Second mediator selection method, detection method, and detection system

Abstract

Provided is a second mediator selection method for selecting a second mediator to be used in a method for detecting a target substance present in a solution by means of an electrochemical measurement method, the method making it possible to detect the target substance without inhibiting a reaction for detection.　The second mediator selection method is for selecting a second mediator to be used in a method for detecting a target substance by measuring an electrochemical characteristic of a solution by means of an electrochemical measurement method, the solution containing at least a target substance not fixed to an electrode, a main mediator, and the second mediator, wherein the main mediator performs charge transfer with a working electrode, and the second mediator varies the charge transfer. The selection method includes (i) a step for selecting compounds that interact with the target substance, and (ii) a step for selecting a compound that satisfies a specific charge transfer resistance ratio in the solution.

Description

Method for selecting, detecting, and detecting system for second mediator

[0001] The present invention relates to a method for selecting a second mediator used in a method for detecting a target substance present in a solution by electrochemical measurement, and a detection method and detection system using the second mediator.

[0002] Field-effect transistors are widely used in biosensors, but they suffer from insufficient sensitivity under physiological conditions due to their short Debye length. Electrochemical measurements, on the other hand, are suitable for biosensing because they allow measurements even under physiological conditions. Electrochemical measurements typically involve adding a few mM of ferricyanide ions or ferrocyanide ions to the test solution, placing a receptor on the electrode surface, and measuring the current and voltage. This method requires the receptor to be immobilized on the sensor surface in advance, and the receptor-immobilized electrode is dedicated to a specific target for that receptor, requiring separate electrodes for each target.

[0003] The present inventors have reported that methylene blue or [Ru(bpy)2DPPZ] is a soluble ion of ferricyanide ion and ferrocyanide ion in a solution containing the ion in an impedance measurement using a carbon electrode. 2+ They discovered that the resistance value can be reduced to 1 / 10 or less by adding a metal complex such as HCl, and found that the material can be used for detecting double-stranded DNA, particularly for quantitative polymerase chain reaction (qPCR) (Patent Document 1).

[0004] International Publication No. 2019 / 026517

[0005] However, according to the investigations of the present inventors, [Ru(bpy)2DPPZ] described in Patent Document 1 2+It has been found that there is room for improvement in the selection of conditions for detecting a target substance, since Patent Document 1 does not disclose guidelines for selecting the type or concentration of impedance-varying substance B in solution, for example, when using , the ligand DPPZ inhibits PCR, and when using methylene blue, the interaction with the double-stranded DNA of the target substance is weak, making qPCR detection difficult. Therefore, an object of the present invention is to provide a method for selecting a second mediator to be used in a method for detecting a target substance present in a solution by electrochemical measurement, and a detection method and detection system that use the second mediator.

[0006] In order to solve the above problems, the present invention provides the following method for selecting a second mediator, and a detection method and detection system using the second mediator. [1] A method for selecting a second mediator used in a method for detecting a target substance by measuring, by electrochemical measurement, the electrochemical properties of a solution containing at least a target substance not immobilized on an electrode, a main mediator, and a second mediator, wherein the main mediator transfers charge between itself and a working electrode, and the second mediator varies the charge transfer, the method comprising the steps of: (i) selecting a compound that interacts with the target substance; and (ii) determining the charge transfer resistance R of a solution containing the main mediator at a unique concentration C1. ct0 The charge transfer resistance R of the solution containing the primary mediator at a concentration C1 and the secondary mediator at a concentration C2 ct The ratio R ct / R ct0a step of selecting a compound that satisfies the following relationship: C2=C1 / C2 (ΔZ 1 / C2) is 0.80 or less. [2] The method for selecting a second mediator according to [1], wherein the second mediator concentration C2 is not more than one tenth of the main mediator concentration C1. [3] The method for selecting a second mediator according to [1], wherein the second mediator concentration C2 is not more than one hundredth of the main mediator concentration C1. [4] The method for selecting a second mediator according to any one of [1] to [3], wherein the target substance is a double-stranded nucleic acid. [5] The method for selecting a second mediator according to [4], wherein the method for detecting a target substance comprises one or more steps of amplifying the target substance. [6] The method for selecting a second mediator according to [4], further comprising: (iii) measuring the impedance of the solution before amplifying the target substance and at each amplification step, and determining (ΔZ max -ΔZ min ) / R ctm A step of selecting a compound whose absolute value exceeds 0.3%. Here, the charge transfer resistance R ct and the charge transfer resistance R just before the target substance amplification process ct When the difference ΔZ between the value of max is the maximum absolute value of ΔZ, and R ctm is ΔZ max The charge transfer resistance R at the cycle number m ct is the value of ΔZ min is ΔZ max [7] In the step (iii), the (ΔZ max -ΔZ min ) / R ctm [8] In the step (iii), the (ΔZ max -ΔZ min ) / R ctm[9] A method for selecting a second mediator used in a method for detecting a target substance by measuring electrochemical properties by electrochemical measurement of a solution containing at least a target substance not immobilized on an electrode, a main mediator, and a second mediator, wherein the target substance is a double-stranded nucleic acid, the method for detecting the target substance includes one or more steps of amplifying the target substance, the main mediator transfers charge between the target substance and a working electrode, and the second mediator changes the charge transfer, the method comprising: (i) a step of selecting a compound that interacts with the target substance; and (iii) measuring the impedance of the solution before amplifying the target substance and at each amplification step, and determining a compound that interacts with the target substance (ΔZ max -ΔZ min ) / R ctm A step of selecting a compound whose absolute value exceeds 0.3%. Here, the charge transfer resistance R ct and the charge transfer resistance R just before the target substance amplification process ct When the difference ΔZ between the value of max is the maximum absolute value of ΔZ, and R ctm is ΔZ max The charge transfer resistance R at the cycle number m ct is the value of ΔZ min is ΔZ max

[10] In the step (iii), the (ΔZ max -ΔZ min ) / R ctm

[11] In the step (iii), the (ΔZ max -ΔZ min ) / R ctmThe method for selecting a second mediator according to [9], wherein a compound having an absolute value of .gtoreq.1.0% is selected from the group consisting of:

[12] The method for selecting a second mediator according to any one of [4] to [9], wherein in step (i), a compound is selected that exhibits at least one interaction with the target substance selected from the group consisting of:

[13] The group consisting of: electrostatic interaction, π-π interaction, intercalation between base pairs of a double-stranded nucleic acid that is the target substance, hydrophobic-π interaction, ion-dipole interaction, stem-loop interaction, conformational interaction, coordinate bond, alkyl-π interaction, intramolecular hydrogen bond, solvation effect, intermolecular water (water-water) interaction, magnetic interaction, van der Waals force (London dispersion force), π-anion interaction, molecular dipole-dipole interaction, induced dipole interaction, allosteric interaction, and covalent bond network. 3- and ferrocyanide ion [Fe(CN)6] 4-

[14] The method for selecting a second mediator according to any one of [1] to

[12] , wherein in step (i), a compound having in one molecule at least a structure represented by the following formula (I) is selected as the compound that interacts with the target substance: (The ring having A in the above formula (I) is a 5- or 6-membered ring fused with an adjacent benzene ring and is a heterocycle having at least one atom selected from a nitrogen atom, an oxygen atom, and a sulfur atom.)

[15] The method for selecting a second mediator according to

[14] , wherein in formula (I), the 6-membered ring (benzene ring) adjacent to the heterocycle having A has a nitrogen atom, an oxygen atom, or a sulfur atom.

[16] The method for selecting a second mediator according to

[14] , wherein formula (I) is any of the following formulas: (In the above formula, A is a nitrogen atom, an oxygen atom, or a sulfur atom.)

[17] The method for selecting a second mediator according to any one of [4] to

[16] , further comprising: (iv) a step of performing sequence-specific double-stranded nucleic acid amplification using a solution containing the target substance, and selecting a second mediator concentration based on the relationship between the second mediator concentration and an amplification rate.

[18] The method for selecting a second mediator according to

[17] , wherein in the step (iv), a second mediator concentration that results in an amplification rate of 1.4 or more is selected.

[19] The method for selecting a second mediator according to any one of [1] to

[18] , wherein the electrochemical measurement method is cyclic voltammetry, chronoamperometry, coulometry, chronopotentiometry, pulse voltammetry, or conductometry.

[20] The electrochemical measurement method is an impedance measurement method, and wherein a charge transfer resistance R ct The method for calculating the charge transfer resistance R is based on EIS (electrochemical impedance spectroscopy). ct or by measuring the impedance at one frequency to calculate the charge transfer resistance R ct or by calculating the charge transfer resistance R by semicircular approximation of the Nyquist plot at two frequencies. ct or the charge transfer resistance R ct or the charge transfer resistance R is calculated by the absolute value of the difference in impedance at two frequencies. ct or by elliptical or arc approximation of the Nyquist plot at three frequencies to calculate the charge transfer resistance R ct

[21] The method for selecting a second mediator according to any one of [1] to

[18] , wherein the charge transfer resistance R is calculated by semicircular approximation of the Nyquist plot at the two frequencies. ct and a method for calculating the charge transfer resistance R by the absolute value of the difference in impedance at the two frequencies. ctand the charge transfer resistance R is calculated by the difference between the real parts of the impedance at the two frequencies. ct

[22] The method for selecting a second mediator according to

[20] , wherein the impedance measurement is performed using a superimposed waveform including frequency components of two frequencies. ct

[23] The method for selecting a second mediator according to any one of

[20] to

[22] , wherein the impedance measurement is performed using a superimposed waveform containing frequency components of three frequencies.

[24] The electrochemical measurement method is applied to at least one sequence-specific double-stranded nucleic acid detection selected from the following group: PCR, LAMP (Loop-mediated isothermal Amplification), SDA (Strand Displacement Amplification), NEAR (Nicking Endonuclease Amplification Reaction), NASBA (Nucleic Acid Sequences Based Amplification), TMA (Transcription Mediated Amplification), RPA (Recombinase Polymerase Amplification), SIBA (Strand-Invasion Based Amplification), HDA (Helicase-dependent Amplification), WGA (Whole Genome Amplification), MDA (Multiple Displacement Amplification), DNA chip, DNA microarray, NGS (Next Generation Sequencing), ChIP (Chromatin Immunoprecipitation), ChIP-on-chip, and ChIP-seq (chromatin immunoprecipitation with high-throughput sequencing), The method for selecting a second mediator according to any one of [1] to

[23] .

[25] The method for selecting a second mediator according to any one of [1] to

[24] , wherein at least the working electrode in the electrochemical measurement method is provided with a porous thin film layer through which the main mediator and the second mediator can pass.

[26] A method for detecting a target substance by measuring electrochemical properties by electrochemical measurement of a solution containing at least a target substance not immobilized on an electrode, a main mediator, and a second mediator, the method comprising one or more steps of amplifying the target substance, the main mediator performs charge transfer between the main mediator and a working electrode, and the second mediator varies the charge transfer, and the second mediator is a compound that interacts with the electrode surface.

[27] The detection method according to

[26] , wherein the second mediator is a compound having at least one structure represented by the following formula in one molecule: (In the above formula, A is a nitrogen atom, an oxygen atom, or a sulfur atom.)

[28] The detection method according to

[26] or

[27] , wherein the target substance is a double-stranded nucleic acid, and the solution in which the target substance is detected further contains a primer and a polymerase.

[29] The detection method according to any one of

[26] to

[28] , wherein the step of amplifying the target substance and the step of measuring the electrochemical property are performed in the same solution.

[30] A target substance detection system comprising a target substance detection device and a main mediator and a second mediator contained in a solution that is a measurement object that may contain the target substance, and which detects the target substance by measuring the electrochemical property of the solution by electrochemical measurement using an electrochemical measurement unit of the target substance detection device, the detection system comprising one or more steps of amplifying the target substance, wherein the main mediator transfers charge between the target substance and a working electrode, and the second mediator varies the charge transfer, and the second mediator is a compound that interacts with the electrode surface.

[31] The detection system according to

[30] , wherein the second mediator is a compound having at least one structure represented by the following formula in one molecule: (In the above formula, A is a nitrogen atom, an oxygen atom, or a sulfur atom.)

[32] The detection system according to

[30] or

[31] , wherein the target substance is a double-stranded nucleic acid, and the solution for detecting the target substance further contains a primer and a polymerase.

[33] The detection system according to any one of

[30] to

[32] , wherein the step of amplifying the target substance and the step of measuring the electrochemical property are carried out in the same solution.

[0007] In a method for detecting a target substance present in a solution using electrochemical measurement, by using a second mediator selected by the selection method of the present invention, it becomes possible to detect the target substance without inhibiting the detection reaction.

[0008] The impedance and charge transfer resistance R depend on the amount of the target substance contained in the solution. ct FIG. 1 is a diagram showing an example of a Nyquist plot showing how the change in the resistance R is caused by the change in the charge transfer resistance R. FIG. 2 is a diagram showing an example of a cyclic voltammogram, which is a response curve of current to an applied potential. FIG. 3 is a graph showing the relationship between the number of PCR cycles and the amplification rate. FIG. 4 is a diagram showing an example of a general equivalent circuit of an object to be measured. FIG. 5 is a schematic diagram showing an example of a Nyquist plot by the EIS method. ct 1 is a schematic diagram showing an example of a change in the Nyquist plot when two frequencies, high and low, are approximated to semicircles, and the diameter of the semicircles is estimated from the impedance, and the charge transfer resistance R ct When ct In the Nyquist plot, the difference between the real parts of the impedances measured at two frequencies, high and low, or the absolute value of the impedance difference (absolute value of the vector difference) is used to calculate the charge transfer resistance R estimated from the impedance. ct In the Nyquist plot, three frequencies are approximated to an ellipse, and the diameter of the ellipse in the real axis direction is estimated from the impedance, which is the charge transfer resistance R ctIn the Nyquist plot, three frequencies are approximated to arcs, and the charge transfer resistance R is estimated from the impedance by the difference between the real parts of the arcs when the imaginary part of the arc is 0Ω. ct 12A is a schematic diagram showing a typical example of the method for calculating the index in step (iii) using PCR detection of double-stranded DNA as an example. FIG. 12A is a graph showing an example of the change in the measured absolute value of impedance with respect to the number of PCR cycles, and FIG. 12B is a graph showing the "amount of change" in the absolute value of impedance for each PCR cycle based on the results of FIG. 12A. 13A-13C are diagrams showing the results of measuring the index for various second mediators in step (ii) in the examples. 13A-13C are diagrams showing the results of measuring the index for various second mediators in step (ii) in the examples. 13A-13C are diagrams showing the results of measuring the index for various second mediators in the cases where SYBR Green I, SYBR Gold, Pico Green, SYBR safe, and Eva Green were used as the second mediator (FIG. 13A), Basic Blue 3, Basic Blue 17, Basic Blue 9 (methylene blue), and [Ru(bpy)2DPPZ] were used, respectively. 2+ (Figure 13B) and Disperse Blue 14, Basic Red 5 (Toluylene Red), Basic Blue 1, Promethazine Hydrochloride, Dipyrido[3,2-a:2',3'-c]phenazine (DPPZ), Melatonin, and [Ru(bpy)3] 2+ 14A to 14D show the results when SYBR Green I (FIG. 14A), SYBR Gold (FIG. 14B), Eva Green (FIG. 14C), and Pico Green (FIG. 14D) were used as the second mediator, respectively. t15A and 15B show the results when KOD FX was used as the polymerase, and FIG. 15B shows the results when Klen Taq was used as the polymerase. It is a diagram showing the measurement results of the indicator of step (iv) in the examples. It is a diagram showing the R ct / R ct0 17A and 17B show the results of measuring the values ​​of .gtoreq.(.gtoreq..times ...

[0009] Preferred embodiments of the present invention will be described below. However, the present invention is not limited to the following description, and various modifications and alterations are possible for those skilled in the art based on the gist of the invention as described in the claims or disclosed in the detailed description. Such modifications and alterations are also within the scope of the present invention. First, an electrochemical measurement method using a second mediator selected by the second mediator selection method of the present invention will be described.

[0010] <Outline of electrochemical measurement methods for detecting target substances>

[0011] Electrochemical measurement methods applicable to the detection of target substances include methods for measuring the electrochemical properties of a solution by impedance measurement and DC polarization measurement using at least two opposing electrodes, and optionally a reference electrode. The present invention is suitable for use in the detection of double-stranded nucleic acids as target substances, and is particularly suitable for sequence-specific double-stranded nucleic acid detection under specified conditions. There are several methods for sequence-specific double-stranded nucleic acid detection, as described below, but the following description will use PCR (Polymerase Chain Reaction) detection as an example.

[0012] In the case of PCR detection by impedance measurement, after the completion of a predetermined temperature cycle, the "charge transfer resistance (R)" calculated from the impedance measurement for each temperature cycle is ct In the change amount of ", the charge transfer resistance R ct The point at which the temperature at which the double-stranded DNA begins to change rapidly can be determined as the temperature cycle at which the double-stranded DNA significantly increases. t ) is called.

[0013] Charge transfer resistance R ct When the impedance spectrum is plotted as a Nyquist plot, the charge transfer resistance R is expressed as the diameter of the semicircular plot, and can be intuitively understood from the Nyquist plot. As shown in Figure 1, when charge transfer is likely to occur, the charge transfer resistance R ct becomes small and is unlikely to occur, the charge transfer resistance R ct becomes larger.

[0014] The charge transfer resistance R ct The method for calculating the charge transfer resistance R is described in detail below. ct or by measuring the impedance at one frequency to calculate the charge transfer resistance R ct A method for calculating the charge transfer resistance R by semicircular approximation of the Nyquist plot at two frequencies ct , and the charge transfer resistance R is calculated by the difference between the real parts of the impedances at the two frequencies. ct The charge transfer resistance R is calculated by the absolute value of the difference in impedance at two frequencies. ct and the charge transfer resistance R by elliptical or arc approximation of the Nyquist plot at three frequencies. ct There are methods for calculating the

[0015] DC polarization measurements include cyclic voltammetry (CV), chronoamperometry (CA), coulometry, chronopotentiometry (CP), pulse voltammetry (PV), conductometry, and the like.

[0016] Cyclic voltammetry is a method for observing changes in current due to redox reactions occurring at the electrode surface, and can easily obtain a wide range of information, such as the electrochemical properties and diffusion coefficients of metal complexes, inorganic and organic molecules, ions, etc. In cyclic voltammetry, the potential of the working electrode is cycled back and forth within a certain range, and the response current is observed.

[0017] Figure 2 shows an example of a cyclic voltammogram, which is a response curve of current to applied potential. In cyclic voltammetry, the peak height of the current and the potential change depending on the amount of the target substance contained in the solution and the state of charge transfer on the electrode surface, so that the target substance can be detected. In other words, the point at which the peak height of the current and the potential change suddenly begins is the temperature cycle where double-stranded DNA significantly increases, C t It can be determined that:

[0018] In addition, in chronoamperometry, potential steps are applied to the electrodes, and the corresponding changes in current over time are tracked to determine the parameters involved in the reaction. In coulometry, quantitative analysis of redox species in a solution is performed from the amount of electricity consumed in electrolysis based on Faraday's law. In chronopotentiometry, a constant current is applied and the change in the potential response over time is measured. In pulse voltammetry, a reference potential at which the sample does not react is applied, and a series of potential steps with amplitudes that are multiples of the pulse value are applied and the current value is measured. In conductometry, the conductivity of the sample solution is measured to obtain physical and chemical information about the system. The point at which the change in the amount of change in these detected values ​​begins to change rapidly is the temperature cycle, C, at which double-stranded DNA significantly increased. t It can be determined that:

[0019] <PCR amplification rate>

[0020] PCR is a well-known conventional technique, so it will be briefly explained using the amplification of DNA (deoxyribonucleic acid) as an example.

[0021] 1. In the thermal denaturation step, the DNA is heated to, for example, 95°C to separate the two strands that make up the DNA and make them into single strands. 2. Next, in the annealing step, the temperature is lowered to, for example, 60°C, and a primer that has been added to the reaction solution beforehand is allowed to bind to the target portion of the single-stranded DNA. 3. Finally, in the extension step, the temperature is raised to, for example, 68°C, and new double-stranded DNA is synthesized by synthesizing a counter strand using the single strand as a template through a polymerase reaction starting from the primer.

[0022] In this way, one cycle of PCR, which consists of three steps: thermal denaturation, annealing, and extension, produces two sets of double-stranded DNA from one set of double-stranded DNA. In other words, if ideal PCR is repeated n cycles, two sets of double-stranded DNA will be produced from one set of double-stranded DNA. n A set of double-stranded DNA can be synthesized.

[0023] However, depending on the concentration and type of the second mediator used in the present invention, PCR may be inhibited.

[0024] For example, consider a case where one cycle of PCR produces an average of only √2 pairs (approximately 1.4 pairs) of double-stranded DNA from one pair of double-stranded DNA. In this case, two cycles of PCR can synthesize two pairs of double-stranded DNA from one pair of double-stranded DNA. In other words, in this case, twice the number of PCR cycles is required compared to ideal PCR.

[0025] Here, in the present invention, a multiple such as "2" when two pairs of double-stranded DNAs are produced from one pair of double-stranded DNA in ideal PCR, or "√2" when an average of √2 pairs (approximately 1.4 pairs) of double-stranded DNAs are produced from one pair of double-stranded DNA as in the above example, will be referred to as the "amplification rate" (in PCR).

[0026] The maximum amplification factor is "2" in the case of ideal PCR. On the other hand, if the amplification factor is 1 or less, double-stranded DNA cannot be increased, so the minimum amplification factor is "1."

[0027] When the amplification rate A is (1 or more and 2 or less), double-stranded DNA is converted into A by n cycles of PCR. n When the amplification factor is A, the number of PCR cycles required is log(2) / log(A) times greater than in ideal PCR. For example, when the amplification factor A is 1.4, 1.6, or 1.8, the number of PCR cycles required is 2.06, 1.47, or 1.18 times greater than in ideal PCR. These relationships can be represented graphically as shown in Figure 3.

[0028] In practice, the amplification factor A is preferably 1.4 or more (the number of PCR cycles required is approximately doubled or less), and more preferably 1.8 or more (the increase in the number of PCR cycles required is approximately 20% or less). The amplification factor varies depending on the concentration and type of the second mediator, so it can be selected so as to achieve the desired amplification factor.

[0029] The amplification rate is the PCR cycle number (threshold cycle: C) at which the amplified double-stranded DNA begins to be detected in PCR measurement. t ) can be known by the ideal amplification C t When the cycle is N, the measured C t If the number of cycles is n, the amplification factor value A is given by the following (Equation 1).

[0030] Since the actual number of PCR cycles is an integer greater than or equal to 1, the measured C t (the PCR cycle number at which the threshold value at which amplified double-stranded DNA begins to be detected is exceeded) is also a natural number. However, the C used to calculate the amplification rate A t It is desirable that the resolution of C is higher. To achieve this, a higher resolution C is obtained by interpolating between the measurement value at the PCR cycle number exceeding the threshold and the measurement value at the PCR cycle number immediately before the threshold. tAs an example, it is possible to obtain a real number C of 1 or more by linear interpolation. t In addition, various interpolation methods such as curve interpolation and intersection with the slope of a straight line are possible, and it is also possible to interpolate using the logarithm of either or both of the PCR cycle number and the measured value.

[0031] <Outline of impedance measurement>

[0032] Fig. 4 shows a typical equivalent circuit of a measurement system in electrochemical impedance measurement using two electrodes. The equivalent circuit shown in Fig. 4 is sometimes called a "Randles circuit including the Warburg impedance W."

[0033] It is well known that a system under test can generally be expressed by an equivalent circuit such as this. However, depending on the type of object under test, the equivalent circuit may differ from the one shown in the figure.

[0034] Solution resistance R s is the electrical resistance component determined by the solution resistance of the measurement system. ct is the resistance caused by charge transfer between the mediator and the electrode at the electrode interface. dl represents the capacitance component due to the electric double layer formed at the electrode interface. The Warburg impedance W represents the diffusion resistance of the mediator in the low frequency region.

[0035] In the measurement system, when the charge transfer performance of the mediator changes as the reaction progresses, the charge transfer resistance R obtained by measuring the impedance Z of the measurement system ct The progress of the reaction in the system being measured can be determined by the change in . An example of such measurement will be given below.

[0036] The impedance Z of the equivalent circuit shown in FIG. 4 is, for example, measured at a high frequency of several kHz or more, dl can be approximated as a short circuit, and the impedance of the equivalent circuit is approximately the solution resistance R s This becomes:

[0037] Electric double layer capacitance C dlThe absolute value of the capacitive reactance is the charge transfer resistance R ct In the frequency range from several hundred Hz to several Hz, for example, where the magnitude of the impedance is not negligible relative to the absolute value of R, the Nyquist plot of impedance shows a semicircle. If a frequency near the apex of the semicircle is selected as the measurement frequency, the imaginary part of the impedance is calculated as the charge transfer resistance R ct That is, the charge transfer resistance R ct It is also possible to calculate R by selecting and measuring multiple frequencies near the vertices of the semicircle and taking the average of the imaginary part of the impedance. ct It is also possible to calculate

[0038] In the low frequency region near the point where the Nyquist plot switches from the right end of the semicircle to the upper right, the electric double layer capacitance C dl approaches the frequency range where it can be approximated as an open circuit, the impedance of the equivalent circuit is s and the charge transfer resistance R in the equivalent circuit of FIG. ct In other words, if such a low frequency is selected as the frequency for measurement, the real part of the impedance approaches the sum of the solution resistance R s and the charge transfer resistance R in the equivalent circuit of FIG. ct Usually the solution resistance R s is sufficiently small, and the charge transfer resistance R ct It is possible to estimate the electric double layer capacitance C dl It is also possible to measure multiple frequencies at which the charge transfer resistance R can be approximated as open, and take the average of the real part of the impedance. ct Furthermore, the absolute value of the impedance is used instead of the real part of the impedance to estimate the charge transfer resistance R ct It can be used as a substitute for the impedance, which may be particularly useful when the imaginary part of the impedance is not zero at the frequency to be measured.

[0039] In the even lower frequency range of a few Hz or less, the impedance changes in such a way that the change due to the Warburg impedance W becomes dominant. In other words, as the frequency decreases, a change occurs in which both the real and imaginary parts of the impedance increase.

[0040] [The charge transfer resistance R ct Method for calculating

[0041] In the present invention, the resistance generated by charge transfer between the mediator and the electrode at the electrode interface, which is determined from the measured impedance, or its estimated value, is broadly referred to as the "charge transfer resistance R ct Impedance is a "vector" expressed by real and imaginary parts, absolute value and phase, whereas charge transfer resistance R ct is the "vector magnitude" of the impedance (unit: [Ω]).

[0042] 5 is a schematic diagram showing an example of a Nyquist plot using the EIS method. In the EIS method, for example, a frequency response analyzer (FRA) is used to apply a sine wave ranging from high to low frequencies (or even in the reverse direction) to an object under test, and the frequency response (the relationship between the applied AC voltage and the AC current flowing through the object under test) is determined.

[0043] In a Nyquist plot, the horizontal axis represents the real number of the frequency response, and the vertical axis represents the negative value of the imaginary number of the frequency response. real is small and the imaginary part of the impedance Z imag The left end of the semicircle where is close to zero corresponds to a high frequency, and as the frequency decreases, the semicircle moves to the right. As the frequency decreases further, the change due to the Warburg impedance W becomes dominant, and the change becomes linear at about 45 degrees diagonally upward to the right. In impedance measurement by the EIS method in PCR, for example, the imaginary part Z of the impedance imag It is advisable to measure over a wide range from a high frequency where is close to zero to a low frequency where it changes linearly in an upward diagonal direction to the right.

[0044] The charge transfer resistance R is calculated by fitting an equivalent circuit from the Nyquist plot of the impedance measured by the EIS method. ct can be calculated.

[0045] As can be seen from the Nyquist plot, the absolute value of the impedance |Z| is the real part of the impedance Z real Therefore, in impedance measurement using the EIS method, the absolute value or real part of the measured impedance is used as the charge transfer resistance R ct The imaginary part of the impedance Z imag The charge transfer resistance R ct (For example, if the Nyquist plot is a semicircle, Z imag The maximum value of the magnitude of the charge transfer resistance R ct Since it is half of the charge transfer resistance R ct The size of can be calculated.)

[0046] [The charge transfer resistance R ct Method for calculating

[0047] As mentioned above, in the EIS method, a sine wave ranging from a high frequency to a low frequency is applied to the object to be measured, and its frequency response is obtained. The charge transfer resistance R is calculated from the absolute value, real part, or imaginary part of the impedance measured by impedance measurement at one frequency. ct Here, a specific example of measuring impedance at one or more frequencies will be described.

[0048] FIG. 6 is a schematic diagram showing an example of a change in the Nyquist plot when the impedance of the object to be measured changes due to temperature repetition in a PCR cycle or the like.

[0049] As can be seen from FIG. 6, even if the impedance of the object to be measured changes, the solution resistance R s (Z real It can be seen that the impedance (approximately 0.7 kΩ) hardly changes. The change in the impedance of the object to be measured is mainly due to the charge transfer resistance R ct This is reflected in changes in

[0050] In the Nyquist plot, at a frequency near the point where the semicircle changes from the right end to the upper right, the impedance of the object being measured is s and the charge transfer resistance R in the equivalent circuit of FIG. ct At such a frequency, as mentioned above, the absolute value of the impedance |Z| and the real part of the impedance Z real There is no significant difference between the solution resistance R s Since there is almost no change, by measuring the impedance at a frequency near the point where the semicircle of the Nyquist plot switches from the right end to the upper right, the charge transfer resistance R ct (For convenience in the present invention, the solution resistance R s With or without the inclusion of ct (hereinafter referred to as "the Company").

[0051] Alternatively, the solution resistance R s is measured in advance, and the solution resistance R s and the charge transfer resistance R in the equivalent circuit of FIG. ct From the sum of the solution resistance R s By subtracting ct It is also possible to calculate

[0052] The frequency can be, for example, 30 Hz or less.

[0053] [The charge transfer resistance R is calculated by the semicircular approximation of the Nyquist plot with two frequencies. ct Method for calculating

[0054] FIG. 7 shows the charge transfer resistance R, which is calculated by approximating a semicircle at two frequencies, high and low, and estimating the diameter of the semicircle from the impedance. ct The charge transfer resistance R ct FIG. 10 is a schematic diagram showing a method for determining the

[0055] In FIG. 7, the solution resistance R s , and a point a-jb on a semicircle whose center point is on the real axis are determined, then b:(a-Rs ) = (R s +R ct -a): b, so the charge transfer resistance R estimated from the impedance measured at two high and low frequencies f is calculated using the following formula: ct The solution resistance R s is obtained by measuring the solution resistance R at least once, for example, by measuring the impedance at a high frequency of 1 kHz or more. s For example, the real part Z of the impedance measurement value at a frequency f = 1 kHz is real It can be said that:

[0056]

[0057] This semicircular approximation method requires measurements at at least two frequencies (two points on the Nyquist plot). s If it is known in advance that both the real and imaginary parts of are sufficiently small and close to zero, it is possible to approximate the semicircle using only the measurement result at another point at a lower frequency.Furthermore, it is also possible to approximate the semicircle using measurement results at more frequencies and then take the average of these.

[0058] At least one of the frequencies may be set to 30 Hz or less, for example.

[0059] [The charge transfer resistance R is calculated by the difference between the real parts of the impedances at two frequencies, or the absolute value of the difference in impedances at two frequencies. ct Method for calculating

[0060] FIG. 8 shows the charge transfer resistance R estimated from the impedance, which is the difference between the real parts of the impedance measured at two frequencies, high and low, or the absolute value of the impedance difference (absolute value of the vector difference). ct FIG. 10 is a schematic diagram showing the above.

[0061] This method also requires measurement results at at least two frequencies (two points on the Nyquist plot). Furthermore, it is also possible to use measurement results at more frequencies and take the average of them.

[0062] At least one of the frequencies may be set to 30 Hz or less, for example.

[0063] [The charge transfer resistance R is calculated by elliptical approximation of the Nyquist plot at three frequencies. ct Method for calculating

[0064] FIG. 9 shows the charge transfer resistance R estimated from the impedance measurement results by approximating an ellipse at three frequencies in the Nyquist plot. ct FIG. 10 is a schematic diagram showing the above.

[0065] In actual measurements of a specific target material, the Nyquist plot often resembles a vertically distorted semicircle, so a more accurate approximation can sometimes be achieved by approximating it to an ellipse. This method requires measurement results at at least three frequencies (three points on the Nyquist plot). It is also possible to use measurement results at more frequencies and take the average of these. Even if the actual measurement results of a specific target material resemble a vertically distorted semicircle (i.e., the diameter in the real axis direction is the minor axis), a similar approximation is possible.

[0066] At least one of the frequencies may be set to 30 Hz or less, for example.

[0067] [The charge transfer resistance R is calculated by approximating the Nyquist plot with three frequencies using an arc. ct Method for calculating

[0068] FIG. 10 shows the charge transfer resistance R estimated from the impedance measurement results by approximating three frequencies to a circular arc at the imaginary part of the arc = 0Ω. ct FIG. 10 is a schematic diagram showing the above.

[0069] In Nyquist plots where the center of a semicircle is close to being below the real axis, a more accurate approximation can sometimes be achieved by approximating it to an arc. This method also requires measurement results at at least three frequencies (three points on the Nyquist plot). Furthermore, it is also possible to use measurement results at more frequencies and take the average of them.

[0070] At least one of the frequencies may be set to 30 Hz or less, for example.

[0071] [Method using a superimposed waveform containing frequency components of multiple frequencies]

[0072] The EIS method is the most accurate method for determining the impedance of an object under test, but it takes a long time to measure the impedance by gradually changing the frequency over a wide range. Furthermore, when the frequency is changed, it takes time to wait until the response from the object under test stabilizes (this time is called the "stabilization time"), which tends to make the measurement time even longer.

[0073] In contrast, as mentioned above, the charge transfer resistance R can be calculated by measuring the impedance at only one frequency. ct This method has the advantage of minimizing measurement time because it does not require frequency switching or stabilization time. This makes it possible to continue measuring while the impedance of the object being measured is changing, making it possible to more reliably capture the changes.

[0074] In the method using two frequencies or the method using three frequencies as described above, when measurements are performed by switching frequencies, frequency switching and stabilization time are required, so the measurement time is longer than that of a method using one frequency, but is still significantly shorter than that of the EIS method.

[0075] On the other hand, when measuring impedance using the two-frequency or three-frequency method, a superimposed waveform containing frequency components of two frequencies or a superimposed waveform containing frequency components of three frequencies can also be used. In this case, frequency switching and stabilization time are not required, so a short measurement time similar to that of the method using one frequency can be achieved. This makes it possible to continue measuring while the impedance of the object being measured is changing, allowing for more accurate capture of the change. However, since it is necessary to simultaneously measure the impedance of two or three frequencies, the measurement circuit becomes more complex than that of the method using one frequency.

[0076] When using a superimposed waveform containing multiple frequency components, many higher frequency periods are included in one cycle of the lowest frequency. In other words, at high frequencies, the effects of noise and disturbances can be reduced by averaging the measurement results of more cycles. This allows the signal amplitude of high frequencies to be smaller than that of low frequencies.

[0077] When using a superimposed waveform containing frequency components of multiple frequencies, if too many frequency components are superimposed, each frequency component will become small, so it is preferable to keep the number of frequency components to a single digit, and it is more preferable to have two or three frequencies.

[0078] Furthermore, when using a superimposed waveform containing frequency components of multiple frequencies, it is preferable to apply an AC voltage of approximately 10 mV to 100 mV in order to enable measurement in the linear region. Note that in this specification, AC voltages expressed in units of "V" are more accurately expressed as "Vrms."

[0079] At least one of the frequencies may be set to 30 Hz or less, for example.

[0080] As described above, the charge transfer resistance R ct There are many methods for estimating or calculating the above, but the present invention is not limited to the methods exemplified above.

[0081] <Target substance detection system>

[0082] FIG. 11 shows an example of the configuration of a target substance detection system 1 according to a preferred embodiment of the present invention.

[0083] The target substance detection system 1 comprises a target substance detection device 2, and a main mediator 5 and a second mediator 6 contained in a solution 8, which is a measurement object that may contain a target substance 7, and detects the target substance 7 by measuring the electrochemical properties of the solution 8 by electrochemical measurement using an electrochemical measurement unit 3 of the target substance detection device 2. The configurations of the main mediator 5 and the second mediator 6 are as described below, but the second mediator is a compound selected by the second mediator selection method of the present invention, which will be described below.

[0084] The target substance detection device 2 also includes a determination unit 4 that determines whether the measurement result obtained by electrochemical measurement of the solution 8 reaches a predetermined value. As an example, the determination unit 4 determines that the target substance 7 has been detected when the measurement result reaches a predetermined value.

[0085] The electrochemical measurement unit 3 includes devices necessary for measuring the electrochemical properties of the solution 8 by an electrochemical measurement method, such as impedance measurement or DC polarization measurement. Known devices can be used as the devices necessary for impedance measurement or DC polarization measurement.

[0086] In impedance measurement, the electrochemical measurement unit 3 includes, for example, electrodes such as a working electrode, a counter electrode, and a reference electrode, an FRA, and a potentiogalvanostat. The potentiogalvanostat controls the electrodes electrochemically, and the FRA connected to the potentiogalvanostat calculates the electrochemical impedance from the potential and current signals. An oscilloscope may also be connected to monitor the input and output signals.

[0087] When the impedance bridge method is used, a bridge circuit including an electrochemical cell is formed using a known resistor, a variable resistor, and a variable capacitor.

[0088] When using the fast Fourier transform (FFT) method, an appropriate input potential signal is applied to a balanced or steady-state electrode, and the resulting output current signal is measured using a potentiogalvanostat. This signal, along with the input signal, is input into a computer using an A / D converter. The computer then performs Fourier transforms on the input potential signal and the output current signal, and determines the impedance spectrum from their cross spectrum.

[0089] In DC polarization measurements, the electrochemical measurement unit 3 is equipped with electrodes, and a potentiostat is used to set a constant potential, and a galvanostat is used to set a constant current. A potential programmer may be used to set a set potential over time. An XY recorder, oscilloscope, memory scope, etc. are used to record the current-potential curve. Recorders include an electrometer, ammeter, coulombmeter, etc.

[0090] <Sequence-specific double-stranded nucleic acid detection> The electrochemical measurement method described above is applicable to sequence-specific double-stranded nucleic acid detection. In addition to the PCR method described above, examples of sequence-specific double-stranded nucleic acid detection include loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), nickeling endonuclease amplification reaction (NEAR), nucleic acid sequences based amplification (NASBA), transcription mediated amplification (TMA), recombinase polymerase amplification (RPA), strand-invasion based amplification (SIBA), helicase-dependent amplification (HDA), whole genome amplification (WGA), multiple displacement amplification (MDA), DNA chips, DNA microarrays, next-generation sequencing (NGS), chromatin immunoprecipitation (ChIP), ChIP-on-chip, and chromatin immunoprecipitation (ChIP-seq). It is also applicable to immunoprecipitation with high-throughput sequencing.

[0091] Next, we will explain each component used in the electrochemical measurement method for detecting a target substance. <Target Substance> Double-stranded nucleic acids can be suitably used as the target substance. Nucleic acids are generally substances having a structure in which nucleotides consisting of bases, sugars, and phosphates are linked via phosphodiester bonds. Examples include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). However, in this specification, nucleic acids also include PNA (peptide nucleic acid) having a structure similar to DNA or RNA, and compounds having a nucleic acid structure within the molecule. Examples of target substances include double-stranded DNA, double-stranded RNA, double-stranded nucleic acids composed of DNA-RNA, double-stranded (artificial) nucleic acids composed of DNA-PNA, double-stranded (artificial) nucleic acids composed of RNA-PNA, and double-stranded PNA. Furthermore, complexes of DNA or RNA with various nucleic acid-binding compounds can also be used as target substances. Nucleic acid-binding compounds include nucleic acid-binding proteins; small molecule compounds such as coenzymes and anticancer drugs; lipids such as cholesterol, phospholipids, and phospholipid derivatives such as lysophosphatidic acid; inorganic polymeric compounds such as silica, zeolite, and metal complexes; and organic polymeric compounds such as polyamines, which may be used alone or in combination of two or more. The target substance detection method or detection system of the present invention can be used to detect sequence-specific nucleic acid interactions on DNA chips and the like when the target substance is a double-stranded nucleic acid, particularly double-stranded DNA, double-stranded RNA, double-stranded nucleic acid composed of DNA-RNA, double-stranded (artificial) nucleic acid composed of DNA-PNA, double-stranded (artificial) nucleic acid composed of RNA-PNA, or double-stranded PNA. Furthermore, the target substance detection method or detection system of the present invention can be used to detect or analyze proteins (including enzymes) when the target substance is a DNA-protein complex or an RNA-protein complex. In the present invention, for example, even if there are only a few double-stranded nucleic acids in solution, the target substance can be detected in PCR amplification. Furthermore, the target substance is present in solution, at least not immobilized on an electrode.When the target substance is a double-stranded nucleic acid and the aim is to detect a sequence-specific double-stranded nucleic acid by PCR, the solution in which the target substance is present can be a buffer solution typically used in PCR, and the solution can contain components typically used in PCR, such as primers, polymerase, buffer electrolytes, etc. Furthermore, when the aim is to detect a sequence-specific double-stranded nucleic acid by a method other than PCR, the solution can contain, in addition to the above-mentioned components, components typically used in the sequence-specific double-stranded detection method.

[0092] <Main Mediator> The main mediator transfers charge between the working electrode and the main mediator. A specific example of the main mediator is ferricyanide ion [Fe(CN)6]. 3- , ferrocyanide ion [Fe(CN)6] 4- and metal complexes that form redox electrolytes, such as ferrocene, and mixtures thereof. In the present invention, the concentration of the main mediator is preferably 0.1 to 10 mM.

[0093] <Electrodes> The electrodes used in electrochemical measurement may be three electrodes consisting of a working electrode, a counter electrode, and a reference electrode, or two electrodes consisting of a working electrode and a counter electrode without using a reference electrode. The latter two-electrode configuration is preferred because it has a simpler configuration. Examples of the reference electrode that may be used as needed include a silver-silver chloride electrode, a silver-silver chloride saturated electrode, and a silver wire made by chlorinating.

[0094] The material of the working electrode used in the present invention is not particularly limited as long as it is an electrode used in electrochemical measurements, and examples thereof include a carbon electrode made of graphite, carbon nanotubes, carbon black, etc., a gold electrode, a platinum electrode, and an ITO (indium-tin oxide) electrode.

[0095] In order to more strongly express the charge transfer fluctuation of the second mediator, it is preferable that the working electrode has a porous thin film layer on its solution-side surface that has pores that are small enough to allow the main mediator and second mediator to pass through, but not to allow nucleic acids, proteins, and other impurities contained in the solution as target substances with higher molecular weights than these compounds to pass through.

[0096] The porous thin film layer, through which the primary mediator and secondary mediator can pass, is preferably formed of a compound that exhibits minimal interaction with target substances, amplification components, biological substances, etc. Examples of compounds that form the porous thin film layer include polymers and organic compounds capable of forming SAM (self-assembled monolayer) films. The compound that forms the porous thin film layer preferably has functional groups such as hydroxyl groups, ether groups, aldehyde groups, carbonyl groups, carboxyl groups, nitro groups, phosphate groups, and sulfonic acid groups, and these functional groups are preferably disposed on the solution-side surface of the electrode. When the compound that forms the porous thin film layer is a polymer, the functional groups may be present in the main chain, side chain, or both of the polymer. Specific examples include polyvinyl alcohol, polyethylene glycol, polyester, polyacrylic acid, polyacrylamide, and polysaccharides. The porous thin film layer can be formed on the electrode surface by known methods, such as electropolymerization, photopolymerization, thermal polymerization, and radical polymerization. The porous thin film layer preferably has a thickness of 0.001 to 10 μm.

[0097] In the present invention, the DC voltage applied to the working electrode is in the range of −1 to 1 V relative to the reference electrode. The AC voltage applied to the working electrode and counter electrode is not particularly limited and is usually in the range of 1 mV to 1 V, preferably 1 to 100 mV, and more preferably 1 to 10 mV. The frequency between the electrodes is usually in the range of 0.1 to 1 MHz, and preferably 1 to 1 kHz.

[0098] <Second Mediator> The second mediator is present in a solution subjected to electrochemical measurement and changes the charge transfer between the main mediator and the working electrode. In one embodiment of the present invention, the second mediator is selected by the following steps (i) and (ii) or the following steps (i), (ii), and (iii). In another embodiment of the present invention, the second mediator is selected by the following steps (i) and (iii). Each step will be described below.

[0099] (i) Step of selecting a compound that interacts with the target substance. Here, "interacting with the target substance" refers to a chemical or physical interaction between the target substance and the second mediator. Therefore, in this step, a second mediator having a structure that can chemically or physically interact with the target substance can be selected depending on the type and structure of the target substance to be detected. Examples of types of interaction include electrostatic interaction and π-π interaction, and further, when the target substance is a double-stranded nucleic acid, examples include insertion (intercalation) between base pairs.

[0100] The interaction with the target substance can be inferred from the chemical structure of the second mediator. As shown in the examples below, the charge transfer resistance R ct This may be confirmed by measuring

[0101] (ii) the charge transfer resistance R of a solution containing the primary mediator at a unique concentration C1 ct0 The charge transfer resistance R of the solution containing the primary mediator at a concentration C1 and the secondary mediator at a concentration C2 ct The ratio R ct / R ct0 The second mediator concentration C2 in this step is not particularly limited, but may be, for example, 1 / 10, 1 / 100, or 1 / 1000 of the main mediator concentration C1. ct and R ct0 It is preferable that each value of R be measured under the same conditions as the solution to be detected. ct and R ct0 Specific examples of the method for measuring each value include the methods shown in the Examples below. ct / R ct0 is 0.80 or less, preferably R ct / R ct0 is 0.50 or less, more preferably R ct / R ct0 is selected to satisfy 0.30 or less.

[0102] (iii) measuring the impedance of the solution containing the target substance before amplification and at each amplification step; and max -ΔZ min ) / R ctm Figure 12 is a diagram illustrating a method for calculating the index in this step, taking PCR detection of double-stranded DNA as an example of sequence-specific double-stranded nucleic acid detection, and includes two graphs, one above the other. This step will be described below with reference to Figure 12.

[0103] The top graph of FIG. 12 (FIG. 12A) shows the charge transfer resistance R ct The horizontal axis represents the number of PCR cycles (1 to 40), and the vertical axis represents the charge transfer resistance R ct As an example, the absolute value of the impedance at 2.5 Hz is measured. The plots of "x" are comparative reference data for a solution that does not contain the double-stranded DNA of the target substance, and the plots of "○" are data for a solution that contains the double-stranded DNA of the target substance. The solution that does not contain the double-stranded DNA of the target substance gradually decreases in charge transfer resistance R with the increase in PCR cycles. ct In contrast to the case where the impedance of a solution containing double-stranded DNA as the target substance increases, the impedance increases sharply after a certain number of PCR cycles.

[0104] The bottom graph of FIG. 12 (FIG. 12B) shows the charge transfer resistance R ct That is, the charge transfer resistance R ct and the charge transfer resistance R in the previous PCR cycle. ct The horizontal axis represents the number of PCR cycles, and the vertical axis represents the difference in the absolute impedance values. "×" and "◯" are the same as in the graph above.

[0105] In this step, the maximum absolute value of ΔZ is max and ΔZ max The charge transfer resistance R at the cycle number m ct The value of R ctm and ΔZ maxThe minimum absolute value ΔZ of ΔZ at a cycle number smaller than the cycle number m min Calculate (ΔZ max -ΔZ min ) / R ctm For example, in the upper graph of FIG. 12 (FIG. 12A), the absolute value of the charge transfer resistance R ct The charge transfer resistance R ct is 76.1 kΩ. In the graph at the bottom of FIG. 12 (FIG. 12B), ΔZ max At cycle number m=26, ΔZ max = 82.7 kΩ - 76.1 kΩ = 6.6 kΩ. Also, ΔZ min is 1.1 kΩ at the 17th PCR cycle. max -ΔZ min = 5.5 kΩ (see the vertical arrow in the graph below (FIG. 12B)), and (ΔZ max -ΔZ min ) / R ctm The absolute value of is calculated as 5.5 kΩ÷82.7 kΩ=6.6%.

[0106] In the above description, the charge transfer resistance R in a certain PCR cycle and the PCR cycle immediately before it is ct The difference between the charge transfer resistance R ct The difference between these two may be taken as ΔZ. ΔZ may also be obtained based on the slope of the approximation curve. In the above description, FIG. 12 shows an example of the results of measuring impedance at a signal frequency of 2.5 Hz, but this is not limiting and other appropriate frequencies may be selected. In the above description, the impedance measurement was performed after the extension step in the repeated cycle of the thermal denaturation step, annealing step and extension step of PCR, but this is not limiting. Furthermore, in the above description, the charge transfer resistance R ct Although the absolute value of the impedance is used as an example, it is not limited to this. ctThere are many methods for obtaining this, and any method can be selected. In the above explanation, the difference in absolute value of impedance is used as ΔZ, but the charge transfer resistance R ct The difference between the two can be used.

[0107] The second mediator may be the above-mentioned (ΔZ max -ΔZ min ) / R ctm The absolute value of (ΔZ max -ΔZ min ) / R ctm The absolute value of (ΔZ max -ΔZ min ) / R ctm The absolute value of which is greater than 3% is selected.

[0108] The second mediator is preferably applied by step (iv) below at a concentration selected based on the relationship between second mediator concentration and amplification rate.

[0109] (iv) A step of performing sequence-specific double-stranded nucleic acid amplification using a solution containing the target substance, and selecting the concentration of the second mediator based on the relationship between the concentration of the second mediator and the amplification rate. The concentration of the second mediator is selected so that the amplification rate of the target substance per step of amplifying the target substance is 1.4 or more and 2.0 or less, preferably 1.8 or more and 2.0 or less. Here, the amplification rate is a value defined by the above (Equation 1). The amplification rate can be calculated by performing qPCR according to a known method prior to implementing the present invention. For example, if the second mediator is a compound that emits fluorescence when incorporated into the target substance, an amplification curve of double-stranded DNA with increasing cycle number can be obtained by monitoring the luminescence of the solution, and C can be calculated from the intersection of the curve with an arbitrary threshold value. t qPCR was performed at various concentrations of the second mediator, and the C at any concentration was determined according to the type and characteristics of the second mediator. t When N is set as N and the second mediator concentration is a certain concentration, C tThe amplification factor value A is calculated by using n as the amplification factor value A. The concentration of the second mediator is determined so that the amplification factor value A calculated in this way is in the range of 1.4 to 2.0, preferably 1.8 to 2.0.

[0110] The inventors have discovered that when the target substance is a double-stranded nucleic acid, the second mediator selected by the method of the present invention is preferably a compound that exhibits at least one of electrostatic interactions, π-π interactions, base pair insertion (intercalation), and other interactions. Examples of other interactions include hydrophobic-π interactions, ion-dipole interactions, stem-loop interactions, conformational interactions, coordinate bonds, alkyl-π interactions, intramolecular hydrogen bonds, solvation effects, intermolecular water (water-water) interactions, magnetic interactions, van der Waals forces (London dispersion forces), π-anion interactions, molecular dipole-dipole interactions, induced dipole interactions, allosteric interactions, and covalent bond networks. When the target substance is a double-stranded nucleic acid, a compound that interacts with the electrode surface is preferred as the second mediator. In particular, compounds having an aromatic ring in the molecule are preferred as second mediators. Furthermore, positively charged compounds may be preferred.

[0111] Therefore, in step (i), it is preferable to select a compound that interacts with the target substance through at least one of electrostatic interaction, π-π interaction, intercalation between base pairs of the target double-stranded nucleic acid, and other interactions. In one embodiment of the present invention, in step (i), a compound having at least one structure represented by the following formula (I) per molecule can be selected: The ring having A in the above formula (I) is a 5- or 6-membered ring fused with an adjacent benzene ring and is a heterocyclic ring having at least one atom selected from a nitrogen atom, an oxygen atom, and a sulfur atom. There may be a plurality of 6-membered rings adjacent to A. The carbon atoms or heteroatoms constituting this heterocyclic ring may have a substituent. Not only the heterocyclic ring having A, but also the 6-membered ring (benzene ring) adjacent to the heterocyclic ring having A may have a substituent, and may have a nitrogen atom, an oxygen atom, or a sulfur atom. The 6-membered ring (benzene ring) adjacent to the heterocyclic ring having A having a nitrogen atom, an oxygen atom, or a sulfur atom may be a heterocyclic ring in which at least one carbon atom constituting the ring is substituted with a nitrogen atom, an oxygen atom, or a sulfur atom, or the substituent bonded to an atom constituting the ring may be a group having a nitrogen atom, an oxygen atom, or a sulfur atom. That is, examples of the substituent that may be possessed by the heterocycle having A or the 6-membered ring (benzene ring) adjacent to the heterocycle having A include hydrocarbon groups such as alkyl groups, alkenyl groups, alkynyl groups, aryl groups, and aralkyl groups; organic groups such as hydroxy groups, carbonyl groups, carboxy groups, ether bonds, ester bonds, amino groups, amide bonds, cyano groups, nitro groups, and sulfo groups; and combinations thereof.

[0112] More preferred examples of the structure represented by formula (I) include the following structures: In the above formula, A is a nitrogen atom, an oxygen atom, or a sulfur atom. The carbon atoms or heteroatoms constituting the fused ring in the above structural formula may have a substituent. Examples of the substituent that the fused ring in the above structural formula may have include the same substituents as those exemplified above. The present inventors have found that when the target substance is a double-stranded nucleic acid, the second mediator selected by the method of the present invention is preferably a compound having at least one of the above four types of fused rings, and preferably two or more types, in one molecule.

[0113] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0114] Step (i) First, a double-stranded nucleic acid was used as the target substance, and a group of compounds known to interact with double-stranded nucleic acid, particularly double-stranded DNA, was selected in step (i). Examples of candidate compounds for the second mediator selected in step (i) are shown in the leftmost column of Table 1.

[0115] Step (ii) [R ct / R ct0 Each of the compounds shown in Table 1 below was used as a second mediator, and impedance measurement was carried out by the EIS method as follows. ct / R ct0 The measurement was carried out under the following conditions: Working electrode: carbon electrode (no porous thin film layer) Reference electrode: silver-silver chloride electrode Counter electrode: carbon electrode AC voltage: 10 mV Frequency: 10 kHz to 0.1 Hz Measurement temperature: 25°C The solution to be measured was 10 mM Tris buffer (pH 8) containing 50 mM KCl, 1.5 mM MgCl2, and [Fe(CN)6] as the main mediator. 3- / 4- A solution containing the above (hereinafter referred to as PCRiF1 solution) was used. ct / R ct0 In the impedance measurement for calculating (I), the concentration C1 of the main mediator in the PCRiF1 solution was set to 1 mM. (1) First, the impedance of the PCRiF1 solution was measured. (2) Next, a PCRiF1 solution containing a second mediator (hereinafter referred to as "second mediator solution") was added to the solution in (1) above in an amount such that the second mediator concentration in the solution became 0.001 μM. After leaving the solution for 30 minutes, the impedance was measured in the same manner as in (1) above. Subsequently, the second mediator solution was added to various concentrations in the range in which the second mediator concentration C2 in the solution became less than 1 mM, the solution was left for 30 minutes, and the impedance measurement in the same manner as in (1) above was repeated. (3) The measurement results were fitted to a Randles circuit to calculate the charge transfer resistance R ct (4) R at each concentration of the second mediator ct The value of the charge transfer resistance R ct The initial value of the PCRiF1 solution, i.e., the R ct0 The value R normalized by ct / R ct0 The vertical axis is (Rct / R ct0 Graphs (Figures 13A to 13C) were obtained by plotting the second mediator concentration on the horizontal axis and the second mediator concentration on the vertical axis. The graphs in Figures 13A to 13C show the results for the cases where SYBR Green I, SYBR Gold, Pico Green, SYBR safe, and Eva Green were used as the second mediator (Figure 13A), Basic Blue 3, Basic Blue 17, Basic Blue 9 (methylene blue), and [Ru(bpy)2DPPZ] were used, respectively. 2+ (Figure 13B) and Disperse Blue 14, Basic Red 5 (Toluylene Red), Basic Blue 1, Promethazine Hydrochloride, Dipyrido[3,2-a:2',3'-c]phenazine (DPPZ), Melatonin, and [Ru(bpy)3] 2+ The figure shows the results when the second mediator concentration C2 in the measurement solution was 1 / 1000 of the main mediator concentration C1, i.e., R ct / R ct0 The results are shown in Table 1. In addition, for the second mediator species measured up to the second mediator concentration C2 of 10 μM, the R ct / R ct0 The results are shown in Table 1. (In Table 1, "-" is entered for cases where measurements were not performed at the same concentration.) ct / R ct0 From the calculation results, values ​​exceeding 0.8 were rated as "C," values ​​exceeding 0.3 but not exceeding 0.8 were rated as "B," and values ​​not exceeding 0.3 were rated as "A," and the results are also shown in Table 1. (Even for substances for which concentration measurements have not been performed and are listed as "-" in Table 1, it is generally possible to determine the concentration from the extrapolated values ​​in the graphs (Figures 13A to 13C).)

[0116]

[0117]

[0118] Step (iii) [(ΔZ max-ΔZ min ) / R ctm Calculation of the absolute value of R ct / R ct0 The charge transfer resistance R was measured by EIS method using several compounds whose value was 0.8 or less as the second mediator. ct Calculate (ΔZ max -ΔZ min ) / R ctm The absolute value of was calculated. Measurements were performed under the following conditions: Working electrode: carbon electrode (having a porous thin film layer coated with polyvinyl alcohol (PVA)) Counter electrode: carbon electrode (having a porous thin film layer coated with polyvinyl alcohol (PVA)) AC voltage: 10 mV Frequency: 2.5 Hz Measurement temperature: 68°C Double-stranded DNA (Treponema denticola genomic DNA, PCR product 180 bp) was used as the target substance, and polymerase (Hemo KlenTaq TM Product number: M0332S (New England Biolabs)), buffer solution (Hemo KlenTaq TM Reaction Buffer (New England Biolabs), composition: 5 mM (NH4)2SO4, 3.5 mM MgCl2, 6% glycerol, 60 mM Tricine, pH 8.7 (25°C), forward primer, reverse primer, and main mediator ([Fe(CN)6] 3- / 4- A solution containing 1 mM of ATP was prepared. PCR was performed with one cycle consisting of a thermal denaturation step (94°C), an annealing step (60°C), and an extension step (68°C), and the impedance was measured after the extension step of each cycle. max -ΔZ min ) / R ctm The absolute value of the charge transfer resistance R may be larger after the annealing step than after the extension step, and can be selected as needed. ctAs an example, the absolute value of the impedance at a single frequency was used, but the present invention is not limited to this, and the method exemplified in the above-mentioned <Outline of Impedance Measurement> can also be applied. ct The changes in ΔZ are shown in each figure in Figure 14 (SYBR Green I (Figure 14A), SYBR Gold (Figure 14B), Eva Green (Figure 14C), and Pico Green (Figure 14D)). max -ΔZ min ) / R ctm The absolute values ​​were as follows: SYBR Green I: 0.09%, SYBR Gold: 6.6%, Eva Green: 1.4%, Pico Green: 5.1%.

[0119] Step (iv) [Calculation of Amplification Rate] In the above step (ii), R ct / R ct0 The PCR amplification rate was calculated from the change in fluorescence intensity in each cycle, and the relationship between the second mediator concentration and the amplification rate was investigated. PCR was performed using a real-time PCR device, Mic qPCR Cycler (BioMolecular Systems), and analysis was performed using the accompanying micPCR software. As the subject substance, double-stranded DNA (1407 bp including a gene (956 bp) encoding a protein involved in NADH production was used as a template, and the PCR product was 110 bp) was used. When KOD FX was used, KOD buffer solution was used, and when Klen Taq was used, Klen Taq buffer solution was used. dNTP was added at 0.4 μM when KOD FX was used, and 0.2 μM when Klen Taq was used, and further forward primer and reverse primer were added at 0.5 μM each. PCR was performed according to the manufacturer's instructions using solutions containing the combinations of polymerase, main mediator, and second mediator shown in Table 2 below (Examples 1 to 5), and the amplification rate was calculated. For fluorescence detection, the solutions contain 0.2 units of SYBR Gold in addition to the second mediator in Examples 1, 2, and 4, and 0.1 units of SYBR Gold in Examples 3 and 5. These concentrations are the minimum concentrations that have minimal effect on PCR and allow fluorescence detection.

[0120] As the second mediator, [Ru(bpy)2DPPZ] 2+ The relationship between the PCR cycle number and the fluorescence intensity in Examples 1 and 2 using the same is shown in Figures 15A (Example 1) and 15B (Example 2). From the graphs in Figures 15A and 15B, the PCR threshold was set to fluorescence intensity = 0.1, and C was calculated from the intersection of the fluorescence signal curve and the threshold. t The value of C at each concentration of the second mediator was calculated. t The values ​​of C and the amplification rate are shown in Table 3 (Example 1) and Table 4 (Example 2). In Examples 1 and 2, the amplification rate was set to 2 when the concentration of the second mediator was zero. This is because substances other than the second mediator in the solution are known to hardly inhibit PCR, so the case where the concentration of the second mediator was zero was set to 2, which is the theoretical maximum amplification rate, and C at the concentration of the second mediator was set to 1. t The value of is N, and the measured C at each concentration of the second mediator t The amplification rate at each concentration of the second mediator was calculated using the above equation (1), where n is the amplification rate.

[0121]

[0122] In the same manner as in Examples 1 and 2, the relationship between the second mediator concentration and the amplification rate in Examples 3 to 5 was calculated. tThe values ​​of and amplification factor are shown in Table 5 (Example 3), Table 6 (Example 4), and Table 7 (Example 5). In Example 3, SYBR Gold itself is a fluorescent substance. Therefore, at zero concentration of the second mediator, no fluorescence is generated, resulting in an apparent minimum amplification factor of 1. Furthermore, when the concentration of the second mediator is low, the fluorescence is weak, making accurate measurements difficult and tending to underestimate the amplification factor. Furthermore, above a certain concentration, the second mediator inhibits PCR, resulting in a decrease in amplification factor. Therefore, with SYBR Gold, the maximum amplification factor is achieved at an intermediate concentration, and with the SYBR Gold in Example 3, the maximum amplification factor of 2 is achieved at an intermediate concentration.

[0123] From the results of Examples 1 and 2 shown in Figure 16, it was found that the influence of the type of DNA polymerase is small when the types of main mediator and second mediator are the same. On the other hand, it was found that the relationship between the second mediator concentration and the amplification rate varies when the type of second mediator is different. Therefore, in the detection method or detection system of the present invention, the type and concentration of the second mediator can be selected so that the amplification rate becomes the desired value.

[0124] The interaction between the second mediator and the target substance was confirmed by the following measurement. The measurement was carried out under the following conditions. Working electrode: carbon electrode (having a porous thin film layer coated with polyvinyl alcohol (PVA)) Reference electrode: silver-silver chloride electrode Counter electrode: carbon electrode (having a porous thin film layer coated with polyvinyl alcohol (PVA)) AC voltage: 10 mV Frequency: 10 kHz to 0.1 Hz Measurement temperature: 25°C (1) First, R ct / R ct0As in the calculation, EIS measurement of the PCRiF1 solution was performed. (2) Next, a second mediator solution was added to the solution (1) above in an amount such that the second mediator concentration in the solution became 1 μM (except that promethazine hydrochloride was added in an amount such that the second mediator concentration in the solution became 5 μM). After leaving the solution for 30 minutes, EIS measurement of this solution was performed. (3) A PCRiF1 solution containing double-stranded DNA (60 bp) (hereinafter referred to as "double-stranded DNA solution") was added to the solution (2) above in an amount such that the double-stranded DNA concentration in the solution became 0.01 μM. After leaving the solution for 30 minutes, EIS measurement was performed. Next, double-stranded DNA solution was added to various concentrations in the range of double-stranded DNA concentration in the solution to 10 μM, and the solution was left for 30 minutes, after which EIS measurement was performed. (4) From the EIS measurement results, the charge transfer resistance R was calculated by fitting to a Randles circuit. ct (5) R at each concentration of double-stranded DNA was calculated. ct The value of the charge transfer resistance R ct The initial value of the PCRiF1 solution, i.e., the R ct0 The value R normalized by ct / R ct0 The vertical axis is (R ct / R ct0 ) and the horizontal axis was the double-stranded DNA concentration, and graphs were obtained (FIGS. 17A-B).

[0125] The R of the PCRiF1 solution (i.e., a solution with zero second mediator concentration and zero double-stranded DNA concentration) in the above measurement procedure (1) ct / R ct0 The value of R for the solution containing the second mediator at a predetermined concentration in the measurement procedure (2) was 1.0 (not shown in FIGS. 17A and 17B). ct / R ct0 The value of R is measured and calculated in step (ii) of the above example, and decreases or increases from 1.0 depending on the type of second mediator. When the compound of the second mediator shown in FIG. 17A is used, R ct / R ct0It can be seen that the value of R increases with increasing double-stranded DNA concentration. The final concentration of double-stranded DNA synthesized by PCR is approximately 1 μM. When SYBR Green I, SYBR Gold, SYBR safe, and Proflabin are used, the R ct / R ct0 The value of R was significantly higher than that at 0 μM, suggesting that the double-stranded DNA PCR product and the second mediator interacted in solution. Furthermore, among the second mediator compounds shown in Figure 17B, Pico Green, Basic Blue 9 (methylene blue), and Basic Blue 17 also exhibited R ct / R ct0 It can be seen that the value of R increases with increasing double-stranded DNA concentration. ct / R ct0 Dipyrido[3,2-a:2',3'-c]phenazine (DPPZ) and promethazine hydrochloride, whose R values ​​are greater than 0.8 at C2 / C1 = 1 / 1000 and C2 / C1 = 1 / 100, respectively, show no significant difference in R even when the concentration of double-stranded DNA in the solution is increased. ct / R ct0 It can be seen that the change in the value of is small.

[0126] DESCRIPTION OF SYMBOLS 1 Target substance detection system 2 Target substance detection device 3 Electrochemical measurement unit 4 Determination unit 5 Main mediator 6 Second mediator 7 Target substance 8 Solution

Claims

1. A method for detecting a target substance by measuring electrochemical properties of a solution containing at least a target substance not immobilized on an electrode, a main mediator, and a second mediator by electrochemical measurement, the method comprising the steps of: (i) selecting a compound that interacts with the target substance; and (ii) determining a charge transfer resistance R of a solution containing a main mediator at a unique concentration C1. ct0 Charge transfer resistance R of a solution containing the primary mediator at a concentration C1 and the second mediator at a concentration C2 ct Ratio of R ct / R ct0 selecting a compound that satisfies the above condition of 0.80 or less.

2. A method for selecting a second mediator as described in claim 1, wherein the second mediator concentration C2 is less than one-tenth of the main mediator concentration C1.

3. A method for selecting a second mediator as described in claim 1, wherein the second mediator concentration C2 is 1 / 100 or less than the main mediator concentration C1.

4. The method for selecting a second mediator according to claim 1, wherein the target substance is a double-stranded nucleic acid.

5. A method for selecting a second mediator according to claim 4, which is a method for detecting a target substance, comprising one or more steps of amplifying the target substance.

6. Furthermore, (iii) measuring the impedance of the solution before amplifying the target substance and at each amplification step, (ΔZ max -ΔZ min ) / R ctm A step of selecting a compound having an absolute value of more than 0.3%. Here, the charge transfer resistance R after a certain target substance amplification step is ct When the difference ΔZ between the impedance value and the impedance value immediately before the target substance amplification step is calculated, ΔZ max is the maximum absolute value of ΔZ, and R ctm is ΔZ max Charge transfer resistance R at cycle number m ct is the value of ΔZ min is ΔZ max 6. The method for selecting a second mediator according to claim 5, wherein the second mediator is selected from the group consisting of:

7. In the step (iii), the (ΔZ max -ΔZ min ) / R ctm The method for selecting a second mediator according to claim 6, further comprising selecting a compound having an absolute value of more than 1%.

8. In the step (iii), the (ΔZ max -ΔZ min ) / R ctm The method for selecting a second mediator according to claim 6, further comprising selecting a compound having an absolute value of more than 3%.

9. A method for selecting a second mediator used in a method for detecting a target substance by measuring electrochemical characteristics by electrochemical measurement of a solution containing at least a target substance not immobilized on an electrode, a main mediator, and a second mediator, the target substance being a double-stranded nucleic acid, the method for detecting the target substance having one or more steps of amplifying the target substance, the main mediator transferring charge between the working electrode and the second mediator varying the charge transfer, the method including: (i) a step of selecting a compound that interacts with the target substance; and (iii) measuring the impedance of the solution before amplifying the target substance and at each amplification step, and determining (ΔZ max -ΔZ min ) / R ctm A step of selecting a compound having an absolute value of more than 0.3%. Here, the charge transfer resistance R after a certain target substance amplification step is ct and the charge transfer resistance R ct When the difference ΔZ between the value of max is the maximum absolute value of ΔZ, and R ctm is ΔZ max Charge transfer resistance R at cycle number m ct is the value of ΔZ min is ΔZ max The method for selecting a second mediator, wherein the second mediator has a minimum absolute value of ΔZ at a cycle number smaller than the cycle number m at which ....

10. In the step (iii), the (ΔZ max -ΔZ min ) / R ctm The method for selecting a second mediator according to claim 9, further comprising selecting a compound having an absolute value of greater than 1%.

11. In the step (iii), the (ΔZ max -ΔZ min ) / R ctm The method for selecting a second mediator according to claim 9, further comprising the step of selecting a second mediator having an absolute value of more than 3%.

12. The method for selecting a second mediator according to claim 4 or 9, wherein in step (i), a compound is selected that exhibits at least one type of interaction selected from the following group of interactions with the target substance: electrostatic interaction, π-π interaction, insertion between base pairs of a double-stranded nucleic acid that is the target substance, hydrophobic-π interaction, ion-dipole interaction, stem-loop interaction, conformational interaction, coordinate bond, alkyl-π interaction, intramolecular hydrogen bond, solvation effect, intermolecular water (water-water) interaction, magnetic interaction, van der Waals force (London dispersion force), π-anion interaction, molecular dipole-dipole interaction, induced dipole interaction, allosteric interaction, covalent bond network.

13. The main mediator is ferricyanide ion [Fe(CN)6] 3- and ferrocyanide ion [Fe(CN)6] 4- The method for selecting a second mediator according to any one of claims 1 to 11, 14. A method for selecting a second mediator as described in claim 13, wherein in step (i), a compound having at least the structure shown in formula (I) below in one molecule is selected as a compound that interacts with the target substance. (The ring having A in the above formula (I) is a 5- or 6-membered ring condensed with the adjacent benzene ring and is a heterocycle having at least one atom selected from a nitrogen atom, an oxygen atom, and a sulfur atom.) 15. The method for selecting a second mediator according to claim 14, wherein in formula (I), the six-membered ring (benzene ring) adjacent to the heterocycle having A has a nitrogen atom, an oxygen atom or a sulfur atom.

16. The method for selecting a second mediator according to claim 14, wherein formula (I) is any of the following formulas: (A in the above formula is a nitrogen atom, an oxygen atom, or a sulfur atom.) 17. A method for selecting a second mediator according to claim 4 or 9, further comprising the step of: (iv) carrying out sequence-specific double-stranded nucleic acid amplification using a solution containing the target substance, and selecting a second mediator concentration based on the relationship between the second mediator concentration and the amplification rate.

18. The method for selecting a second mediator according to claim 17, wherein in step (iv), a second mediator concentration that results in an amplification rate of 1.4 or more is selected.

19. The method for selecting a second mediator according to any one of claims 1 to 11, wherein the electrochemical measurement method is cyclic voltammetry, chronoamperometry, coulometry, chronopotentiometry, pulse voltammetry, or conductometry.

20. The electrochemical measurement method is an impedance measurement method, and the charge transfer resistance R ct The method for calculating the charge transfer resistance R from EIS is ct or by measuring the charge transfer resistance R ct or the charge transfer resistance R by a semicircular approximation of the Nyquist plot at two frequencies. ct or the charge transfer resistance R ct or the charge transfer resistance R is calculated by the absolute value of the impedance difference at two frequencies. ct or by calculating the charge transfer resistance R by elliptical or circular arc approximation of the Nyquist plot at three frequencies. ct The method for selecting a second mediator according to any one of claims 1 to 11, which is a method for calculating 21. The charge transfer resistance R is calculated by approximating the Nyquist plot of the two frequencies ct A method for calculating the charge transfer resistance R by the absolute value of the difference in impedance at the two frequencies. ct and calculating the charge transfer resistance R by the difference between the real parts of the impedance at the two frequencies. ct 21. The method of claim 20, wherein the impedance measurement is performed using a superimposed waveform that includes frequency components at two frequencies.

22. The charge transfer resistance R is calculated by elliptical or circular arc approximation of the Nyquist plot for the three frequencies. ct 21. The method of claim 20, wherein the impedance measurement is performed using a superimposed waveform that includes frequency components at three frequencies.

23. The method of selecting a second mediator according to claim 20, wherein any of said frequencies is below 30 Hz.

24. The electrochemical measurement method is applied to at least one sequence-specific double-stranded nucleic acid detection selected from the following group: PCR, LAMP (Loop-mediated isothermal Amplification), SDA (Strand Displacement Amplification), NEAR (Nicking Endonuclease Amplification Reaction), NASBA (Nucleic Acid Sequences Based Amplification), TMA (Transcription Mediated Amplification), RPA (Recombinase Polymerase Amplification), SIBA (Strand-Invasion Based Amplification), HDA (Helicase-dependent Amplification), WGA (Whole Genome Amplification), MDA (Multiple Displacement Amplification), DNA chip, DNA microarray, NGS (Next Generation Sequencing), ChIP (Chromatin Immunoprecipitation), ChIP-on-chip, and ChIP-seq (chromatin immunoprecipitation with high-throughput sequencing), A method for selecting a second mediator according to any one of claims 1 to 11.

25. A method for selecting a second mediator described in any one of claims 1 to 11, wherein at least the working electrode of the electrochemical measurement method is provided with a porous thin film layer through which the main mediator and the second mediator can pass.

26. A method for detecting a target substance by measuring the electrochemical characteristics of a solution containing at least a target substance not fixed to an electrode, a main mediator, and a second mediator, by electrochemical measurement, the method comprising one or more steps of amplifying the target substance, the main mediator transferring charge between the main mediator and a working electrode, and the second mediator varying the charge transfer, the second mediator being a compound that interacts with the electrode surface.

27. The detection method according to claim 26, wherein the second mediator is a compound having at least one of the structures represented by the following formulas in one molecule: (A in the above formula is a nitrogen atom, an oxygen atom, or a sulfur atom.) 28. The detection method according to claim 26 or 27, wherein the target substance is a double-stranded nucleic acid, and the solution for detecting the target substance further contains a primer and a polymerase.

29. The detection method according to claim 26, wherein the step of amplifying the target substance and the step of measuring the electrochemical property are carried out in the same solution.

30. A target substance detection system comprising a detection device for a target substance, and a main mediator and a second mediator contained in a solution that is a measured object that may contain the target substance, and which detects the target substance by measuring the electrochemical characteristics of the solution by an electrochemical measurement method using an electrochemical measurement unit of the target substance detection device, the detection system having one or more steps of amplifying the target substance, wherein the main mediator transfers charge between the target substance and a working electrode, and the second mediator fluctuates the charge transfer, and the second mediator is a compound that interacts with the electrode surface.

31. The detection system according to claim 30, wherein the second mediator is a compound having at least one of the structures represented by the following formulas in one molecule: (A in the above formula is a nitrogen atom, an oxygen atom, or a sulfur atom.) 32. The detection system according to claim 30 or 31, wherein the target substance is a double-stranded nucleic acid, and the solution for detecting the target substance further comprises a primer and a polymerase.

33. The detection system according to claim 30, wherein the step of amplifying the target substance and the step of measuring the electrochemical property are carried out in the same solution.

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