JP3882038B2 - A highly sensitive method for measuring the amount of double-stranded DNA - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrochemical measurement method and a measurement kit for measuring the total amount of double-stranded DNA contained in a sample with high sensitivity and ease.
[0002]
[Prior art]
Conventionally, the target of DNA measurement is based on the determination of the presence or absence of DNA having a specific base sequence, which is too large for technological development to enable simple and highly sensitive measurement of the total amount of DNA in a sample. Effort has not been paid.
On the other hand, in the method for quantifying the target component in the sample, there is an electrochemical method as a simple and optimum method for achieving high sensitivity, and this method can cope with changes in the concentration of the target component or target component in the solution. Thus, the specific component whose concentration changes is converted and output as a current signal on the electrode.
[0003]
Of the bases that make up DNA, guanine is known to be susceptible to electrolytic oxidation, and an electrochemical method using this property has been tried, but it is still 1.3 for the oxidation of guanine in double-stranded DNA. When a high potential with V (to silver-silver chloride electrode) is required (Non-patent Document 1), when such a high potential is applied, many of the oxidizing substances coexisting in the sample are oxidized, resulting in a high back-up. Since it gave a ground, high-sensitivity measurement was accompanied by difficulties. That is, such a measurement method based on direct electrolytic oxidation of DNA has not been practical.
[0004]
On the other hand, among the intercalators that can be inserted into the base pair binding region of double-stranded DNA, the presence of substances that cause redox under relatively mild conditions, such as methylene blue, is known and is also difficult to redox It is known that an intercalator can be easily detected electrochemically by binding a redox functional molecule such as ferrocene to the intercalator. However, the analytical use of the electrochemical reaction of these intercalators is based on the fact that single-stranded DNA oligomers are bound on the electrode, and the single-stranded DNA having a complementary base sequence existing in the sample solution. A DNA having a specific base sequence is measured by measuring the oxidation or reduction current of the intercalator inserted into this duplex, that is, the intercalator immobilized in the vicinity of the electrode. It was limited to the purpose of determining the presence or absence.
[0005]
[Non-Patent Document 1]
B. Meric et al, Electroanalysis, vo. 14, no. 18, p. 1245 (2002)
[0006]
[Problems to be solved by the invention]
Under such circumstances, the object of the present invention is to measure and measure the amount of double-stranded DNA in a sample simply and with high sensitivity, and more specifically, by intercalator DNA insertion. In addition to causing changes in electrochemical properties, a new system has been constructed that can detect and amplify these property changes without being affected by contaminants. It is intended to measure the amount easily and with high sensitivity.
[0007]
[Means for Solving the Problems]
The inventors of the present invention have made various studies on a method that can be measured by using an intercalator having a reversible oxidation-reduction ability or a derivative thereof without being greatly affected by an oxidizing or reducing substance in a sample. As a result, we noticed that the magnitude of the electrolysis current on the electrode changed significantly before and after the intercalator was inserted into the double-stranded DNA. When measuring this electrolysis current change, By coupling electrolytic reduction or electrolytic oxidation with oxidation or reduction by enzymatic reaction, it is possible to amplify and measure the change in the electrochemical activity of the intercalator, and thus the amount of double-stranded DNA that causes the change. Has been found to be capable of being measured with high sensitivity, and the present invention has been completed.
[0008]
That is, the present invention relates to the following (1) to (4).
(1) In the method for measuring the amount of double-stranded DNA contained in a sample, the concentration of unintercalated intercalator is obtained by intercalating an intercalator having reversible redox ability into double-stranded DNA in the sample. change, and amplified on the working electrode fixed oxidoreductase and measuring, double-stranded DNA of measurement methods.
(2) An enzyme electrode formed by immobilizing an oxidoreductase on the electrode is used as a working electrode, and an intercalator having reversible redox ability without insertion of double-stranded DNA is oxidized or reduced by the oxidoreductase at the working electrode. A current cycle generated at the electrode is amplified and measured by forming a reaction cycle comprising a reaction that comprises a reaction to reduce and oxidize and regenerate the intercalator by an electrode reaction (1) ) For measuring the amount of double-stranded DNA.
(3) The measuring method according to (1) or (2) above, wherein the intercalator having reversible redox ability is a polycyclic aromatic compound.
(4) The measuring method according to (3) above, wherein the polycyclic aromatic compound is methylene blue.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The measurement system for measuring the amount of double-stranded DNA in the present invention comprises a working electrode on which an oxidoreductase is immobilized, and an intercalator having a reversible redox ability as a measurement reagent.
Here, the intercalator refers to a substance that intercalates. That is, since a DNA molecule has a double helix structure having a complementary base pair, a molecule having a planar structure (polycyclic aromatic compound) is inserted between the base pair stacking. This is called intercalation, and this intercalating substance is called intercalator.
[0010]
The measurement principle of the double-stranded DNA of the present invention will be described below.
When a solution containing a certain amount of intercalator is added to a sample containing double-stranded DNA or when the sample is added to a solution containing a certain amount of intercalator, depending on the amount of double-stranded DNA in the sample, A calator is inserted into the DNA. The intercalator inserted into this DNA and the non-inserted intercalator have different electrochemical characteristics and can be distinguished.
Therefore, by measuring the amount of uninserted intercalator, it becomes possible to know the amount of DNA in the sample corresponding to the amount of intercalator inserted.
In order to measure the amount of the uninserted intercalator with higher sensitivity, the present invention uses an intercalator having a reversible redox ability as an intercalator and uses an electrode on which an oxidoreductase is immobilized as an electrode. . The intercalator having the reversible redox ability is an intercalator that can be oxidized or reduced, in other words, can reversibly take the oxidized and reduced states by the exchange of electrons. Basically, the amount of intercalator that has not been inserted is measured by measuring the current flowing through the electrodes that is generated by the exchange of electrons, and the amount of double-stranded DNA is grasped. This measurement current is amplified and is characterized in that it can be measured with high sensitivity.
[0011]
That is, among the intercalators added to the sample solution, the non-inserted intercalator that does not intercalate with the double-stranded DNA is oxidized or reduced on the electrode to which a constant potential is applied, and shifts to the oxidized or reduced form. The current flowing through the electrode changes due to the exchange of electrons accompanying the electrode reaction. Furthermore, the intercalator that has shifted to the oxidized or reduced form by the electrode reaction is regenerated again to its original state (reduced or oxidized form) by the oxidoreductase immobilized on the electrode. The oxidation, reduction, and regeneration reaction through the intercalator form a reaction cycle, and the current flowing through the electrode is amplified by repeating the reaction cycle. Therefore, even if the amount of uninserted intercalator is very small, the measurement current is amplified, and this makes it possible to measure the amount of double-stranded DNA with high sensitivity.
According to the measurement principle of the present invention, the intercalator or oxidoreductase may be any substance as long as it can form the reaction cycle as described above, and is limited to a specific substance or a specific oxidoreductase. It should be understood that it should not.
[0012]
The material of the electrode used in the present invention is not particularly limited, but a noble metal such as platinum or gold or a carbon-based material is advantageously used from the viewpoint of corrosion resistance.
As the intercalator used in the present invention, a compound having both an insertion function into double-stranded DNA and a reversible redox ability, such as methylene blue, is used, but not limited thereto, ethidium bromide, benzopyrene, etc. Even a substance that has an intercalator function but does not have reversible redox ability can be used by introducing a group having reversible redox ability such as ferrocene or quinone as a side chain of the molecule. .
[0013]
The oxidoreductase is not particularly limited as long as the reaction proceeds smoothly using the above intercalator or its derivative as an electron acceptor or donor, but a stable and inexpensive one is advantageously used. Specifically, when methylene blue is used as an intercalator, a peroxidase using this as an electron donor for hydrogen peroxide reduction is used. In an intercalator having ferrocene or quinone as a side chain, this is used during substrate oxidation. An oxidase used as an electron acceptor, for example, glucose oxidase, lactate oxidase, glutamate oxidase, choline oxidase and the like is conveniently used.
The method for immobilizing the enzyme on the electrode is not particularly limited, but a carrier material and an immobilization method that can maintain the enzyme highly active and do not inhibit the permeation of the substrate and the intercalator are selected. Used. For example, methods that are known to have good permeability, such as a method of binding an enzyme to a porous carrier formed on the electrode surface, a method of forming an enzyme-fixed cross-linked membrane with albumin and glutaraldehyde on the electrode surface, Select used.
[0014]
Next, an example of an embodiment of the present invention will be described in detail. First, a predetermined pretreatment such as polishing is performed on an electrode serving as a substrate, and then an oxidoreductase immobilization film is provided on the electrode. The enzyme electrode thus prepared is used as a working electrode and is inserted into an aqueous electrolyte solution containing an intercalator in combination with an appropriate counter electrode and reference electrode. The concentration of the intercalator is not particularly limited, but a concentration of about 0.1 to 10 μmol / liter is convenient as a concentration that can provide a clear current response by amplification measurement using an enzyme electrode. The concentration and type of the electrolyte solution to be used are not particularly limited, but a neutral pH buffer solution is preferably used in order to keep the enzyme activity stable and to keep the redox potential of the intercalator constant, for example, around pH 7 A phosphate buffer solution having a concentration of around 0.1 mol / liter is conveniently used. When a potential necessary for the oxidation or reduction of the intercalator is applied to the enzyme electrode, a current accompanying the oxidation or reduction flows, but this current value is small because the intercalator concentration is relatively low.
[0015]
Next, when a substrate is added to the solution, a cyclic reaction of reduction or oxidation of the intercalator as an electron acceptor or donor, reoxidation on the electrode, or re-reduction proceeds with substrate oxidation or reduction by an enzymatic reaction. The electrolysis current becomes extremely large and reaches a level at which accurate measurement is easy.
[0016]
For example, when methylene blue is used as the intercalator and peroxidase is used as the enzyme, the following reaction occurs on the electrode when a potential of -0.15 V (vs. silver-silver chloride electrode) is applied at pH 6.
(A) Methylene blue oxidized type + 2e → methylene blue reduced type Further, when hydrogen peroxide as an enzyme substrate is added thereto, the following enzyme reaction occurs.
(B) Methylene blue reduced form + hydrogen peroxide → methylene blue oxidized form + water Therefore, the methylene blue oxidized form is regenerated by this enzyme reaction, and the cycle of electrode reaction and enzyme reaction is repeated. Due to this cyclic reaction, an increase in current of about 10 to 1000 times is observed with addition of hydrogen peroxide compared to before addition of hydrogen peroxide. In this reaction cycle,
Methylene blue repeats oxidation and reduction, but the total amount of oxidation and reduction does not change, and methylene blue also functions as a mediator. Further, the amount of 2e in the above reaction formula, that is, the current at the electrode depends on the amount of methylene blue involved in this reaction if the amount of hydrogen peroxide to be added is excessive.
[0017]
On the other hand, in the double-stranded DNA in the sample, a part of the intercalator is inserted into the binding region of the base pair of the double-stranded DNA. Since this intercalator inserted with double-stranded DNA is in a state of binding to high molecular DNA, the diffusion rate in the solution is remarkably reduced, that is, it becomes difficult to reach the vicinity of the electrode from the solution bulk. However, even if it reaches the electrode surface, it is blocked by the DNA strand and the speed of electron transfer with the electrode decreases. Therefore, an intercalator inserted into double-stranded DNA effectively gives no reduction or oxidation current.
[0018]
That is, only the uninserted intercalator repeats the cycle of the electrode reaction and the enzyme reaction, and the current at this time is almost proportional to the concentration of the uninserted intercalator. Therefore, the current decreases as the concentration of double-stranded DNA in the sample increases, that is, the concentration of inserted intercalator increases and the concentration of uninserted decreases. In other words, the amount of added double-stranded DNA can be determined from the degree of decrease in current.
In addition, the double-stranded DNA measurement kit of the present invention includes at least an oxidoreductase-immobilized electrode and an intercalator having reversible redox ability. Furthermore, a substrate for the enzyme immobilized on the electrode, a pH adjuster, a current measuring device, or the like, such as members or reagents for constructing the measurement system of the present invention, may be combined to form a measurement kit.
[0019]
The measurement method and measurement kit of the present invention are used in fields related to separation, extraction, analysis, use, and the like of biological components. For example, in the manufacture of vaccines, it is necessary to remove components that may cause harmful effects when inoculated into living organisms from the liquid obtained by inoculating cells with viruses. Considering the danger of foreign DNA on living organisms, it is necessary to remove this, and in order to determine whether the removal has been performed as intended, a method for simply measuring a small amount of DNA is necessary. is there. In addition, even in genetically modified drugs, a small amount of foreign DNA derived from the host may be mixed. Therefore, in the present invention, it is extremely effective in measuring these DNAs.
[0020]
Next, the present invention will be described in more detail with reference to examples.
【Example】
(Measurement of DNA with peroxidase-immobilized enzyme electrode)
Peroxidase 0.5% (w / v) aqueous solution (pH adjusted to 7) 0.5 microliters, bovine serum albumin 0.5% (w / v) aqueous solution (pH adjusted to 7) 1 microliters on 1mm diameter glassy carbon electrode A peroxidase-immobilized electrode was prepared by adding 0.5 microliters of a 0.1% (w / v) aqueous solution of glutaraldehyde (pH adjusted to 7) dropwise and allowing to stand at room temperature for 2 hours or more and drying. This enzyme electrode was inserted into a 0.1 M phosphate buffer solution at pH 6.1, and a potential of -0.15 V (vs. silver-silver chloride electrode) was applied. Hydrogen peroxide (2 mM), a peroxidase substrate, and methylene blue (5 micromol / l), which functions as a mediator and intercalator, are added, and salmon sperm is derived every 5 minutes at 5 microg / ml. Of DNA was added. The results are shown in FIG. In FIG. 1, the horizontal axis represents time, the vertical axis represents the electrolysis current value, the solid line represents the measurement value of the reduction current, and the arrows represent the time points at which 5 microg / ml DNA was added, respectively. As is clear from FIG. 1, a 1.1 microampere hydrogen peroxide reduction current was observed at the time when salmon sperm-derived DNA was not added, but 5 μg / ml of salmon sperm-derived DNA was added thereto. Every time, a clear reduction of the reduction current was observed. According to this result, the current decrease when 5 μg / ml of DNA was initially added was 55 nA, and when S / N = 3, it was estimated that 20 pg / ml of DNA could be quantified. It was lost.
[0021]
[Comparative example]
(Measurement of DNA on electrodes without immobilized enzyme)
A glassy carbon electrode with a diameter of 1 mm, to which no enzyme was immobilized, was inserted into a 0.1 M phosphate buffer solution at pH 6.1, and a potential of -0.15 V (vs. silver-silver chloride electrode) was applied. When methylene blue (5 micromol / l) was added here, as shown by the broken line in FIG. 1, a reduction current of only 30 nA and about 1/40 of that in the case of the peroxidase-immobilized electrode was given. When salmon sperm-derived DNA is added in increments of 5 microg / ml, the reduction current reduction cannot be displayed on the scale shown in FIG. In fact, when 5 microg / ml of DNA is initially added, the current decrease is only 1 nA, and if S / N = 3, 1 microg / ml of DNA can still be quantified. It was.
[0022]
【The invention's effect】
As is apparent from the above, the present invention is to measure the amount and concentration of double-stranded DNA in a sample using an enzyme-immobilized electrode and an intercalator having reversible redox ability. In this method, the decrease in the non-inserted intercalator based on the intercalation with respect to the double-stranded DNA in the sample is measured as a decrease in current through the electrode. In particular, by using an enzyme-immobilized electrode, Compared to the case where the change in concentration is directly measured by an electrode, it is possible to amplify the side constant current and to capture it, and as a result, provide a method for measuring DNA with high sensitivity.
[0023]
That is, the intercalator of the present invention is oxidized or reduced on the electrode, but is regenerated by an enzyme reaction, and the electrode reaction and the enzyme reaction are repeated. For this reason, an intercalator of one molecule moves many times and gives a large current signal. This gives an amplified signal as compared to the case of only an electrochemical reaction system that does not involve an enzyme reaction. Such signal amplification is possible only by providing an enzyme membrane on the electrode. Therefore, this method is useful as a simple and highly sensitive DNA measurement method.
[Brief description of the drawings]
FIG. 1 is a graph showing the measurement of each electrolysis current in the presence of hydrogen peroxide and methylene blue when using a peroxidase-immobilized electrode and when using an electrode without immobilized enzyme.

Claims (4)

In a method for measuring the amount of double-stranded DNA contained in a sample, an intercalator having a reversible redox ability is intercalated with a double-stranded DNA in the sample, and the concentration change of the uninserted intercalator is changed. and amplified on the working electrode fixed oxidoreductase and measuring, double-stranded DNA of measurement methods. An enzyme electrode formed by immobilizing an oxidoreductase on the electrode is used as a working electrode, and at the working electrode, an intercalator having a reversible redox ability without insertion of double-stranded DNA is oxidized or reduced by a oxidoreductase. 2. The current signal generated at the electrode is amplified and measured by forming a reaction cycle comprising a reaction of reducing or oxidizing and regenerating the intercalator by an electrode reaction. A method for measuring the amount of double-stranded DNA. The measuring method according to claim 1 or 2, wherein the intercalator having reversible redox ability is a polycyclic aromatic compound. The measurement method according to claim 3, wherein the polycyclic aromatic compound is methylene blue.

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