode The modified electrode used in the embodiment of the present invention can be obtained by binding the polynucleobase probe to a gold electrode or a conductor such as graphene oxide or gold nanoparticles on a screen print electrode. The method of the present invention may optionally include a step of preparing a polynucleobase-modified electrode by bonding a polynucleobase probe comprising a nucleic acid-binding redox marker low-binding nucleobase to a conductor.

3. One embodiment of the present invention is a method for electrochemically detecting a target nucleic acid in a test sample,
A nucleic acid-binding redox marker, a polynucleobase-modified electrode formed by bonding a polynucleobase probe comprising a low-binding nucleobase to a conductor, and a liquid composition containing a test sample, and
The nucleic acid binding redox marker is brought into contact with the polynucleobase-modified electrode,
The present invention relates to a method for electrochemically detecting a target nucleic acid, which detects the transfer of electrons to the polynucleobase-modified electrode by a redox reaction of the nucleic acid-binding redox marker on the polynucleobase-modified electrode.

  In this method, a target nucleic acid having a sequence complementary to the probe bound to the electrode surface binds, and the nucleic acid-binding redox marker binds to the nucleic acid. The emission or acquisition of electrons by the redox reaction of the bound nucleic acid binding redox marker manifests itself as a current or voltage electrical signal at the electrode. That is, by transferring electrons from the redox marker to the electrode, the bound target nucleic acid is detected. Electrochemical detection methods using oxidation-reduction markers are widely known. For example, methods using methylene blue are described in the above non-patent documents 6 and 7 (however, in these non-patent documents, Using DNA as a probe). That is, when methylene blue is used, the transfer of electrons between the methylene blue and the electrode indicates the binding of the target nucleic acid.

  Therefore, the present method includes as essential components the binding of the target nucleic acid to the electrode surface, the binding of the redox marker to the target nucleic acid, and the electrochemical detection of the redox reaction of the redox marker bound to the target nucleic acid. Each of the reactions constituting the present method can be performed sequentially or simultaneously. For example, the modified electrode can be immersed in a liquid composition containing both the test sample in which the presence of the target nucleic acid is to be confirmed and the redox marker, and the binding measurement can be performed while reacting. Alternatively, after binding a test sample for which the presence of the target nucleic acid on the electrode surface is desired to be confirmed, the redox marker is brought into contact with the target nucleic acid and bound, and then the redox reaction of the redox marker bound to the target nucleic acid is performed. It may be detected electrochemically. Alternatively, the sample to be tested for the presence of the target nucleic acid on the electrode surface and the redox marker are simultaneously contacted and bound, and then the redox reaction of the redox marker bound to the target nucleic acid is electrochemically detected. Is also good. In the case of performing stepwise, a step of cleaning between each step may be included. For example, after binding a test sample for which it is desired to confirm the presence of the target nucleic acid on the electrode surface, the electrode may be washed in a liquid containing no sample (such as a buffer solution), and then contacted with a redox marker. . Alternatively, after contacting the redox marker, the electrode is washed with a liquid containing no redox marker (such as a buffer), and then the redox reaction of the redox marker bound to the target nucleic acid is electrochemically detected. You may. In particular, by performing a washing operation before detection, a non-specific background electric signal due to a redox marker in the liquid can be suppressed. Therefore, the washing operation is preferably performed before detection.

The concentration of the nucleic acid contained in the test sample is not particularly limited, but may be, for example, 10 ⁻⁴ to 10 ⁻¹⁶ M. Since the method is performed to determine the presence of a target nucleic acid in a test sample, the test sample does not necessarily need to contain the target nucleic acid. Further, the test sample may contain a nucleic acid molecule other than the target nucleic acid. Furthermore, the test sample may have a high or low purification level, may contain only nucleic acids as a substance derived from the test sample, or may contain impurities (sugar chains, proteins, etc.) other than nucleic acids. Good. For example, a test sample can be prepared using a target sample for which the presence of a target nucleic acid to be detected is to be examined. For example, for the purpose of diagnosis, the sample is not particularly limited as long as it is a sample from which DNA or RNA can be detected. Lymph, blood (serum, plasma), urine, stool, saliva, cerebrospinal fluid, tears, biopsy, hair, Body fluids, cells, such as skin, nails, exudates, cells (e.g., cells derived from circulating tumor cells (CTC), vascular endothelial cells, ES cells, iPS cells, muse cells, or pluripotent stem cells); Tissues; exosomes; or cell-free DNA can be used. These samples are appropriately prepared as samples suitable for detection of DNA or RNA.

The reaction between the test sample and the electrode is performed in a liquid, and is not particularly limited as long as the condition allows binding of the nucleic acid-binding redox marker and low-binding polynucleobase to the target nucleic acid. For example, as a condition for hybridization, Molecular Cloning, a Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012), or a method described in Current Protocol, Molecular Biology, etc., which can be described in a method described in Current Protocol, Molecular Biology, etc. For example, in 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0.2 M NaH ₂ PO ₄ , 20 mM EDTA · 2Na, pH 7.4) at 38-42 ° C. For 10 minutes to 1 hour.

The binding of the redox marker to the target nucleic acid can be appropriately determined according to the type of the redox marker. For example, it can be carried out by adding a redox marker to a phosphate buffer containing NaClO ₄ , bringing it into contact with an electrode to which a target nucleic acid has been bound, and allowing it to stand for 1 to 30 minutes. The concentration of the redox marker to be used is not particularly limited as long as it is a concentration that efficiently binds to the nucleic acid without causing nonspecific binding. For example, in the case of methylene blue, the concentration is 1 to 100 μM. be able to. At the time of detection measurement, it is preferable not to include a redox marker in the measurement solution. That is, it is preferable that after binding the redox marker to the target nucleic acid hybridized to the probe, washing is performed, and the transfer of electrons occurring when a potential is applied to the redox marker bound to the target nucleic acid is detected. If the measurement solution contains a redox marker, it becomes difficult to distinguish between the transfer of electrons generated from the redox marker in the solution and the transfer of electrons from the redox marker bound to the target nucleic acid, resulting in lower detection sensitivity. there's a possibility that.

  The electrochemical measurement of the oxidation-reduction reaction can be performed using, for example, a three-electrode system device such as a potentiostat or a galvanostat. The measurement of the oxidation-reduction reaction can be performed by voltammetry such as CV; AC impedance (EIS); Faraday impedance spectroscopy; and coulometry.

4. Another aspect of the present invention is a method for electrochemically quantifying a target nucleic acid in a test sample, comprising:
A nucleic acid-binding redox marker, a polynucleobase-modified electrode formed by bonding a polynucleobase probe comprising a low-binding nucleobase to a conductor, and a liquid composition containing a test sample, and
The nucleic acid binding redox marker is brought into contact with the polynucleobase-modified electrode,
Measuring the level of electron transfer to the polynucleobase-modified electrode by a redox reaction of the nucleic acid binding redox marker on the polynucleobase-modified electrode,
The present invention relates to a method for electrochemically quantifying a target nucleic acid, wherein a level of the target nucleic acid in the test sample is calculated from a level of electron transfer to the polynucleobase-modified electrode detected.

  This method of quantifying a target nucleic acid by an electrochemical measurement method can be performed using the same method as the above-described method of electrochemically detecting a target nucleic acid. However, in the above-described method, the measurement is performed by "measuring" the level of the transfer of the target nucleic acid or the electron instead of "detecting" the transfer of the bound target nucleic acid or the electron. In the present application, “level” means an index relating to abundance quantified, and includes, for example, the concentration or amount of a target nucleic acid or an index that can be used as an alternative. Therefore, the level may be a measured value of an electric signal or the like, or a value converted into a concentration or the like. The level may be an absolute value (abundance, abundance per unit area / volume, etc.) or a relative value as compared with a comparative control set as necessary. Is also good.

  The calculation of the level of the target nucleic acid in the test sample from the detected level of transfer of electrons to the polynucleobase-modified electrode can be appropriately performed according to the employed measurement method or the like. For example, the calculation of the level of the target nucleic acid can be performed by applying the measured value to a predetermined standard curve. Alternatively, a standard solution whose concentration is known in advance is measured at the same time as the sample, and the measured value of the sample can be compared with the measured value of the standard solution.

  Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto. All documents cited throughout the application are incorporated herein by reference in their entirety.

(Example 1) Oxidation-reduction potential of methylene blue The bare gold electrode was subjected to plasma irradiation for 2 minutes at 10 mA using an oxygen plasma apparatus to wash the surface of the gold electrode. Then, 0.1 mM methylene blue + 0.25 mM phosphate buffer (sodium, pH7.0) + 0.5mM NaClO _4, a gold electrode and an aqueous reference electrode in 1 mL (Ag / AgCl BAS Inc. RE-1B) and Pt counter electrode ( (Manufactured by BAS Corporation), and the SWV was measured using a potentiostat from an initial potential of 0 mV to a final potential of -500 mV, under the conditions of a step potential of 5 mV, a pulse amplitude of 50 mV, and a cycle of 20 Hz. CV was measured by sweeping in the negative direction at an initial potential of 0 mV, a high potential of 0 mV, a low potential of -500 mV, and a scan speed of 500 mV / sec. Methylene blue is a positively charged redox marker and was shown to have a redox potential near -250 mV. FIG. 1 is a graph showing the oxidation-reduction peak of methylene blue. The upper diagram shows a graph of potential-current when CV is measured, and the lower diagram shows a graph of potential-current when SWV is measured. The vertical axis represents current (nA), and the horizontal axis represents potential (V).

(Example 2) CV measurement using potassium ferrocyanide The bare gold electrode was subjected to plasma irradiation with an oxygen plasma apparatus at 10 mA for 2 minutes to wash the surface of the gold electrode. On the washed gold electrode, 10 μL of 10 μM PNA (TTCAGTTATCACAGACTCTGTA-o-Cys) (SEQ ID NO: 1) dissolved in 0.05% TFA was applied, and allowed to stand at 25 ° C. for 30 minutes. Thereafter, the substrate was washed with ultrapure water, 1 mM HHT was applied on the gold electrode, and the plate was allowed to stand at 25 ° C. for 30 minutes. Before hybridization, a PNA probe and an HHT-modified gold electrode in 1 mL of 1 mM potassium ferrocyanide + 0.25 mM phosphate buffer (sodium, pH 7.0) +0.5 mM NaClO ₄ , an aqueous reference electrode (Ag / AgCl BAS RE- 1B) and a Pt counter electrode (manufactured by BAS) were immersed, and the CV was measured using a potentiostat by sweeping in the positive direction at an initial potential of −150 mV, a high potential of 500 mV, a low potential of −150 mV, and a scan speed of 500 mV / sec. After washing with ultrapure water, a ^10-7 M target nucleic acid (UACAGUACUGUGUAUAACUGAA) dissolved in 1 × SSC (0.15 M NaCl + 0.015 M trisodium citrate) + 20% DMSO was placed on a gold electrode modified with a PNA probe and HHT. (SEQ ID NO: 3) 10 μL of the solution was added dropwise, and hybridization was carried out at 40 ° C. for 40 minutes. After washing the gold electrode after hybridization in 100 mL of ultrapure water at 50 ° C. for 2 minutes, hybridization was performed in 1 mL of 1 mM potassium ferrocyanide + 0.25 mM phosphate buffer (sodium, pH 7.0) +0.5 mM NaClO _4. The gold electrode, the aqueous reference electrode (Ag / AgCl BAS Co., RE-1B) and the Pt counter electrode (BAS Co.) were immersed, and the CV was set to an initial potential of −150 mV and a high potential of 500 mV using a potentiostat. The measurement was performed by sweeping in the positive direction at a low potential of -150 mV and a scan speed of 500 mV / sec. The potassium ferrocyanide was detected by performing CV measurement in a potassium ferrocyanide-containing measurement solution before and after hybridization, and calculating as a ratio of the peak current values. The current value at the same potential after hybridization, when the peak current value of the CV waveform before hybridization was 1, was calculated as a ratio. The results are shown in FIG. It is a graph of the potential-current at the time of CV measurement before and after hybridization with potassium ferrocyanide. The upper figure shows the case where the target nucleic acid does not exist, and the lower figure shows the case where the ^10-7M target nucleic acid exists. When potassium ferrocyanide is used as a redox marker, the difference between the current values, particularly the decrease in the current value, indicates the amount of the target nucleic acid, so that measurement before and after hybridization is required.

(Example 3) Background current of PNA probe and DNA probe The bare gold electrode was subjected to plasma irradiation for 2 minutes at 10 mA using an oxygen plasma apparatus to wash the surface of the gold electrode. On the washed gold electrode, 10 μL of 10 μM PNA (TTCAGTTATCACAGACTCTGTA-o-Cys) (SEQ ID NO: 1) dissolved in 0.05% TFA was applied, and allowed to stand at 25 ° C. for 30 minutes. Thereafter, the PNA probe-modified gold electrode was washed with ultrapure water. Further, 10 μL of 10 μM DNA (TTCAGTTATCACAGTACTGTA-SH) (SEQ ID NO: 2) dissolved in 0.05% TFA was applied on the gold electrode after the plasma cleaning, and the mixture was allowed to stand at 25 ° C. for 30 minutes. Thereafter, the DNA probe-modified gold electrode was washed with ultrapure water. Both probe-modified gold electrodes were immersed in 1 mL of 50 μM methylene blue + 0.25 mM phosphate buffer (sodium, pH 7.0) +0.5 mM NaClO _{4 and} allowed to stand for 5 minutes. Then 0.25mM phosphate buffer (sodium, pH 7.0) + 0.5 mM washed with NaClO _4, 0.25mM phosphate buffer (sodium, pH7.0) + 0.5mM NaClO ₄ aqueous reference electrode in 1 mL (Ag / The sample was immersed together with AgCl BAS RE-1B) and a Pt counter electrode (BAS), and SWV was measured using a potentiostat from an initial potential of 0 mV to a final potential of -500 mV, a step potential of 5 mV, a pulse amplitude of 50 mV, and a cycle of 20 Hz. Was performed under the following conditions. FIG. 4 shows the background current value of the PNA probe and the DNA probe-modified gold electrode measured by SWV. In FIG. 4, the upper diagram shows a graph of potential-current when the background current of the PNA probe is measured, and the lower diagram shows a graph of potential-current when the background current of the DNA probe is measured. The vertical axis represents current (nA), and the horizontal axis represents potential (V). The background current value of the PNA probe-modified gold electrode was 4.55 nA, while the background current value of the DNA probe-modified gold electrode was 58.49 nA, which was about 13 times the background value. If the background value is small, it is easy to detect a difference in the current value of several nA or less, but if the background value is too high as in the case of a DNA probe, even if the background value is increased by several nA, the detected value will vary in the background value. It is buried and leads to a decrease in detection sensitivity. Therefore, it was shown that the PNA probe is more suitable for detecting a small amount of nucleic acid than the DNA probe.

(Example 4) Measurement of nucleic acid binding to PNA probe The bare gold electrode was irradiated with plasma at 10 mA for 2 minutes using an oxygen plasma apparatus to wash the surface of the gold electrode. On the washed gold electrode, 10 μL of 10 μM PNA (TTCAGTTATCACAGACTCTGTA-o-Cys) (SEQ ID NO: 1) dissolved in 0.05% TFA was applied, and allowed to stand at 25 ° C. for 30 minutes. Thereafter, the substrate was washed with ultrapure water, 1 mM HHT was applied on the gold electrode, and the plate was allowed to stand at 25 ° C. for 30 minutes. Before hybridization, a PNA probe and an HHT-modified gold electrode in 1 mL of 1 mM potassium ferrocyanide + 0.25 mM phosphate buffer (sodium, pH 7.0) +0.5 mM NaClO ₄ , an aqueous reference electrode (Ag / AgCl BAS RE- 1B) and a Pt counter electrode (manufactured by BAS) were immersed, and the CV was measured using a potentiostat by sweeping in the positive direction at an initial potential of −150 mV, a high potential of 500 mV, a low potential of −150 mV, and a scan speed of 500 mV / sec. After washing with ultrapure water, a ^10-7 to ^10-10 M target dissolved in 1 × SSC (0.15 M NaCl + 0.015 M trisodium citrate) + 20% DMSO was placed on a gold electrode modified with a PNA probe and HHT. 10 μL of a nucleic acid (UACAGUACUGUGUAUACUGAA) (SEQ ID NO: 3) solution was added dropwise, and hybridization was performed at 40 ° C. for 40 minutes. After washing the gold electrode after hybridization in 100 mL of ultrapure water at 50 ° C. for 2 minutes, hybridization was performed in 1 mL of 1 mM potassium ferrocyanide + 0.25 mM phosphate buffer (sodium, pH 7.0) +0.5 mM NaClO _4. The gold electrode, the aqueous reference electrode (Ag / AgCl BAS Co., RE-1B) and the Pt counter electrode (BAS Co.) were immersed, and the CV was set to an initial potential of −150 mV and a high potential of 500 mV using a potentiostat. The measurement was performed by sweeping in the positive direction at a low potential of -150 mV and a scan speed of 500 mV / sec. The potassium ferrocyanide was detected by performing CV measurement in a potassium ferrocyanide-containing measurement solution before and after hybridization, and calculating as a ratio of the peak current values. The current value at the same potential after hybridization, when the peak current value of the CV waveform before hybridization was 1, was calculated as a ratio.

Thereafter, 0.25 mM phosphate buffer (sodium, pH 7.0) + 0.5 mM washed with NaClO _4, 50 [mu] M methylene blue + 0.25 mM phosphate buffer (sodium, pH7.0) + 0.5mM NaClO ₄ post-hybridization in 1mL Was immersed and allowed to stand for 5 minutes. Then 0.25mM phosphate buffer (sodium, pH 7.0) + 0.5 mM washed with NaClO _4, 0.25mM phosphate buffer (sodium, pH7.0) + 0.5mM NaClO ₄ 1mL gold electrode after hybridization in And a water-based reference electrode (Ag / AgCl, RE-1B manufactured by BAS) and a Pt counter electrode (manufactured by BAS) are immersed. Using a potentiostat, SWV is measured from an initial potential of 0 mV to a final potential of -500 mV, and a step potential of 5 mV. , A pulse amplitude of 50 mV and a cycle of 20 Hz. Further, CV measurement was performed by sweeping in the negative direction at an initial potential of 0 mV, a high potential of 0 mV, a low potential of -500 mV, and a scan speed of 500 mV / sec. In this experiment, if methylene blue is added to the test solution, it may not be possible to distinguish between the transfer of electrons generated from methylene blue in the test solution and the transfer of electrons from methylene blue bound to nucleic acid, resulting in lower detection sensitivity. In consideration of the above, the measurement was performed under the condition that methylene blue was not present in the measurement solution.

The results are shown in FIGS. FIG. 5 is a graph showing the relationship between the target nucleic acid concentration and the peak current value ratio when potassium ferrocyanide is used as the redox marker. FIG. 6 shows the target nucleic acid concentration and SWV measurement when methylene blue is used as the redox marker. It is a graph which shows a peak current value. FIG. 5 shows the relationship between the ratio of the peak current value (vertical axis) and the target nucleic acid concentration [M] (horizontal axis). In FIG. 6, the horizontal axis represents the target nucleic acid concentration [M], and the vertical axis represents the current value [nA]. Is shown. Methylene blue had a lower detection limit of about one digit lower than that of potassium ferrocyanide, a negatively charged redox marker, and was detectable at 10 ⁻⁹ M. The dynamic range was 10 ⁻⁹ to 10 ⁻⁷ M or more, with a width of two digits or more. In this method, the dynamic range is widened and the resolution can be seen finely because the current flows when the nucleic acid hybridizes. Further, since there was almost no background, it was shown that nucleic acid measurement can be performed without performing measurement before hybridization.

Claims (11)

A method for electrochemically detecting a target nucleic acid in a test sample,
A nucleic acid-binding redox marker, a polynucleobase-modified electrode formed by bonding a polynucleobase probe comprising a low-binding nucleobase to a conductor, and a liquid composition containing a test sample, and
The nucleic acid binding redox marker is brought into contact with the polynucleobase-modified electrode,
A method for electrochemically detecting a target nucleic acid, comprising detecting transfer of electrons to the polynucleobase-modified electrode by a redox reaction of the nucleic acid-binding redox marker on the polynucleobase-modified electrode.   The method for electrochemically detecting a target nucleic acid according to claim 1, wherein the polynucleobase probe comprises PNA or MNA.   3. The method for electrochemically detecting a target nucleic acid according to claim 1, wherein the nucleic acid binding redox marker is methylene blue, a viologen analog, a ruthenium complex or a cobalt complex.   The method for electrochemically detecting a target nucleic acid according to any one of claims 1 to 3, wherein the conductor is a gold electrode.   The method for electrochemically detecting a target nucleic acid according to any one of claims 1 to 4, wherein a nucleic acid non-binding substance is bound to a polynucleobase probe non-binding region of the polynucleobase-modified electrode.   The method for electrochemically detecting a target nucleic acid according to claim 5, wherein the nucleic acid non-binding substance is HHT.   The method for electrochemically detecting a target nucleic acid according to any one of claims 1 to 6, wherein the target nucleic acid is DNA or RNA.   The method for electrochemically detecting a target nucleic acid according to claim 7, wherein the target nucleic acid is a miRNA.   The method for electrochemically detecting a target nucleic acid according to claim 8, wherein the measurement of the redox reaction of the nucleic acid-binding redox marker is performed using a potentiostat or a galvanostat.   The method for electrochemically detecting a target nucleic acid according to claim 9, wherein the measurement of the redox reaction of the nucleic acid-binding redox marker is performed by a voltammetry method (CV or SWV) or an alternating current impedance method (EIS). A method for electrochemical quantification of a target nucleic acid in a test sample, comprising:
A nucleic acid-binding redox marker, a polynucleobase-modified electrode formed by bonding a polynucleobase probe comprising a low-binding nucleobase to a conductor, and a liquid composition containing a test sample, and
The nucleic acid binding redox marker is brought into contact with the polynucleobase-modified electrode,
Measuring the level of electron transfer to the polynucleobase-modified electrode by a redox reaction of the nucleic acid binding redox marker on the polynucleobase-modified electrode,
A method for electrochemically quantifying a target nucleic acid, comprising calculating a level of the target nucleic acid in the test sample from a level of electron transfer to the polynucleobase-modified electrode detected.