Method for measuring changes in pyrophosphate ion concentration in polymerase chain reactions by using electrochemical methods

Abstract

The present invention relates to a method for measuring changes in pyrophosphate ion concentration in polymerase chain reactions by using electrochemical methods. According to the present invention, the disclosed PCR amplification measurement method comprises the steps of: injecting, into a PCR container, a PCR sample including a template nucleic acid and a PCR reagent; mounting, in the PCR container, a connector to which an electrode is connected; performing PCR in the PCR container; and using an electrochemical sensor device to measure an electrochemical signal generated in the step of performing PCR. The present invention can sensitively and accurately measure DNA amplification. In addition, amplification can be measured regardless of structural shape such as a double or single strand of nucleic acid, and since measurement is performed using EIS, which is an electrochemical method, equipment can be miniaturized and gene amplification can be accurately detected during PCR. In particular, since changes in pyrophosphate exponentially generated in a PCR amplification process can be measured, sensitivity significantly higher than that of a conventional electrochemical measurement method can be achieved. Therefore, the PCR amplification measurement method of the present invention solves the limitations of a conventional rapid antigen diagnosis method and fluorescence measurement-based PCR gene diagnosis method, and thus can be utilized as a foundational technique for electrochemical PCR gene diagnosis.

Description

Method for measuring changes in pyrophosphate ion concentration in polymerase chain reaction using an electrochemical method

[0001] This application claims the benefit of priority to Korean Patent Application No. 10-2023-0069187, filed May 30, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a method for measuring changes in pyrophosphate ion concentration in a polymerase chain reaction using an electrochemical method.

[0003] In general, PCR (Polymerase Chain Reaction) is a method that uses a double-stranded DNA template to simultaneously amplify a specific base sequence at a primer annealing site. Specifically, PCR is a method that amplifies a desired DNA molecule in a test tube by repeatedly synthesizing a specific DNA region. This is considered a groundbreaking technology in molecular biology because it can amplify a large amount of DNA using a very small amount of DNA.

[0004] Through this type of PCR, DNA of a specific region can be extracted in vitro by 10 5 ~ 10 8 PCR can amplify DNA in a short period of time, and the amplified DNA can be used in a variety of experiments. In particular, PCR is widely used in genetic disease diagnosis, early cancer gene detection, early viral gene detection, genetic engineering research, and infectious disease diagnosis, and its scope of application is gradually expanding.

[0005] In recent years, in the event of an infectious disease pandemic like COVID-19, accurate and rapid diagnosis of infected individuals is crucial. While rapid antigen tests exist, their low sensitivity limits their accuracy. In contrast, PCR tests, which bind complementary DNA primers to template DNA extracted from a suspected positive individual and amplify specific base sequences, enable diagnosis even with small amounts of antigen. In particular, fluorescent optics-based real-time PCR technology has been widely used in COVID-19 diagnosis due to its high accuracy and utility in genetic detection.

[0006] However, fluorescence-based real-time PCR technology requires expensive, dedicated fluorescence monitoring equipment, complex optical systems, and large, heavy equipment, making on-site diagnosis difficult. Furthermore, in addition to conventional primers, the design of a probe specifically for the target genetic sequence and conjugated with a fluorescent dye is required, leading to high costs for PCR performance and nucleic acid detection.

[0007] Therefore, there is a need for a PCR amplification technology based on an electrochemical signal sensor that is easy to manufacture, economical, and miniaturizable.

[0008] Under this background, the present inventors designed a PCR amplification measurement method that can electrochemically detect gene amplification using pyrophosphatase (PPase), and measured the change according to the interaction of PCR reagents and PCR by-products during PCR using electrochemical impedance spectroscopy (EIS) to determine the charge transfer resistance (R) as the PCR cycle increases. ct) was confirmed to be able to sensitively measure DNA amplification by changing the PCR amplification measurement method of the present invention. In addition, the present invention was completed by confirming that the result of the PCR amplification measurement method of the present invention is similar to that of the existing real-time polymerase chain reaction (real-time PCR, quantitative PCR, qPCR).

[0009] Accordingly, the purpose of the present invention is to provide a PCR amplification measurement method and a real-time PCR device that have excellent stability and sensitivity over a long period of time and can indirectly measure PCR amplification products.

[0010] The various problems to be solved by the present invention are not limited thereto, but can be clearly understood by a person having ordinary knowledge in the field to which the present invention pertains through the matters described in the embodiments and claims described below.

[0011] Additionally, terms not specifically defined herein should be understood to have the meanings commonly used in the technical field to which the present invention pertains. Furthermore, unless specifically defined in context, the singular includes the plural, and vice versa.

[0012] According to the present invention for achieving the above object, a PCR amplification measurement method is provided, comprising the steps of: injecting a PCR sample containing a template nucleic acid and a PCR reagent into a PCR vessel; mounting a connector connected to an electrode on the PCR vessel; performing PCR using the PCR vessel; and measuring an electrochemical signal generated in the step of performing the PCR using an electrochemical sensor device.

[0013] Here, the electrode may be at least one selected from the group consisting of gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes, graphene, and carbon.

[0014] Additionally, the PCR reagent may include at least one selected from the group consisting of a redox reagent, magnesium chloride, dNTP (deoxynucleotide triphosphate), a primer pair, Taq polymerase, a PCR buffer, and pyrophosphatase (PPase).

[0015] Additionally, the redox reagent may be at least one selected from the group consisting of ferricyanide, ferrocyanide, ferrocene derivatives, ruthenium derivatives, osmium derivatives, and methylene blue.

[0016] In addition, the pyrophosphatase may be at least one selected from a thermostable inorganic pyrophosphatase and a pyrophosphatase that operates at room temperature.

[0017] Additionally, the ferricyanide and ferrocyanide may be included in the PCR reagent at a concentration of 0.1 to 10.0 mM.

[0018] Additionally, the magnesium chloride may be included in the PCR reagent at a concentration of 0.1 to 10.0 mM.

[0019] Additionally, the dNTP may be included at a concentration of 0.1 to 10.0 mM.

[0020] Additionally, the PCR reagent may have a concentration ratio of magnesium chloride / dNTP of 0.5 to 5.

[0021] In addition, the PCR sample may be one or more selected from the group consisting of saliva, urine, tears, sweat, blood, and interstitial fluid. The PCR sample contains template nucleic acid, and the template nucleic acid can be extracted from the PCR sample using a general cell lysis and nucleic acid extraction method known in the art to which the present invention pertains.

[0022] Additionally, the electrochemical sensor device may be selected from the group consisting of a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV), a differential pulse voltammetry (DPV), and an impedance meter.

[0023] In addition, the step of mounting a connector on the PCR container may be such that a connector having an electrode protruding from the PCR container into which the PCR sample and PCR reagent are injected is mounted so that the end of the electrode connected to the electrochemical sensor device is immersed in the PCR sample and PCR reagent.

[0024] In addition, the electrochemical signal may be an electrical measurement that changes as PCR is performed. Here, the electrical measurement may be at least one of voltage, current, and resistance measured by an electrochemical sensor device, and preferably, charge transfer resistance (R ct ) may be.

[0025] Additionally, the step of performing the PCR may be to change the denaturation step temperature, the annealing step temperature, and the extension step temperature of the PCR in the PCR container, and this may be performed repeatedly, for example, two or more times.

[0026] PCR amplification measured by the method according to the present invention includes, but is not limited to, PCR amplification product, or quantitative amount or degree thereof.

[0027] In addition, the PCR amplification measurement method according to the present invention may be, for example, a pyrophosphate measurement method.

[0028]

[0029] Meanwhile, according to the present invention, a PCR method performed by the PCR amplification measurement method is proposed. In the present invention, the PCR method may include general PCR, isothermal amplification PCR (LAMP, Loop-Mediated Isothermal Amplification), RCA (Rolling Circle Amplification), RPA (Recombinase Polymerase Reaction), and CRISPR-related Amplifications (Kang, Tongjia, et al. "Advances in nucleic acid amplification techniques (NAATs): COVID-19 point-of-care diagnostics as an example." Biosensors and Bioelectronics (2022): 114109).

[0030] Meanwhile, according to the present invention, a real-time PCR device is provided, including: a PCR container into which a PCR sample containing a template nucleic acid and a PCR reagent are injected; a connector having an electrode provided so as to protrude into the interior of the PCR container into which the PCR sample and the PCR reagent are injected so that the terminal is immersed in the PCR sample and the PCR reagent; and an electrochemical sensor device electrically connected to the electrode and measuring an electrochemical signal generated inside the PCR container.

[0031] Here, the real-time PCR device may be configured to change the denaturation step temperature, the annealing step temperature, and the extension step temperature of the PCR container, and this may be performed repeatedly, for example, two or more times.

[0032] Additionally, the electrode may be at least one selected from the group consisting of gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes, graphene, and carbon.

[0033] Additionally, the PCR reagent may include at least one selected from the group consisting of a redox reagent, magnesium chloride, dNTP, a primer pair, Taq polymerase, a PCR buffer, and pyrophosphatase.

[0034] Additionally, the redox reagent may be at least one selected from the group consisting of ferricyanide, ferrocyanide, ferrocene derivatives, ruthenium derivatives, osmium derivatives, and methylene blue.

[0035] Additionally, the ferricyanide and ferrocyanide may be included in the PCR reagent at a concentration of 0.1 to 10.0 mM.

[0036] Additionally, the magnesium chloride may be included in the PCR reagent at a concentration of 0.1 to 10.0 mM.

[0037] Additionally, the dNTP may be included at a concentration of 0.1 to 10.0 mM.

[0038] Additionally, the PCR reagent may have a concentration ratio of magnesium chloride / dNTP of 0.5 to 5.

[0039] Additionally, the PCR sample may be one or more selected from the group consisting of saliva, urine, tears, sweat, blood, and interstitial fluid.

[0040] Additionally, the electrochemical sensor device may be selected from the group consisting of a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV), a differential pulse voltammetry (DPV), and an impedance meter.

[0041] As described above, according to the present invention, since DNA is not involved in the measurement mechanism, DNA amplification can be measured sensitively and accurately regardless of the adsorption of macromolecules to the electrode surface. In addition, amplification can be measured regardless of the structural shape of the nucleic acid, such as double or single strands, and since the measurement is performed using an electrochemical method, EIS, miniaturization of the equipment is possible and gene amplification can be accurately detected during PCR. In particular, since the change in pyrophosphate generated exponentially during the PCR process can be measured, significantly higher sensitivity can be achieved than the existing electrochemical measurement method. Therefore, the PCR amplification measurement method of the present invention can be utilized as a basic technology for electrochemical PCR gene diagnosis by overcoming the limitations of the existing rapid antigen diagnosis method and fluorescence measurement-based PCR gene diagnosis method.

[0042] The effects of the present invention are not limited to the above, and other effects of the present invention can be clearly understood by a person having ordinary skill in the art to which the present invention pertains through the matters described in the following examples and claims.

[0043] Figure 1 illustrates the PCR container, sensor electrode, and connector of the present invention. (A) Assembled photo (B) Disassembled photo.

[0044] Figure 2 schematically illustrates a real-time PCR measurement mechanism using pyrophosphatase (PPase) of the present invention.

[0045] Figure 3 is an electrophoresis photograph confirming the presence or absence of DNA amplification according to the concentration of ferricyanide and ferrocyanide of the present invention.

[0046] Figure 4 shows the charge transfer resistance (R) according to the concentration of ferricyanide, ferrocyanide, and magnesium chloride of the present invention. ct ) shows the change in the reduction rate. (■: [Fe(CN)6] 3- / 4- 1mM / 1mM, ●: [Fe(CN)6] 3- / 4- 3mM / 3mM, ▲: [Fe(CN)6] 3- / 4- 5mM / 5mM).

[0047] Figure 5 is a Nyquist plot and R showing the impedance change due to dNTP, pyrophosphate ion (PPi), and phosphate ion (Pi) of the present invention. ct This graph shows the increase rate of [Fe(CN)6]. (A) Nyquist plot showing the impedance change due to each molecule. 3- / 4-When 1 mM / 1 mM is present, from the bottom graph, a: Add 1.5 mM MgCl2, b: Add 1.5 mM MgCl2 and 2 mM Pi, c: Add 1.5 mM MgCl2 and 1 mM dNTP, d: Add 1.5 mM MgCl2 and 1 mM PPi. (B) R by each molecule ct The growth rate of .

[0048] Figure 6 shows photographs and graphs showing the feasibility of a real-time PCR measurement mechanism using pyrophosphatase (PPase) of the present invention. The experiment was conducted with a composition of 1.5 mM MgCl2 and 1 mM dNTP. (A) Electrophoresis photographs showing the degree of DNA amplification for samples of each cycle. (B) Nyquist plot for samples of each cycle. (C) R according to the presence or absence of Lambda template. ct Comparison graph of reduction rate data and fluorescence data measured using qPCR. (▲: w / lambda template (1 ng), ●: w / o lambda template, solid line: w / lambda template (1 ng) using qPCR).

[0049] Figure 7 shows the DNA amplification efficiency and R according to the change in the concentration of magnesium chloride of the present invention. ct It shows the change in the rate of decrease.

[0050] [Fe(CN)6] 3- / 4- The experiment was conducted with a composition of 1 mM / 1 mM, dNTP 1 mM, and PPase 1 U (Unit). (A) Mg 2+ Electrophoresis images showing the extent of DNA amplification at different concentrations. (B) Mg 2+ R varies depending on concentration ct Decrease rate graph.

[0051] Figure 8 shows the DNA amplification efficiency and R according to the change in dNTP concentration of the present invention. ct This shows the change in reduction rate. [Fe(CN)6] 3- / 4-The experiment was conducted with a composition of 1 mM / 1 mM, 1.5 mM MgCl2, and 1 U of PPase. (A) Electrophoresis images showing the degree of DNA amplification according to dNTP concentration. (B) R varying with dNTP concentration. ct Decrease rate graph.

[0052] Figure 9 is a graph comparing real-time PCR measurement and qPCR quantitative analysis using the phosphatase of the present invention. [Fe(CN)6] 3- / 4- The experiment was conducted with a composition of 1 mM / 1 mM, MgCl2 1.5 mM, dNTP 1 mM, and PPase 1 U. (A) R according to the PCR cycle ct Graph showing the growth rate. (◆: w / o PPase, w / lambda template (1ng), ▲: w / PPase, w / lambda template (1ng), ■: w / PPase, w / o lambda template). (B) Actual R corrected according to the correction formula. ct Comparison graph of reduction rate data and fluorescence data measured using qPCR.

[0053] Figure 10 shows the R of the present invention according to the amount of DNA amplification. ct This graph shows the change in the rate of decrease. (■: 1.83*10 7 copies, ▲: 1.83*10 8 copies, ●: 1.83*10 9 copies).

[0054] To facilitate understanding of the present invention, specific examples will be provided below. However, the following examples are intended to illustrate the present invention more specifically and should not be construed as limiting the present invention. Modifications common to those skilled in the art are possible within the scope of the present invention.

[0055] In addition, terms, techniques, etc. described in this specification should be understood as having the meanings commonly used in the technical field to which the present invention belongs, unless there are special limitations.

[0056] The present invention relates to a method for measuring changes in pyrophosphate ion concentration in a polymerase chain reaction using an electrochemical method.

[0057]

[0058] Example 1

[0059] 1-1. Preparation of materials and reagents

[0060] K3Fe(CN)6 and K4Fe(CN)6, MgCl2, Na4P2O7, and Na2HPO4 were purchased from Deoksan General Science (Korea). PCR 10x buffer (Tris 100 mM, KCl 400 mM, pH 9.0), dNTP 10 mM, and Taq Polymerase used as PCR reagents were purchased from Bioneer (Korea). Pyrophosphatase (PPase) used for electrochemical real-time PCR measurement was purchased from Enzynomics (Korea). Thin-film platinum single electrode used in the feasibility study was purchased from Micrux (Spain), and 0.2 mm diameter, 99.99% Pt wire used for real-time PCR measurement using PPase was purchased from MadeLAB (Korea). Distilled water with a resistivity of 18.2 MΩ cm was used in all experiments.

[0061]

[0062] 1-2. PCR (Polymerase chain reaction)

[0063] All PCR solutions were prepared according to the protocol provided by Bioneer. Except for dNTPs and MgCl2, the remaining ingredients were used at the same concentrations as listed in Table 1 below. Lambda DNA was used as the template DNA for all PCR experiments, and the sequences of the primer pairs (Forward: SEQ ID NO: 1, Reverse: SEQ ID NO: 2) are as follows.

[0064] Forward: 5′-GAAGCGTTTATGCGGAAGAG

[0065] Reverse: 5′-TGACTCCTGTTGATAGATCCAGT

[0066] First, DNA was denatured at 90°C for 3 minutes, and each cycle consisting of three steps (denaturation step: 90°C for 30 seconds, annealing step: 55°C for 30 seconds, extension step: 72°C for 30 seconds) was performed approximately 30 times. Then, an additional extension step was performed at 72°C for 3 minutes.

[0067] ComponentTotal reaction volume: 50μLTris-HCl (pH 9)10mMKCl40mMMgCl21.5mMEach: Ferricyanide / Ferrocyanide1mMEach: dNTP (dATP, dCTP, dGTP, dTTP)250μMTemplate DNA1ngEach: Forward primer, Reverse primer10pmoleTaq polymerase5UPyrophosphatase1U

[0068]

[0069] 1-3. Electrophoresis

[0070] Experiments were conducted using an electrophoresis device from Bio-Rad Laboratories, Inc. (US). Agarose gels were prepared using 1.5 wt% EtBr 40,000x. Electrophoresis was performed at 100 V for 1 hour. Electrophoresis results were analyzed using DuxGeldoc from Biomedux (Korea).

[0071]

[0072] 1-4. Electrochemical Impedance Spectroscopy (EIS) measurements

[0073] All electrochemical measurements were performed using a VersaSTAT3 (AMETEK, Inc. US) cyclic voltammetry device with a two-electrode setup at an AC voltage amplitude of 10 mV and a voltage frequency range of 10 kHz to 1 Hz. All EIS measurements were performed on [Fe(CN)6] 3- / 4- It was measured with added concentrations, and different concentrations were used depending on the experiment.

[0074]

[0075] 1-4-1. Verification of the Measurement Mechanism Using a Thin-Film Platinum Single Electrode

[0076] Measurements were performed using a Thin-Film Platinum Single Electrode from Micrux Technologies (Spain). Experiments were conducted with 30 μL samples placed in a dedicated chamber. To enhance the reliability of the experimental results, the following cleaning process was performed, based on literature review on changes in electrode stability depending on cleaning method.

[0077] First, the electrodes were incubated for 30 minutes with 1% SDS, followed by sonication in 10% (v / v) nitric acid for 10 minutes. After drying with nitrogen gas, UV ozone cleaning was performed for 10 minutes. In all experiments, the electrodes were used immediately after cleaning.

[0078]

[0079] 1-4-2. Electrochemical real-time PCR measurement

[0080] To measure EIS during real-time PCR, a system capable of repeated measurements within a conventional thermocycler was required. Therefore, we developed and used an electrode guide (Figure 1) that secures the electrodes within the PCR vessel and an electrode connector that connects the electrodes to the measuring device outside the PCR vessel.

[0081] A 0.2 mm diameter Pt wire was cut to 4 cm and used as the working and counter electrodes, respectively. To prevent evaporation of the solution during PCR, 30 μL of mineral oil purchased from Sigma-Aldrich (US) was used.

[0082] To increase the reliability of the experimental results, the Pt wire electrode was also subjected to the same cleaning process as the Thin-film Platinum Single Electrode above before the experiment, and was used immediately after cleaning.

[0083]

[0084] 1-5. qPCR real-time measurement

[0085] Measurements were performed using the CFX Connect Real-Time PCR Detection System from Bio-rad (US). SYBR® Green I nucleic acid gel stain purchased from Sigma-Aldrich (US) was added to the PCR reaction solution to measure real-time fluorescence amplification. All qPCR processes were performed identically to the preceding PCR process.

[0086]

[0087] Example 2

[0088] 2-1. Principle of real-time PCR measurement mechanism using PPase

[0089] Figure 2 shows a mechanism for measuring DNA amplification in real time using PPase. To understand this mechanism, Mg 2+ And we need to learn about the interactions between the four molecules.

[0090]

[0091] 2-1-1. Mg 2+ and [Fe(CN)6] 3- / 4- Interaction of

[0092] [Fe(CN)6] 3- / 4- When there is K + As the concentration of increases, the charge transfer resistance (R) ct ) decreases. Similarly, Mg 2+ [Fe(CN)6] 3- / 4- Interact with R ct Reduces .

[0093]

[0094] 2-1-2. Mg 2+ Interactions of dNTP, PPi, and Pi

[0095] Mg 2+ It strongly interacts with dNTPs required for the PCR process and pyrophosphate ion (PPi), a byproduct of PCR. Mg2+ The strong interaction between PPi and Mg 2+ It can be inferred from the fact that PPi forms a precipitate. Therefore, [Fe(CN)6] 3- / 4- Wow Mg 2+ As the concentration of dNTP or PPi in the solution increases, Mg 2+ This [Fe(CN)6] 3- / 4 In addition, it interacts strongly with dNTP and PPi, thereby forming R ct increases. However, unlike dNTP and PPi, phosphate ion (Pi) is Mg 2+ Because it has a weak interaction with R ct The rate of increase is not large.

[0096] The interactions described above serve as the core concepts of the real-time PCR measurement mechanism. The real-time PCR measurement mechanism using PPase consists of the following three steps, which enable sensitive measurement of DNA amplification.

[0097] Step 1 (Figure 2 Step 1):

[0098] This step simply represents the PCR reaction solution before DNA amplification. Mg 2+ dNTP and [Fe(CN)6] 3- / 4- interacts competitively with a relatively large R ct is measured.

[0099] Second step (Fig. 2 Step 2):

[0100] When DNA is synthesized, dNTPs are used as reactants and PPi is produced as a product. Like dNTPs, Mg 2+ PPi and [Fe(CN)6] 3- / 4- interacts competitively with a relatively large R ct is measured. Therefore, R similar to the previous step ct is measured.

[0101] Third step (Figure 2 Step 3):

[0102] When PPase hydrolyzes PPi to Pi, Mg 2+ is [Fe(CN)6] than Pi 3- / 4- It interacts more strongly with R ct is lowered. Therefore, R is lowered every time one cycle of PCR passes. ct is reduced, and the degree of DNA amplification can be sensitively measured.

[0103]

[0104] 2-2. Verification of the real-time PCR measurement mechanism using PPase

[0105] To verify the operation of the real-time PCR measurement mechanism using PPase [Fe(CN)6] 3- / 4- We investigated the effect of Mg on PCR. In addition, Mg, which plays a key role in the real-time PCR measurement mechanism, 2+ and [Fe(CN)6] 3- / 4- , dNTP, PPi, and Pi were investigated. Finally, the feasibility of real-time PCR measurement using PPase was investigated using a thin-film platinum single electrode.

[0106]

[0107] 2-2-1. [Fe(CN)6] in the PCR process 3- / 4- The impact of

[0108] [Fe(CN)6] 3- / 4- There are cases where [Fe(CN)6] was applied to the PCR system, but 3- / 4- There is no study yet specifically addressing the effect of [Fe(CN)6] on the PCR system. 3- / 4- In this study, [Fe(CN)6] can inhibit DNA amplification in PCR solution. 3- / 4-It is important to determine the effect of different concentrations on the PCR system. Therefore, PCR was performed by adding 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM of ferricyanide and ferrocyanide to the PCR solution, respectively, and the results are shown in Figure 3.

[0109] 3 mM ferricyanide, 3 mM ferrocyanide ([Fe(CN)6] 3- / 4- It can be seen that DNA amplification is inhibited from 3mM / 3mM (Fig. 3). This is because Mg is used as a cofactor for Taq polymerase. 2+ and high concentration of [Fe(CN)6] 3- / 4- Due to the interaction of Mg 2+ This phenomenon appears to occur because Mg fails to act as a cofactor. 2+ It appears to occur by a mechanism similar to that of EDTA, Tannic acid, etc., which act as PCR inhibitors by capturing DNA. Therefore, in order to prevent DNA amplification from being inhibited in a real-time PCR measurement system, 3 mM ferricyanide and 3 mM ferrocyanide ([Fe(CN)6] 3- / 4- Less than 3mM / 3mM) should be applied, for example, less than 2mM ferricyanide and less than 2mM ferrocyanide.

[0110]

[0111] 2-2-2. Mg 2+ and [Fe(CN)6] 3- / 4- The interaction of R ct Impact on

[0112] [Fe(CN)6] 3- / 4- is generally sensitive to cations in the system. The thermal and electrochemical properties of this phenomenon are investigated, or Li using cyclic voltammetry (CV). + , K + Although research has been conducted on Mg 2+There has been no study conducted on [Fe(CN)6] using EIS method. Therefore, for the purpose of this study, 3- / 4- Mg by concentration 2+ R by concentration ct There is a need to investigate the rate of decline.

[0113] Mg in Fig. 4 2+ This [Fe(CN)6] 3- / 4- Interact with R ct You can see that it is lowering. The important thing to note in this graph is that [Fe(CN)6] 3- / 4- Mg depending on concentration 2+ R by concentration ct The difference is the reduction rate. [Fe(CN)6] 3- / 4- The higher the concentration of R ct The absolute value of the slope of the reduction rate decreases. This is because [Fe(CN)6] 3- / 4- High concentration of Mg 2+ Instead of being able to measure resistance changes over a range of concentrations, the measurement sensitivity [ΔR ct Decrease rate / ΔMg 2+ The concentration] decreases, so Mg 2+ This means that the concentration change of [Fe(CN)6] cannot be measured sensitively. Conversely, [Fe(CN)6] 3- / 4- When the concentration is low, the measurement sensitivity [ΔR ct Decrease rate / ΔMg 2+ [Concentration] increases and becomes sensitive to Mg 2+ Instead of being able to measure changes in concentration, a narrow Mg 2+ This means that resistance changes can only be measured over a range of concentrations.

[0114]

[0115] 2-2-3. [Fe(CN)6] 3- / 4- When Mg is present, 2+ The interaction between dNTP, PPi, and Pi is R ct Impact on

[0116] [Fe(CN)6] 3- / 4- When 1mM / 1mM is in solution, Mg2+ R by interaction of dNTP and PPi ct The change in R of the sample containing only MgCl2 was investigated. As can be seen in the c and d graphs of Fig. 5 (A), the R of the sample containing only MgCl2 ct (R of the sample with 1 mM dNTP added compared to a) in Fig. 5 (A) ct (c in Fig. 5 (A)) increased by 132.9±10.4%, and R of the sample with 1 mM PPi added ct (d in Fig. 5 (A)) increased by 197.4±10.8% (Fig. 5 (B)). R shown here ct The increase of [Fe(CN)6] 3- / 4- Interact with R ct Mg, which played a role in lowering 2+ This phenomenon appears to be caused by a stronger interaction between dNTP and PPi. In contrast, even when 2 mM of Pi is added (b in Fig. 5 (A)), R ct The increase rate is not large at 32.7±3.1% (Fig. 5 (B)). This means that Pi is not as Mg as dNTP or PPi. 2+ It appears to be a phenomenon that occurs due to the inability to interact strongly with R. Therefore, as DNA amplification occurs by the real-time PCR measurement mechanism using PPase described above, ct It was confirmed that the decrease was

[0117]

[0118] 2-2-4. Feasibility Study on a Real-Time PCR Measurement Mechanism Using PPase

[0119] To confirm the feasibility of the above mechanism, three different experiments were conducted to compare and verify the results. The comparison was conducted through electrophoresis, qPCR, and electrochemical measurements, and [Fe(CN)6] was added to the PCR solution. 3- / 4-The experiment was conducted by adding 1 mM / 1 mM and PPase 1 U (Unit). The PCR was stopped every 3 cycles at the annealing stage (55°C), and the samples taken out (0, 3, 6, …, 30 cycles) were subjected to electrophoresis and EIS measurements. The fluorescence intensity increased at the 9th cycle in the electrophoresis image, and it was maintained from the 21st cycle (Fig. 6 (A)). The R of each cycle ct Looking at Figure 6 (C) showing the reduction rate, the trend of change in electrophoretic fluorescence intensity is similar to that of R from 9 cycles (-1.76±0.97%) to 21 cycles (-18.5±1.2%). ct We can see that the rate of decrease changes rapidly. Furthermore, this graph shape is similar to that seen in commercial qPCR (Figure 6 (C)). This demonstrates that the real-time PCR measurement mechanism using PPase is in operation.

[0120]

[0121] 2-3. Optimization of experimental variables

[0122] 2-3-1. Mg 2+ Concentration optimization

[0123] Mg, which plays a key role in the real-time PCR measurement mechanism using PPase to improve the measurement sensitivity 2+ Optimization of the concentration of Mg is required. Therefore, 2+ After PCR amplification by concentration of R ct The reduction rate was measured. In the electrophoresis image, Mg 2+ As the concentration increases, the amount of DNA amplification by PCR increases (Fig. 7 (A)). However, R ct It can be seen that the reduction rate is not proportional to the amount of DNA amplification (Fig. 7 (B)).

[0124] First, Mg 2+ When the concentration of MgCl2 is 0.5 mM [MgCl2 / dNTP= 0.5], Mg 2+It appears that the concentration of R is low and therefore amplification is not possible. ct The decrease rate is -0.07%, and we can see that there is almost no change in resistance. Mg 2+ In the range of 1~4mM [MgCl2 / dNTP = 1~4], the amount of DNA amplification increased, but R ct It can be confirmed that the reduction rate is decreasing from -27.2±0.23% to -1.32±1%. This phenomenon is due to [Fe(CN)6] not being captured by dNTP. 3- / 4- Mg interacting with 2+ The more this happens, the more R ct The experimental results of Example 2-2-2, in which the rate of change is rapidly reduced, and 1 mM ferricyanide and 1 mM ferrocyanide ([Fe(CN)6] of Fig. 4 3- / 4- It was found to be consistent with the range of MgCl2 1 mM or more at 1 mM / 1 mM. In conclusion, it was found that the amplification efficiency was not affected in the PCR solution, and R ct Mg with a high reduction rate 2+ It can be seen that the concentration is 1 mM [MgCl2 / dNTP = 1].

[0125]

[0126] 2-3-2. Optimizing dNTP concentration

[0127] It is well known that high concentrations of dNTPs act as inhibitors in PCR systems. For this reason, optimization by increasing the concentration of dNTPs may seem unnecessary. However, as the concentration of dNTPs increases, [Fe(CN)6] 3- / 4- Mg that can interact with 2+ Because decreasing the concentration can positively affect measurement sensitivity, a process of increasing the dNTP concentration to find the optimal value is necessary. Therefore, PCR was performed with 0.5–2.5 mM dNTPs, followed by electrophoresis and EIS measurements.

[0128] When the concentration of dNTP is 2 mM or 2.5 mM, DNA amplification is inhibited and R ct It can be confirmed that the reduction rate is small at -3.18±1.4% and -3.26±0.55% (Fig. 8 (A), (B)). In contrast, at 0.5 to 1.5 mM dNTP, R increases as the concentration increases. ct It can be confirmed that the reduction rate also increases from -12.2±0.86% to -27.3±0.3% (Fig. 8 (B)). What should be noted here is that, although the concentrations of MgCl2 and dNTP are different from those in the experiment conducted in 2-3-1 above, the largest R is obtained when the ratio of MgCl2 / dNTP is 1. ct The point is that it shows a decreasing rate. Through this result, R ct It can be inferred that the maximum reduction rate is determined not by a specific concentration of MgCl2 or dNTP, but by the ratio of the two substances. Another point to note is that in both the experiments conducted in Example 2-3-1 and Example 2-3-2, PCR was completely inhibited when MgCl2 / dNTP was less than 1. This means that the concentration of dNTP was Mg 2+ When the concentration of dNTP is greater than (MgCl2 / dNTP < 1), dNTP is converted to Mg 2+ It appears that the MgCl2 / dNTP ratio is more than 1 for PCR to occur, and the maximum R ct It was confirmed that the MgCl2 / dNTP ratio must be 1 to obtain a reduction rate.

[0129]

[0130] 2-4. Real-time PCR measurement using PPase

[0131] By applying the above results, EIS was measured in real time while PCR was being performed. ct Changes were observed. R by PCR cycle in the previous feasibility study ctIt can be seen that the reduction rate graph (Fig. 6 (C)) and the real-time PCR measurement graph using PPase (Fig. 9 (A)) are different. This appears to be due to the measurement being performed at high temperatures, resulting in the adsorption of large molecules such as DNA onto the electrode surface.

[0132] Actual R ct Rate of change = w / PPase R ct Rate of change - w / o PPase R ct rate of change

[0133] If the change rate is corrected by the above formula to remove the effect of adsorption, a graph modification like Figure 9 (B) can be obtained. This is similar to the fluorescence graph obtained using qPCR. In addition, the R for each PCR cycle of the feasibility study ct It can be confirmed that the graph shape is the same as the reduction rate graph (Figure 6 (C)).

[0134] This type of electrode adsorption of macromolecules has been demonstrated in numerous papers. Furthermore, various methods (protein coating, polymer coating) have been proposed to prevent electrode adsorption noise. However, the measurement mechanism in this study operates independently of macromolecule electrode adsorption. Therefore, this experiment verified that it can ignore electrode adsorption noise and sensitively measure DNA amplification.

[0135] Measure EIS in real time to determine R for each amount of amplified DNA ct The change was confirmed. The sensitivity of the PCR amplification measurement method of the present invention is 10 in only 25 cycles of amplification. 7 It was possible to detect up to copy (Fig. 10).

[0136] The sensitivity of the existing electrochemical detection method is not very high due to the limitations of the detection method. However, according to the results of a previous paper developed by measuring the concentration of hydrogen ions rather than pyrophosphate, it is 10 8DNA could be detected even in copies. [Veigas, Bruno, et al. "Ion sensing (EIS) real-time quantitative monitoring of isothermal DNA amplification." Biosensors and Bioelectronics 52 (2014): 50-55.]

[0137] The sensitivity of the present invention is 10 times higher than this with only 25 cycles of amplification. 7 It was possible to detect even copy.

[0138]

[0139] In the present invention, a system capable of electrochemically detecting gene amplification using PPase was designed and verified. [Fe(CN)6] was detected through EIS measurement. 3- / 4- , dNTP, PPi, Pi, and Mg 2+ The interaction between R and PPase was confirmed, and the measurement mechanism was verified by applying PPase to the PCR system. In conclusion, as the PCR cycle increases, R ct It was confirmed that the amount of DNA amplification could be sensitively measured by reducing the amount of DNA. In addition, the results analyzed using this measurement mechanism were confirmed to be similar to those of conventional quantitative real-time polymerase chain reaction (qPCR).

[0140] This measurement mechanism can sensitively and accurately measure DNA amplification regardless of the adsorption of macromolecules to the electrode surface, as DNA is not involved in the measurement mechanism. Furthermore, amplification can be measured regardless of the structural configuration of the nucleic acid, such as double or single strands. Furthermore, because the measurement utilizes electrochemical electrochemical staining (EIS), it can accurately detect gene amplification during PCR. Finally, the measurement sensitivity of this system is not dependent on the specific concentrations of MgCl2 and dNTPs, but rather on the ratio of MgCl2 and dNTPs. This allows for flexible adjustment of the concentrations according to the user's PCR environment.

[0141] Therefore, the PCR amplification measurement method of the present invention can be utilized as a basic technology for electrochemical PCR genetic diagnosis by overcoming the limitations of the existing rapid antigen diagnosis method and the fluorescence measurement-based PCR genetic diagnosis method.

Claims

1. A step of injecting a PCR sample containing a template nucleic acid and a PCR reagent into a PCR container; A step of mounting a connector to which an electrode is connected to the PCR container; A step of performing PCR using the above PCR container; and A step of measuring an electrochemical signal generated in the step of performing the above PCR using an electrochemical sensor device; A PCR amplification measurement method comprising:

2. In paragraph 1, A PCR amplification measurement method, wherein the electrode is at least one selected from the group consisting of gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes, graphene, and carbon.

3. In paragraph 1, A PCR amplification measurement method, wherein the PCR reagent comprises at least one selected from the group consisting of a redox reagent, magnesium chloride, dNTP (deoxynucleotide triphosphate), a primer pair, Taq polymerase, PCR buffer, and pyrophosphatase.

4. In paragraph 3, A PCR amplification measurement method, wherein the redox reagent is at least one selected from the group consisting of ferricyanide, ferrocyanide, ferrocene derivatives, ruthenium derivatives, osmium derivatives, and methylene blue.

5. In paragraph 4, A PCR amplification measurement method, wherein the above ferricyanide and ferrocyanide are included in a PCR reagent at a concentration of 0.1 to 10.0 mM.

6. In paragraph 3, A PCR amplification measurement method, wherein the above magnesium chloride is included in a PCR reagent at a concentration of 0.1 to 10.0 mM.

7. In paragraph 3, A PCR amplification measurement method, wherein the above dNTP is included at a concentration of 0.1 to 10.0 mM.

8. In paragraph 3, A PCR amplification measurement method, wherein the above PCR reagent has a concentration ratio of magnesium chloride / dNTP of 0.5 to 5.

9. In paragraph 1, A PCR amplification measurement method, wherein the PCR sample is at least one selected from the group consisting of saliva, urine, tears, sweat, blood, and interstitial fluid.

10. In paragraph 1, A PCR amplification measurement method, wherein the electrochemical sensor device is selected from the group consisting of a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV), a differential pulse voltammetry (DPV), and an impedance meter.

11. In paragraph 1, A method for measuring PCR amplification, wherein the step of mounting a connector on the PCR container comprises mounting a connector with an electrode protruding from the PCR container into which the PCR sample and PCR reagent are injected so that the terminal of the electrode connected to the electrochemical sensor device is immersed in the PCR sample and PCR reagent.

12. In paragraph 1, A PCR amplification measurement method, wherein the above electrochemical signal is an electrical measurement value that changes as PCR is performed.

13. In paragraph 1, A PCR amplification measurement method, wherein the step of performing the above PCR is to repeatedly change the denaturation step temperature, the annealing step temperature, and the extension step temperature of the PCR using the PCR container.

14. A PCR method performed using the PCR amplification measurement method of Article 1.

15. PCR container into which PCR sample containing template nucleic acid and PCR reagent are injected; A connector having an electrode provided so that the terminal protrudes into the interior of a PCR container into which the PCR sample and PCR reagent are injected so that the terminal is immersed in the PCR sample and PCR reagent; and An electrochemical sensor device electrically connected to the electrode and measuring an electrochemical signal generated inside the PCR vessel; A real-time PCR device comprising:

16. In paragraph 15, The above real-time PCR device is a real-time PCR device that repeatedly changes the denaturation step temperature, the annealing step temperature, and the extension step temperature of the PCR container.

17. In paragraph 15, A real-time PCR device, wherein the electrode is at least one selected from the group consisting of gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes, graphene, and carbon.

18. In paragraph 15, A real-time PCR device, wherein the PCR reagent comprises at least one selected from the group consisting of a redox reagent, magnesium chloride, dNTP, a primer pair, Taq polymerase, a PCR buffer, and pyrophosphatase.

19. In paragraph 18, A real-time PCR device, wherein the redox reagent is at least one selected from the group consisting of ferricyanide, ferrocyanide, ferrocene derivatives, ruthenium derivatives, osmium derivatives, and methylene blue.

20. In paragraph 19, A real-time PCR device, wherein the above ferricyanide and ferrocyanide are included in a PCR reagent at a concentration of 0.1 to 10.0 mM.

21. In paragraph 19, A real-time PCR device, wherein the magnesium chloride is included in a PCR reagent at a concentration of 0.1 to 10.0 mM.

22. In paragraph 19, A real-time PCR device wherein the above dNTP is included at a concentration of 0.1 to 10.0 mM.

23. In paragraph 18, A real-time PCR device wherein the PCR reagent has a concentration ratio of magnesium chloride / dNTP of 0.5 to 5.

24. In paragraph 15, A real-time PCR device, wherein the PCR sample is at least one selected from the group consisting of saliva, urine, tears, sweat, blood, and interstitial fluid.

25. In paragraph 15, A real-time PCR device, wherein the electrochemical sensor device is selected from the group consisting of a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV), a differential pulse voltammetry (DPV), and an impedance meter.

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