JP2020058244A - Electrochemical unlabeled nucleic acid detection method - Google Patents

Abstract

To provide methods that can suppress electrostatic repulsion between nucleic acids and secure the Debye length, which is a distance from the electrode where the negative charges inherent in nucleic acids can be detected, longer than the molecular size so that the increase in charge density of double-stranded nucleic acid formation based on the negative charge of the phosphate group originally possessed by nucleic acid can be detected as a change without using nucleic acid-specific labeling reagents in electrochemical formulas.SOLUTION: By using a low-concentration organic polyvalent cation molecule aqueous solution of biological origin instead of a high-concentration sodium chloride aqueous solution in order to suppress electrostatic repulsion between nucleic acids and to suppress ionic strength, and by fixing the nucleic acid for capture at a distance of about 1 nm from the electrode in order to bring the double-stranded nucleic acid forming region closer to the electrode, negative charges of nucleic acids can be measured as changes in charge density without being blocked by aqueous solution ions.SELECTED DRAWING: Figure 4

Description

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

  DNA and RNA, which are called nucleic acids, are used for genetic diagnosis and identification of bacterial flora because they contain genetic information of the living body. Recently, RNA (micro RNA) present in serum, urine, and saliva has been shown to be usable as an index for cancer diagnosis, and the practical application of nucleic acid detection technology as a sensor that can be used at home for healthcare applications. Is desired.

  Up to now, the technique for detecting nucleic acids has been to mix a capture nucleic acid that interacts with a target nucleic acid to be detected in a solution, or to allow the target nucleic acid to act on a substrate on which the capture nucleic acid is immobilized, thereby forming an interacting double-stranded nucleic acid. A method has been used in which a fluorescent labeling substance that acts specifically or a fluorescent substance that is labeled in advance with a nucleic acid and whose fluorescence changes with interaction is detected using an optical device. It is unsuitable as a sensor for home use because it has a large detection sensitivity, but requires a large, expensive, and precise optical device.

  In addition, sensor development of a principle of detecting an electron mediator labeling substance that specifically acts on a double-stranded nucleic acid in which a target nucleic acid and a capture nucleic acid interact with each other has been reported (see Patent Documents 1 and 2). However, while the electrochemical method is advantageous for downsizing the device, it has a problem that a nucleic acid-specific reagent and a label are still necessary. Fluorescent and electron mediator reagents and labeling substances that act specifically on double-stranded nucleic acids cannot be denied potential carcinogenic potential, and safety is a problem when used at home, including handling and disposal. Becomes

  As a technique for detecting nucleic acids without labeling, there are methods of measuring impedance (Patent Documents 3 and 4) and measuring transfer characteristic change of FET (Non-Patent Document 1). However, in order to measure the impedance and the change in the transfer characteristic of the FET, a large device capable of controlling the current and the voltage at the same time and measuring with high accuracy is required, and it is difficult to make a small device for home use. .

  The reason why a nucleic acid sensor that can be used at home has not been realized so far is because of the above problems.

  Nucleic acid has a negatively charged ion in the phosphate group, and if the negative charge can be measured electrochemically, the nucleic acid can be detected without using a labeling substance. However, when the target nucleic acid interacts with the capture nucleic acid on the sensor substrate and is detected as a double-stranded nucleic acid, it is generally used to suppress the electrostatic repulsion between the nucleic acids due to the negatively charged ions of the phosphate groups of the nucleic acids and to bind them. Specifically, it is necessary to carry out the treatment in the presence of sodium chloride (NaCl) at a concentration of 100 to 200 mM, which is similar to that of physiological saline.

When measuring the change in charge density at the electrode solution interface in an ionic solution in electrochemistry, the ions in the solution move to create a Debye shield that shields the electric field, which is the distance from the electrode where the shield is effective. The length depends on the ionic strength in the solution. The Debye length is expressed by Equation 1. δ is the Debye length, ε is the relative permittivity, ε ₀ is the permittivity of vacuum, k is the Boltzmann constant, T is the absolute temperature, q is the charge, and I is the ionic strength.

  Regarding the distance from the sensor substrate, the biosensor generally uses avidin-biotin binding method, which is a method of immobilizing biomolecules on the substrate, and biotinylated 10 bases as a capture nucleic acid via avidin protein 2 on the surface of sensor substrate 1. FIG. 1 shows an approximate relationship between the size of each molecule and the Debye length δ when the nucleic acid 3 is immobilized and the 10-base nucleic acid 4 is allowed to act as the target nucleic acid.

  The avidin protein is an ellipsoidal sphere having a size of about 6 nm × 3 nm, and the length per 10 bases of the double-stranded nucleic acid is 3.4 nm. The measurement needs to be detected within the range of 6.4 nm or 9.4 nm from the sensor substrate. On the other hand, the Debye length δ in a sodium chloride (NaCl) solution having a concentration of 100 to 200 mM is calculated from Equation 1 to be 0.7 nm to 1 nm. The interaction of the two nucleic acids occurs outside the Debye shield, and the increase in negatively charged ions of the phosphate groups of the nucleic acid due to duplex formation cannot be detected as an increase in charge density. Even if the 10-base nucleic acid 5 as the capture nucleic acid can be directly immobilized on the sensor substrate, more than two-thirds of the nucleic acid cannot be accurately measured because it goes out of the Debye shield.

JP, 2000-125865, A Japanese Patent No. 4018672 International Publication No. 2003/024954 International Publication No. 2018/075085

"Advanced Technology and Applications of Biosensors", pp. 213-226, CMC Publishing

  As described above, when double-stranded nucleic acid formation is detected as a method for detecting nucleic acids, it is necessary to interact in a high-concentration sodium chloride (NaCl) solution in order to reduce electrostatic repulsion due to the negative charge between nucleic acids. However, the distance from the electrode that can detect the charge electrochemically and the Debye length become shorter than the molecular size, and the nucleic acid can be detected as a change in the charge density based on the negative charge of the phosphate group inherent in the nucleic acid. There is a trade-off problem that cannot be done.

  An object of the present invention is to provide a method capable of securing a detectable Debye length of a nucleic acid while suppressing electrostatic repulsion between the nucleic acids to form a double-stranded nucleic acid, thereby using a nucleic acid-specific labeling reagent in an electrochemical formula. It is to make it possible to detect nucleic acid by general-purpose potentiometric measurement without any need.

  The measuring method for detecting the double-strand formation of a nucleic acid in the present invention is a method for measuring a nucleic acid as a change in charge density using a negatively charged ion of a phosphate group which a nucleic acid originally has, without using a labeling reagent, An organic polyvalent cation molecule derived from a living body, such as spermidine, was used in place of sodium chloride to suppress electrostatic repulsion between nucleic acids, and the captured nucleic acid was separated from the electrode by a distance of about 1 nm in order to shorten the distance from the electrode. It was fixed directly in.

  The present invention uses an organic polyvalent cation molecule derived from a living body to act on a phosphate anion of a nucleic acid by a multipoint interaction even with a low concentration of positively charged ions of about 1 mM, so that the phosphate anion of the nucleic acid is electrostatically charged. The target nucleic acid was immobilized by directly fixing the capture nucleic acid at a distance of about 1 nM from the electrode while suppressing the repulsion and ensuring the Debye length to the same level as the molecular size by reducing the ionic strength in the solution. It has become possible to measure the binding of the above as a change in charge density on the electrode based on the negatively charged ions originally present in the nucleic acid with a general-purpose potentiometer without using a labeling reagent.

  Further, since the reagent used is highly safe, it can be used not only in the field of research and development but also as a nucleic acid sensor for daily diagnosis in a hospital or a general home.

The figure which shows the relationship between the size of each molecule | numerator and Debye length (delta) at the time of fixing DNA to an electrode using an avidin-biotin binding method and observing the bond of the DNA which interacts with it. The schematic diagram which shows the surface design of the DNA sensor of embodiment of this invention. The figure which shows the connection of the experimental apparatus used by the measuring method of this invention. The figure which compared the result observed when each of the detection of the DNA by the measuring method of this invention and the detection of the DNA which has 1 base mismatch was performed.

Hereinafter, embodiments of the present invention will be described.
FIG. 2 is a schematic diagram showing the surface design of a nucleic acid sensor in which 10-base DNA 7 having an arbitrary sequence as a capture nucleic acid is immobilized on the electrode 6. Gold is used for the surface material of the electrodes. The DNA 7 having an amino group at the 5'end is used to fix the DNA 7 to the surface of the gold electrode 6 with an alkylthiol 8 having a carboxyl group at the end of a carbon chain length of 2 via an amide bond. Gold and alkyl thiol are fixed by gold-thiol adsorption. The amide bond between the alkylthiol and the capture DNA was generated by an amine coupling reaction using a water-soluble condensing agent. The alkyl thiol 9 having a hydroxyl group is fixed to the end of the carbon chain length 2 in the gap of the gold electrode on which the DNA 7 is not fixed. The distance from the electrode surface to the end of the DNA is about 4.4 nm.

  FIG. 3 shows a schematic diagram of an experimental apparatus for performing electrochemical measurement. A general-purpose potentiometer 10 is connected to a sensor electrode 11 and a glass reference electrode 12 on which capture DNA is fixed. The sensor electrode 11 and the glass reference electrode 12 are immersed in a beaker 13 filled with the aqueous solution, and the aqueous solution is stirred by a stirrer 14. The experiment for detecting the target DNA as an aqueous solution is performed in a solution containing 200 μM of spermidine as an organic polyvalent cation molecule instead of sodium chloride.

  Changes in the charge density due to the interaction between the capture DNA and the target DNA whose sequence is complementary or the mismatch DNA in which only one base is not complementary are measured by a potentiometer as changes in the potential difference from the reference electrode. In comparing the respective measurement results, the difference in the sequence is detected from the difference in the binding behavior resulting from the mismatch of one base in the nucleic acid.

[Example 1]
Examples of the method for detecting unlabeled nucleic acid using the assay method of the present invention will be shown below.
As the nucleic acid, DNA of 10 bases having the following sequence (manufactured by Eurofin Genomics) was used.
Capture _{DNA NH 2 -5'-AGCTTGGGAA-3} '
Target DNA 3'-TCGAACCTT-5 '
Mismatch DNA 3'-TCGA C CCCTT-5 '

  For cleaning the surface of the gold electrode, a Piranha solution (sulfuric acid: hydrogen peroxide solution = 3: 1) was dropped and left for 5 minutes, and then rinsed with ultrapure water. Further, this operation was repeated twice. A mixed aqueous solution containing 4 mM 3,3'-Dithiodipropionic acid and 40 mM Bis (2-hydroxyethyl) disulfide was prepared, and the mixed aqueous solution was added dropwise to the washed gold electrode and left at room temperature for 30 minutes. Then, the gold electrode was washed with ultrapure water. 0.52M (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide aqueous solution and 0.87M N-hydroxysuccinimide aqueous solution were mixed in equal amounts, and the mixed solution was added dropwise to the gold surface for 30 minutes at room temperature. After the reaction, the reaction mixture was washed with ultrapure water, 10 mM HEPES-NaOH (pH 8.0) buffer containing 1 μM of captured DNA was added dropwise, and an amine coupling reaction was performed for 60 minutes. After that, an equal amount of 10 mM HEPES-NaOH (pH 8.0) buffer containing 5 mM ethanolamine was added and left for 10 minutes, after washing the sensor surface with ultrapure water, a reference electrode (manufactured by BAS, RE-1B). And a potentiometer (34405A manufactured by Agilent) were set together with 5 mL of an aqueous solution containing 200 μM spermidine in a beaker. The temperature of the solution was adjusted to 20 ° C., and the target DNA was added to the measurement beaker so that the final concentration (fc) was 50 nM After the measurement, the gold electrode was washed with ultrapure water and the beaker 13 was used again. Was replaced with an aqueous solution containing 200 μM spermidine, and the mismatch DNA was added.

The measurement results are shown in FIG.
When the target DNA was added, a decrease 15 in the electrode potential due to an increase in negative charge density due to binding was observed. On the other hand, it was observed that the electrode potential 16 was not changed by the addition of mismatched DNA. Therefore, it is possible to measure the state of binding the nucleic acid to be detected to the captured DNA over time with a general-purpose potentiometer without the use of a labeling substance, and it will be a nucleic acid sensor that can identify nucleic acids that differ only in one base sequence. Was done.
As described above, the nucleic acid sensor that can detect nucleic acid electrochemically unlabeled is a general-purpose, inexpensive and simple potentiometric system that does not use a potentially carcinogenic labeling substance. Since a difference in base mismatch can be detected, a safe, small-sized and inexpensive nucleic acid sensor can be realized and can be used as a method of a nucleic acid sensor not only for use in a dedicated facility but also for home healthcare applications.

1 Sensor Substrate 2 Avidin Protein 3 10-base Biotinylated Nucleic Acid for Capture 4 Target 10-base Nucleic Acid 5 10-base Nucleic Acid for Capture 6 Gold Electrode 7 10-base Nucleic Acid 8 Alkylthiol with a carbon chain length of 2 that is bound to DNA by amine coupling 9 End Is an alkylthiol having a carbon chain length of 2 hydroxyl group 10 potentiometer 11 sensor electrode 12 glass reference electrode 13 beaker 14 stirrer 15 potential difference response due to addition of target DNA to capture DNA 16 potential difference due to addition of one base mismatch DNA to capture DNA response

Claims (5)

  A method for detecting nucleic acid using an organic polyvalent cation molecule having a size smaller than that of the nucleic acid to be detected in order to suppress electrostatic repulsion of the phosphate anion of the nucleic acid.   The method for detecting nucleic acid according to claim 1, which is based on forming a double-stranded nucleic acid with a nucleic acid whose nucleotide sequence is known.   The method for detecting nucleic acid according to claim 1 or 2, wherein the nucleic acid having a known base sequence is immobilized at the end at a distance of 1 nm or less from the sensor surface.   The method for detecting nucleic acid according to claim 3, wherein a portion where the nucleic acid is not fixed is covered with a hydroxyl group.   The method for detecting nucleic acid according to claim 1, wherein a potentiometer is used.