KR20140029627A

KR20140029627A - Pcr chip for detecting electrochemcial signal comprising heating block of repetitively disposed heater unit, real-time pcr device comprising the same, and real-time pcr using the same

Info

Publication number: KR20140029627A
Application number: KR1020120094678A
Authority: KR
Inventors: 김성우; 박현규; 이정환; 이유진; 김덕중; 원병연; 백송이
Original assignee: 나노바이오시스 주식회사; 한국과학기술원
Priority date: 2012-08-29
Filing date: 2012-08-29
Publication date: 2014-03-11
Also published as: WO2014035167A1; KR101983593B1

Abstract

An embodiment of the present invention relates to a PCR chip having a heating bock with repetitiously arranged heater units, for detecting electrochemical signals, a real-time PCR device including same, and a real-time PCR method using the same. According to the embodiment of the present invention, the device not only analyzes multiple samples at a high speed via the heating block of repetitiously arranged heater units and PCR chips in a shape of a panel, but also contributes significantly to micro-miniaturization and enhances portability of products by simple modularization allowing the easy detection of continuous electrochemical signals generated in the process of nucleic acid amplification.

Description

PCB chip for detecting an electrochemical signal including a heat block in which the heater unit is repeatedly arranged, a PCR device including the same, and a real-time PCR method using the same {PCR chip for detecting electrochemcial signal comprising heating block of repetitively disposed heater unit, Real -time PCR device comprising the same, and Real-time PCR using the same}

One embodiment of the present invention relates to a PCR chip capable of detecting and measuring an electrochemical signal according to an amplified nucleic acid in real time, a real time PCR apparatus including the same, and a real time PCR method using the same.

Polymerase chain reaction, or PCR (Polymerase Chain Reaction), is a technology that repeatedly heats and cools a specific site of a template nucleic acid, thereby serially replicating the specific site and exponentially amplifies a nucleic acid having the specific site. It is widely used for analysis and diagnostic purposes in science, genetic engineering and medical fields. Recently, various PCR apparatuses for performing the PCR have been developed. One example of a conventional PCR apparatus is equipped with a container containing a sample solution containing a template nucleic acid in one reaction chamber, and repeatedly heating and cooling the container to perform a PCR reaction. However, although the overall structure is not complicated because the PCR apparatus has one reaction chamber, it is necessary to have a complicated circuit for accurate temperature control, and the overall PCR execution time due to repeated heating and cooling of one reaction chamber. There is a problem with this lengthening. Further, another example of the conventional PCR apparatus is equipped with a plurality of reaction chambers having a PCR progression temperature, and PCR is performed by flowing a sample solution containing nucleic acid through one channel passing through these reaction chambers. However, since the PCR apparatus uses a plurality of reaction chambers, a complicated circuit for accurate temperature control is not required, but a long flow path for passing a high and low temperature reaction chamber is necessary, so that the overall structure is complicated. There is a need for a separate control device for controlling the flow rate of a sample solution comprising nucleic acid flowing in a channel through the chamber. On the other hand, the PCR apparatus has recently been developed to open an efficient method for grasping PCR progress in real time as well as efforts to improve PCR yield. Such a technique for real-time understanding of PCR progress is called "real-time PCR", and a real-time PCR device inputs a fluorescent material into a PCR chamber to detect an optical signal generated by coupling with an amplification product. The measuring technique is adopted. However, in this case, the real-time PCR apparatus has a complex structure such as a separate light source module for activating an optical signal from a fluorescent material, a light detection module for detecting an optical signal obtained from amplified nucleic acid, and a reflector for adjusting other optical paths. Bar must be adopted, there is a problem that it is difficult to miniaturize the device, it is difficult to utilize a portable.

Therefore, there is a need for a real-time PCR device that can reduce the PCR time and at the same time obtain a reliable PCR yield, further miniaturization and portability of the product.

In order to solve the above problems of the background art, an embodiment of the present invention provides a PCR chip capable of reasonably improving PCR time and yield, further miniaturizing and carrying a product, a real time PCR device including the same, and real time PCR using the same. I would like to suggest a method.

According to a first embodiment of the present invention, a heater group including one or more heaters, two or more heater groups, and the two or more heater groups include two or more heater units spaced apart from each other such that mutual heat exchange does not occur. A thermal block having a contact surface of a PCR chip on which at least one surface accommodates a sample and a reagent; A column electrode unit having a column electrode connected to supply electric power to heaters provided in the column block; And at least one reaction channel disposed at an upper portion of the thermal block and having both an inlet and an outlet formed at both ends, and repeatedly spaced apart across the cross section in the longitudinal direction of the reaction channel. A capture probe formed at the surface thereof and capable of complementarily binding to one region of the amplifying target nucleic acid, the capture probe having a fixed surface-treated layer and a detection electrode formed at the other region inside the reaction channel to detect an electrochemical signal. A PCR comprising a plate-shaped PCR reaction unit, comprising a complex having a metal nanoparticle and a signal probe connected to the metal nanoparticle and complementarily binding to another region of the amplification target nucleic acid. Provide Polymerase Chain Reaction) chip.

In the PCR chip according to the first embodiment of the present invention,

The metal nanoparticles are selected from the group consisting of zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu), gallium (Ga), indium (In), gold (Au), and silver (Ag). Can be selected.

The electrochemical signal may be due to a change in current generated as the amplification target nucleic acid complementarily binds to the capture probe and the signal probe of the complex.

The detection electrode is at least one selected from the group consisting of gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes, graphene, and carbon. Can be.

The amplification target nucleic acid, the capture probe, and the signal probe may be single stranded DNA.

The electrode is a two-electrode module having a working electrode in which an oxidation or reduction reaction occurs and a reference electrode in which no oxidation or reduction reaction occurs, or the indicator electrode, the reference electrode, and the indicator electrode It can be implemented as a three-electrode module having a counter electrode (counter electrode) for adjusting the electronic balance generated from.

The thermal block may have two to four heater groups.

The thermal block has two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group is PCR annealing Maintain extension step temperature and the second heater group may maintain PCR denaturation step temperature.

The thermal block has three heater groups, the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing step temperature, and the third heater group maintains the PCR extension step temperature. Or the first heater group maintains a PCR annealing step temperature and the second heater group maintains a PCR extension step temperature and the third heater group maintains a PCR denaturation step temperature, or the first heater group Maintaining the PCR extension step temperature and the second heater group may maintain the PCR denaturation step temperature and the third heater group may maintain the PCR annealing step temperature.

The one or more reaction channels may be extended so as to pass in a straight longitudinal direction through the upper corresponding part of the heater most disposed among the heater unit and the upper corresponding part of the heater disposed last.

The PCR reaction unit comprises a first plate provided with the detection electrode; A second plate disposed on the first plate and provided with the one or more reaction channels; And a third plate disposed on the second plate and provided with the inlet and the outlet.

A second embodiment of the present invention is a PCR chip according to a first embodiment of the present invention; A power supply unit for supplying power to the column electrode unit; A chip holder mounted with the PCR chip, the chip holder having a connection port configured to be electrically connected to the detection electrode end of the PCR chip; And an electrochemical signal measuring module electrically connected to the connection port of the chip holder to measure in real time an electrochemical signal generated in the reaction channel of the PCR chip.

In the real-time PCR device according to a second embodiment of the present invention,

The electrochemical signal measuring module includes an anodic stripping voltammetry (ASV), a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV), and a pulse voltage It may be selected from the group consisting of differential pulse voltammetry (DPV), and impedance.

The PCR chip may be implemented detachably to the chip holder.

It may further comprise a pump arranged to provide a positive or negative pressure to control the flow rate and flow rate of the fluid flowing in the one or more reaction channels.

According to a PCR chip according to an embodiment of the present invention, a real-time PCR apparatus including the same, and a real-time PCR method using the same, a plurality of samples are simultaneously carried out at high speed through a heat block and a plate-shaped PCR chip in which a heater unit is repeatedly arranged. In addition to being able to analyze, a simple modular implementation that can easily detect the continuous electrochemical signals generated during nucleic acid amplification can contribute significantly to the miniaturization and portability of the product.

1 to 4 show a column block and a column electrode portion of a PCR chip according to a first embodiment of the present invention.
5 schematically shows a specific structure of a PCR chip according to the first embodiment of the present invention.
6 to 9 illustrate a coupling between a capture probe and a signal probe and an amplification target nucleic acid occurring in a reaction chamber of a PCR reaction unit of a PCR chip according to a first embodiment of the present invention, and an electrochemical signal generation process accordingly.
10 to 12 show the detailed components of the PCR reaction unit of the PCR chip according to the first embodiment of the present invention.
13 to 14 are enlarged views of one horizontal section of a PCR reaction unit of a PCR chip according to a first embodiment of the present invention.
15 shows a chip holder of a real-time PCR device according to a second embodiment of the present invention.
16 shows a real-time PCR device according to a second embodiment of the present invention having a PCR chip, a power supply, and a pump.
17 illustrates a nucleic acid amplification process by a real time PCR apparatus according to a second embodiment of the present invention, and a process of detecting and measuring a nucleic acid amplification signal in real time.
18 illustrates a series of procedures for detecting and measuring nucleic acid amplification and nucleic acid amplification signals in real time using a real time PCR apparatus according to a second embodiment of the present invention.

Hereinafter, embodiments according to the present invention will be described in detail with reference to the accompanying drawings. The following description is merely intended to facilitate understanding of embodiments of the present invention and is not intended to limit the scope of protection.

PCR device according to an embodiment of the present invention refers to a device used for PCR (Polymerase Chain Reaction) for amplifying a nucleic acid having a specific base sequence. For example, in order to amplify deoxyribonucleic acid, a PCR apparatus is designed to amplify a solution containing PCR sample and reagent containing double stranded DNA, which is a template nucleic acid, at a specific temperature, for example, about 95 < 0 & A denaturing step of separating the double stranded DNA into single strand DNA by heating, and an oligonucleotide primer having a sequence complementary to the nucleotide sequence to be amplified, wherein the isolated single strand DNA Annealing step (annealing step) of cooling the DNA to a specific temperature, for example, 55 ° C to bind the primer to a specific base sequence of the single strand DNA to form a partial DNA-primer complex, The solution is maintained at an appropriate temperature, for example, 72 ° C, and a primer of the partial DNA-primer complex is prepared by a DNA polymerase (Or amplification) step of forming double stranded DNA as a base, and repeating the above three steps 20 to 40 times, for example, to exponentially amplify the DNA having the specific nucleotide sequence . Optionally, the PCR device may simultaneously perform the annealing step and the extension (or amplification) step, wherein the PCR device performs two steps, comprising the extension step and the annealing and extension (or amplification) steps , Thereby completing the first circulation. Therefore, the real-time PCR device 1 according to an embodiment of the present invention refers to a device including modules for performing the above steps, detailed modules not described herein are disclosed in the prior art for performing PCR It is assumed that all of them are provided or are provided in the obvious range.

According to a first embodiment of the present invention, a heater group including one or more heaters, two or more heater groups, and the two or more heater groups include two or more heater units spaced apart from each other such that mutual heat exchange does not occur. A thermal block having a contact surface of a PCR chip on which at least one surface accommodates a sample and a reagent; A column electrode unit having a column electrode connected to supply electric power to heaters provided in the column block; And at least one reaction channel disposed at an upper end of the thermal block and having inlets and outlets at both ends, and repeatedly spaced apart across the cross section in the longitudinal direction of the reaction channel, in one region within the reaction channel. And a capture probe formed on the surface-treated fixed layer and the other region inside the reaction channel, the capture probe being formed and capable of complementarily binding to one region of the amplification target nucleic acid, the detection electrode being configured to detect an electrochemical signal. PCR comprising a plate-shaped PCR reaction unit, comprising a complex comprising a metal nanoparticle and a signal probe connected to the metal nanoparticle and complementary to the other region of the amplification target nucleic acid, PCR (Polymerase) Chain Reaction) chip.

1 to 4 show a column block and a column electrode portion of a PCR chip according to a first embodiment of the present invention.

The thermal block 100 is a module implemented to supply heat to a sample and a reagent at a specific temperature to perform a PCR, and has at least one surface of a contact surface of a PCR chip containing a sample and a reagent, One side of the PCR chip to be described is contacted to heat the sample and reagents present in one or more reaction channels to perform PCR. The thermal block 100 may be implemented using a substrate as a body. The substrate may be made of any material such that physical and / or chemical properties thereof do not change due to heating and temperature maintenance of a heater disposed in the substrate, and mutual heat exchange does not occur between two or more heaters spaced apart in the substrate. Can be. For example, the substrate may be formed of a material such as plastic, glass, silicon, or the like, or may be implemented to be transparent or translucent. The thermal block 100 may be implemented in a plate shape as a whole, but is not limited thereto. The thermal block 100 includes a heater group including one or more heaters, two or more heater groups, and the two or more heater groups are repeatedly disposed at least two heater units spaced apart from each other so that mutual heat exchange does not occur. In addition, the contact surface of the PCR chip is implemented on at least one surface of the thermal block 100, various shapes for efficiently supplying heat to the PCR chip containing the sample and reagents, for example, to increase the surface area of the contact surface It may be implemented in a planar shape or a pillar shape (pillar).

The heaters 111, 112, 121, 122, 131, and 132 are heat generating elements, and may be implemented such that a hot wire (not shown) is disposed therein. The heating wire may be operably connected with various heat sources to maintain a constant temperature, and may be operably connected with various temperature sensors for monitoring the temperature of the heating wire. The heating wire may be disposed to be symmetrical in the vertical direction and / or the horizontal direction with respect to the surface center point of the heater in order to maintain a constant internal temperature of the heater as a whole. Also, the heater may have a thin film heater (not shown) disposed therein. The thin film heaters may be disposed at regular intervals in the vertical direction and / or the left and right directions with respect to the center point of the heater surface in order to maintain the internal temperature of the heater as a whole. In addition, the heater is a heating element, and may itself be a metal material, for example, chromium, aluminum, copper, iron, silver, and the like, for even heat distribution and rapid heat transfer over the same area. In addition, the heater is a light-transmitting heating element, for example, conductive nanoparticles including an oxide semiconductor material or a material added with impurities selected from the group consisting of In, Sb, Al, Ga, C and Sn to the oxide semiconductor material, And at least one selected from the group consisting of indium tin oxide, conductive polymeric materials, carbon nanotubes, and graphene.

The heater groups 110, 120, and 130 are units including the one or more heaters, and are regions that maintain a temperature for performing a denaturation step, annealing step, and / or extension step for PCR. Two or more heater groups are disposed in the thermal block 100, and the two or more heater groups are spaced apart from each other so that mutual heat exchange does not occur. Two to four heater groups may be included in the thermal block 100. That is, the thermal block includes two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group Maintaining the PCR annealing / extension step temperature and the second heater group may maintain the PCR denaturation step temperature. The heat block includes three heater groups, the first heater group maintains the PCR denaturation temperature, the second heater group maintains the PCR annealing temperature, and the third heater group maintains the PCR extension temperature , Or the first heater group maintains the PCR annealing step temperature, the second heater group maintains the PCR extension step temperature, the third heater group maintains the PCR denaturation step temperature, or the first heater group Group can maintain the PCR extension step temperature, the second heater group can maintain the PCR denaturation step temperature, and the third heater group can maintain the PCR annealing step temperature. Preferably, the heater group may be disposed three times in the thermal block 100 to maintain three temperatures for performing PCR, that is, a temperature for performing a denaturation step, an annealing step, and an extension step, and more preferably, The heater group may be disposed twice in the thermal block 100 to maintain two temperatures for performing PCR, that is, a temperature for performing a denaturation step and an annealing / extension step, respectively, but is not limited thereto. The heater group is disposed in the heat block 100 twice, and when performing two steps for performing PCR, that is, denaturation step and annealing / extension step, three steps for performing PCR, that is, denaturation step, annealing step and extension step It is possible to reduce the reaction time than to perform, there is an advantage of simplifying the structure by reducing the number of heaters. In this case, in the three steps for performing the PCR, the temperature for performing the denaturation step is 85 ℃ to 105 ℃, preferably 95 ℃, the temperature for performing the annealing step is 40 ℃ to 60 ℃, preferably 50 ℃, the temperature for performing the extension step is 50 ℃ to 80 ℃, preferably 72 ℃, in two steps for performing the PCR, the temperature for performing the denaturation step is 85 ℃ to 105 ℃, preferably 95 ° C., and the temperature for performing the annealing / extension step is 50 ° C. to 80 ° C., preferably 72 ° C. However, the specified temperature and temperature range for performing the PCR can be adjusted within a range feasible in performing the PCR. On the other hand, the heater group may further include a heater that serves as a temperature buffer.

The heater units 10 and 20 are units including the two or more heater groups including the one or more heaters, and the first circulation including the denaturation step, annealing step, and / or extension step for performing PCR is completed. Area. The heater unit is repeatedly arranged at least two in the thermal block (100). Preferably, the heater unit may be repeatedly arranged in the heat block 100 10 times, 20 times, 30 times, or 40 times, but is not limited thereto.

According to FIG. 1, the heat block 100 includes heater units 10 and 20 repeatedly arranged, two heater groups 110 and 120 included therein, and one heater 111 and 121 respectively included therein. By providing a two-step temperature for performing the PCR, that is, one temperature of the denaturation step and one temperature of the annealing / extension step are repeatedly provided sequentially. For example, the first heater 111 maintains one temperature in the range of 85 ° C. to 105 ° C., preferably 95 ° C., so that the first heater group 110 provides a temperature for performing the modification step. The second heater 121 maintains one temperature in the range of 50 ° C. to 80 ° C., preferably 72 ° C. such that the second heater group 120 provides a temperature for performing an annealing / extension step. 100 sequentially and repeatedly provides two-step temperature for performing PCR in the first heater unit 10 and the second heater unit 20.

According to FIG. 2, the thermal block 100 includes heater units 10 and 20 repeatedly arranged, two heater groups 110 and 120 included therein, and two heaters 111 and 112 respectively included therein. 121, 122) to provide a two-step temperature for performing PCR, that is, two temperatures of the denaturation step and two temperatures of the annealing / extension step. For example, the first heater 111 has one temperature in the range of 85 ° C to 105 ° C, and the second heater 112 has one temperature that is the same as or different from the temperature of the first heater 111 in the range of 85 ° C to 105 ° C. By maintaining the first heater group 110 provides a temperature for performing the modification step, the third heater 121 is one temperature in the range of 50 ℃ to 80 ℃, the fourth heater 122 is 50 ℃ to The thermal block 100 is maintained by maintaining a temperature equal to or different from the temperature of the third heater 121 in an 80 ° C range so that the second heater group 120 provides a temperature for performing an annealing / extension step. Is sequentially and repeatedly provided two-step temperature for performing PCR in the first heater unit 10 and the second heater unit 20.

According to FIG. 3, the heat block 100 may include heater units 10 and 20 repeatedly arranged, three heater groups 110, 120, and 130 included therein, and one heater 111, respectively included therein. 121, 131, sequentially provide three steps of temperature for performing PCR, that is, one temperature of the denaturation step, one temperature of the annealing step, and one temperature of the extension step. For example, the first heater 111 maintains one temperature in the range of 85 ° C. to 105 ° C., preferably 95 ° C., so that the first heater group 110 provides a temperature for performing the modification step. The second heater 121 maintains one temperature in the range of 40 ° C. to 60 ° C., preferably 50 ° C., so that the second heater group 120 provides a temperature for performing the annealing step, and the third heater 131. Is maintained at a temperature in the range of 50 ° C to 80 ° C, preferably 72 ° C, so that the third heater group 130 provides a temperature for performing the extension step, whereby the thermal block 100 is provided with a first heater unit. 10 and the second heater unit 20 sequentially and repeatedly provide three step temperatures for performing PCR.

According to FIG. 4, heater units 10 and 20 repeatedly arranged, three heater groups 110, 120, and 130 included therein, and two heaters 111, 112, 121, 122, and 131 respectively included therein , 132) to provide three steps of temperature for performing PCR, that is, two temperatures of the denaturation step, two temperatures of the annealing step, and two temperatures of the extension step. For example, the first heater 111 has one temperature in the range of 85 ° C to 105 ° C, and the second heater 112 has one temperature that is the same as or different from the temperature of the first heater 111 in the range of 85 ° C to 105 ° C. By maintaining the first heater group 110 provides a temperature for performing the modification step, the third heater 121 is in the temperature range of 40 ℃ to 60 ℃ 1, the fourth heater 122 is 40 ℃ to The second heater group 120 provides a temperature for performing the annealing step by maintaining one temperature equal to or different from the temperature of the third heater 121 in a range of 60 ° C, and the fifth heater 131 is 50 ° C. The third heater group 130 extends by maintaining one temperature equal to or different from the temperature of the fifth heater 131 in a temperature range of 50 ° C. to 80 ° C., and the sixth heater 132. By providing a temperature for performing the step, the thermal block 100 is configured in three stages for performing PCR in the first heater unit 10 and the second heater unit 20. The system temperature is repeatedly provided sequentially.

1 to 4, by repeatedly disposing two or more heaters maintaining a constant temperature, the rate of change of temperature can be significantly improved. For example, according to the conventional single heater method using only one heater, while the temperature change rate is within the range of 3 ° C to 7 ° C per second, according to the repeated heater arrangement method according to an embodiment of the present invention, the heater The rate of change of temperature between them is within the range of 20 ℃ to 40 ℃ per second can greatly shorten the reaction time. In the nucleic acid amplification reaction in which the heaters are spaced apart from each other so that mutual heat exchange does not occur and as a result, they can be greatly affected even by a minute temperature change, the denaturation step, the annealing step and the extension step (or the denaturation step and annealing / Denaturation step), and it is possible to maintain a desired temperature or a temperature range only at a position where heat is supplied from the heaters. In addition, two or more heater units are repeatedly arranged in the thermal block 100, and the number of repetitive batches of the heater units 10 and 20 may vary according to a user or a kind of sample and reagent to be PCR. have. For example, when the PCR apparatus according to an embodiment of the present invention is to be applied to a PCR having 10 cycles, the heater unit may be repeatedly arranged 10 times. That is, the heater unit may be repeatedly arranged in 10 times, 20 times, 30 times, 40 times, 50 times, etc. in consideration of the PCR circulation cycle according to the user who intends to perform the PCR or the type of the sample and the reagent. It is not limited. On the other hand, the heater unit may be repeatedly arranged with a half number of a predetermined PCR cycle period. For example, when the PCR apparatus according to an embodiment of the present invention is applied to PCR in which the circulation cycle is 20 cycles, the heater unit can be repeatedly arranged 10 times. In this case, the sample and reagent solutions may be repeated 10 times of the PCR cycle from the inlet to the outlet in one or more reaction channels to be described in detail below, followed by 10 times of the PCR cycle from the outlet to the inlet. Can be run repeatedly

5 schematically shows a specific structure of a PCR chip according to the first embodiment of the present invention.

According to FIG. 5, a PCR chip according to a first embodiment of the present invention includes a column block 100 and column electrodes 210 and 220 connected to supply power to heaters provided in the column block 100. It includes a column electrode 200, and a PCR reaction unit 900 disposed on the thermal block 100. Specifically, in the thermal block 100 according to an embodiment of the present invention in thermal contact with the PCR reaction unit 900, the upper end of Figure 5 shows a vertical cross-sectional view of the PCR chip, the lower end of Figure 2 A horizontal cross sectional view of the thermal block 100 is shown. According to FIG. 5, the thermal block 100 includes a heater unit repeatedly disposed ten times, and the heater unit includes a first heater group and a second heater group, and the first heater group and the second heater group. Each includes one heater, that is, the first heater 110 and the second heater 120. The heaters, heater groups, heater units and heat blocks according to FIG. 5 are as described above. The column electrode part 200 is a module for heating the heat block 100 by supplying power to the heat block 100 from a power supply unit (not shown), and the heaters provided in the heat block 100. And column electrodes 210 and 220 connected to supply power thereto. According to FIG. 5, the first column electrode 210 of the column block 100 is connected to supply power to the first heater 110, and the second column electrode 220 is connected to the second heater ( It is connected to supply power to 120, but is not limited thereto. If the first heater 110 maintains the PCR denaturation step temperature, for example 85 ℃ to 105 ℃ and the second heater 120 maintains the PCR annealing / extension stage temperature, for example 50 ℃ to 80 ℃ When the first column electrode 210 is supplied with power for maintaining the PCR denaturation step temperature from the power supply, the second column electrode 220 is supplied with power for maintaining the PCR annealing / extension step temperature from the power supply I can receive it. According to FIG. 5, the first column electrode 210 and the second column electrode 220 are respectively provided to the first heater 110 and the two or more second heaters 120 repeatedly arranged in the column block 100. Can be connected. The first column electrode 210 and the second column electrode 220 may be conductive materials such as gold, silver, and copper, and are not particularly limited. The PCR reaction unit 900 will be described later.

6 to 9 illustrate a coupling between a capture probe and a signal probe and an amplification target nucleic acid occurring in a reaction chamber of a PCR reaction unit of a PCR chip according to a first embodiment of the present invention, and an electrochemical signal generation process accordingly.

According to Figure 6, the PCR reaction unit 900 is a nucleic acid, for example, a template nucleic acid double-stranded DNA PCR sample, an oligonucleotide primer having a sequence complementary to a specific base sequence to be amplified PCR reagent, DNA polymerase, A solution comprising deoxyribonucleotide triphosphates (dNTP) and PCR reaction buffer can be accommodated. The PCR reaction unit 900 includes an inlet 931 for introducing the sample and the reagent, an outlet 932 for discharging the nucleic acid amplification reaction solution, and a nucleic acid amplification reaction of the sample and the reagent And has a reaction channel 921. According to FIG. 6, the reaction channel 921 extends so as to pass through the upper corresponding portion of the first heater and the upper corresponding portion of the second heater in the longitudinal direction. When the outer surface of the PCR reaction unit 900 is in thermal contact with the thermal block 100, the heat is received from the thermal block 100 and included in the reaction channel 921 of the PCR reaction unit 900. PCR samples and reagents can be heated and maintained. Also, the PCR reaction unit 900 is implemented in a plate shape as a whole so as to increase thermal conductivity and to have two or more reaction channels 921. In addition, the external structure of the PCR reaction unit 900 is implemented to be fixedly mounted in the inner space of the chip holder 300 so as not to be separated from the chip holder 300 to be described later. In addition, the PCR reaction unit 900 may be implemented as a plastic material of a transparent or opaque material, the thickness of the plastic material can be easily adjusted to increase the heat transfer efficiency only by adjusting the thickness, the manufacturing process is simple chip The manufacturing cost can be reduced.

According to FIG. 7, the reaction chamber 921 is a space in which PCR is performed by a PCR sample and a reagent, and is formed in the PCR reaction unit 900. The reaction chamber 921 may be implemented in various shapes and structures, such as a hollow cylinder shape, a bar shape, and a square pillar shape. Specifically, FIG. 7 is a detailed view of the reaction chamber 921 and the complex 29 accommodated in the PCR reaction unit 900 of the PCR chip according to the first embodiment of the present invention. The reaction chamber 921 is disposed in one region therein, and the fixed layer 940 surface-treated with the capture probe 24 capable of complementarily binding to one region of the amplifying target nucleic acid, and the other work therein. The detection electrode 950 is disposed in the area and is configured to detect an electrochemical signal. The fixed layer 940 and the detection electrode 950 may be disposed at various positions within the reaction chamber 921. As illustrated in FIG. 7, it is preferable to be disposed to face each other up and down or left and right. In addition, the reaction chamber 921 is connected to the metal nanoparticles 27 and the metal nanoparticles 27 therein, the signal probe 28 that can be complementarily coupled to another region of the amplification target nucleic acid (28) Housed with a composite 29 having a. In this case, the complex 29 may be previously contained in the reaction chamber 921 before introduction of a PCR sample including a template nucleic acid and the like, and the reaction chamber may be included in a PCR reagent including a primer, a polymerase, and the like. 23 may be introduced together. The pinned layer 940 is formed of various materials, for example, silicon, plastic, glass, metal, or the like, so that the capture probe 24 is deposited and exposed on one surface thereof. In this case, the surface of the pinned layer 940 may be first surface-treated with a material such as amine NH ₃ ⁺ , aldehyde COH, carboxyl group COOH, or the like before the capture probe 24 is deposited. The capture probe 24 is implemented to complementarily bind to a portion (region) of the amplification target nucleic acid, and combines with the metal nanoparticles 27 to form a complex 29. The metal nanoparticles 27 may vary, but zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu), gallium (Ga), indium (In), gold (Au), and silver One or more from the group consisting of (Ag). The signal probe 28 is implemented to bind to a region of the amplification target nucleic acid complementarily, in this case, in the amplification target nucleic acid, the complementary binding region of the signal probe 28 is the capture probe ( Different from the complementary binding region of 24). Thus, the capture probe 24 and signal probe 28 may bind complementarily to the amplification target nucleic acid (see FIG. 8). Referring to FIG. 8, the left figure shows a case where the amplification target nucleic acid is not present before the PCR is performed, that is, the right figure shows the case where the amplification target nucleic acid is present after the PCR is performed. When the target nucleic acid is amplified inside the amplification target nucleic acid 2 as shown in the right figure, the amplification target nucleic acid 2 is complementarily bound to the capture probe 24 surface-treated in the fixed layer 940, and to the metal nanoparticle 27 Complementarily binds to the connected signal probe 28 to concentrate the metal nanoparticles 27 in a region proximate the pinned layer 940. As a result, the metal nanoparticles 27 do not reach the detection electrode 26 and cause a current change (decrease) between the metal nanoparticles 27 and the detection electrode 26 to cause A detectable electrochemical signal is generated upon amplification. Meanwhile, the amplification target nucleic acid 2, the capture probe 24, and the signal probe 28 may be single stranded DNA.

The detection electrode 950 is disposed in at least one region of the reaction chamber 921 and is implemented to detect an electrochemical signal generated in the reaction chamber 921. The detection electrode 950 may be made of various materials to perform the above functions, but for example, gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes (carbon) nanotube), graphene, graphene, and carbon. In addition, the detection electrode 950 may be implemented in various shapes and structures in order to efficiently detect the electrochemical signal generated inside the reaction chamber 921, but for example, the reaction chamber 921 as shown in FIG. 7. ) It may be implemented in a plate shape of a metal material disposed along the inner surface. On the other hand, the electrochemical signal is measured by the electrochemical signal measuring module to be described later, the electrochemical signal measuring module may vary, but anodizing stripping voltammetry (ASV), a time-based ammeter (chronoamperometry, CA) ), Cyclic voltammetry, square wave voltammetry (SWV), differential pulse voltammetry (DPV), and impedance. The electrochemical signal may be due to a change in current that occurs as the amplification target nucleic acid complementarily binds with the capture probe 24 and the signal probe 28. 9 illustrates an electrochemical signal generation process of a real-time PCR device according to an embodiment of the present invention. According to FIG. 9, step S1 is a complex including a capture probe 24, a signal probe 28, and metal nanoparticles 27 surface-treated in a gold matrix, before the PCR is started. (29, indicating the original state of the signaling probe-AuNP), the step S2 is the current change (signal, signal generated by the reduction or oxidation between the detection electrode (950, GC electrode) and the metal nanoparticles (27, yellow particles) , And step S3 is after initiation of PCR, wherein the amplification target nucleic acid (2, H1N1 DNA) is a signal probe (28, Signaling probe-AuNP) of the capture probe (24) and the complex (29, Signaling probe-AuNP) ) And a process that causes a decrease in current change (signal reduction). Specifically, when a reduction voltage is applied to the metal nanoparticles 27 (AuNP) of the complex 29 (Signaling probe-AuNP), the metal nanoparticles 27 (AuNP) are close to the detection electrode 950 (GC electrode). The accumulation of AuNP, which is accumulated on the surface, is reduced (Accumulation of AuNP), and when a voltage is applied to the detection electrode 950 (GC electrode), the reducing metal nanoparticles 27 and AuNP are oxidized (Stripping). That is, a signal is generated (Signal), the current change can be easily measured by the voltage value indicated by the oxidation current peak (S2). In this case, the current change value, that is, the electrochemical signal, represents the maximum value in the reaction chamber 921. In addition, since the current change is different for each type of metal nanoparticles 27 and AuNP, simultaneous signal detection for two or more samples is also possible when two or more metal nanoparticles 27 and AuNP are used. After PCR, the target nucleic acid (2, H1N1 DNA) is amplified from the template nucleic acid, and the amplified target nucleic acid (2, H1N1 DNA) is captured by the capture probe (24) and the complex (29, signaling probe). Accumulation of the electrode surface of the metal nanoparticles 27 (AuNP) of the complex (29, Signaling probe-AuNP) as described above by hybridizing with a signaling probe (28, Signaling probe) of AuNP) of AuNP), decrease the current value, and further increase the amount of the amplification target nucleic acid (2, H1N1 DNA) as the PCR cycle proceeds, so that the capture probe (24) and the complex (29, The complementary target DNA of the signaling probe (AuNP) and the signal probe 28 (Signal Probe 28) also increase, so that the current value (signal) is further reduced. Therefore, real-time PCR can be implemented by detecting and measuring the current reduction phenomenon, that is, the electrochemical signal.

10 to 12 show the detailed components of the PCR reaction unit of the PCR chip according to the first embodiment of the present invention.

The PCR reaction unit 900 is disposed above the thermal block 100, and at least one reaction channel 921 having an inlet part 931 and an outlet part 932 at both ends thereof, and the reaction channel. Fixed capture surface disposed in the longitudinal direction of the (921) is repeatedly spaced apart across the cross-section, the capture probe is formed in a region inside the reaction channel 921, the capture probe capable of complementarily binding to one region of the amplification target nucleic acid And a detection electrode 950 formed in the layer 940 and another region inside the reaction channel 921 and configured to detect an electrochemical signal.

10 to 12, the pinned layer 940 and the detection electrode 950 are repeatedly spaced apart across the cross section in the longitudinal direction of the reaction channel 921, but are in thermal contact with the thermal block 100. The fixed layer 940 and the detection electrode 950 are implemented to be disposed between the two or more heater groups 110, 120, and 130. According to FIG. 10, which shows a plan view of the PCR reaction unit 900, the fixed layer 940 and the detection electrode 950 have a reaction channel 921 region from the inlet 931 to the outlet 932. It is repeatedly spaced at regular intervals in the, through such a structure it is possible to repeatedly detect the electrochemical signal from the nucleic acid sequentially amplified while passing through the reaction channel 921 in the longitudinal direction. In addition, according to FIGS. 11 to 12, which show vertical cross-sectional views of the PCR reaction unit 900, the fixed layer 940 and the detection electrode 950 are disposed to face each other in the cross section of the reaction channel 921. As can be seen, the positions of the pinned layer 940 and the detection electrode 950 may be changed up and down.

11 to 12, the PCR reaction unit 900 may be largely divided into three layers based on the vertical cross-sectional view. 11 to 12, the PCR reaction unit 900 includes a first plate 910 provided with the detection electrode 950; A second plate (920) disposed on the first plate (910) and having the at least one reaction channel (921); And a third plate 930 disposed on the second plate 920 and provided with the fixing layer 940, the inlet 931, and the outlet 932. In this case, the detection electrode 950 may be disposed on the third plate 930, and the fixing layer 940 may be disposed on the first plate 910.

The upper surface of the first plate 910 provided with the detection electrode 950 is adhered to the lower surface of the second plate 920. The first plate 910 is adhered to the second plate 920 having the reaction channel 921 so that a space for the reaction channel 921 is ensured and at least a space for the reaction channel 921 is secured The detection electrode 950 is disposed in one region (surface). The first plate 910 may be formed of a variety of materials, but preferably includes polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmethacrylate , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET) And the like. In addition, a hydrophilic material (not shown) may be processed on the upper surface of the first plate 910 to facilitate PCR. A single layer containing a hydrophilic substance may be formed on the first plate 910 by the treatment of the hydrophilic substance. The hydrophilic material may be a variety of materials but is preferably selected from the group consisting of a carboxyl group (-COOH), an amine group (-NH2), a hydroxyl group (-OH), and a sulfone group (-SH) The treatment of the hydrophilic material can be carried out according to methods known in the art.

The upper surface of the second plate 920 is disposed in contact with the lower surface of the third plate 930. The second plate 920 includes the reaction channel 921. The reaction channel 921 is connected to a portion corresponding to the inlet 931 and the outlet 932 formed in the third plate 910 so that the inlet 931 and the outlet 932 are implemented at both ends Thereby completing at least one reaction channel 921. Therefore, after the PCR sample and the reagent are introduced into the reaction channel 921, the PCR proceeds. In addition, the reaction channel 921 may exist in two or more depending on the purpose and scope of the PCR apparatus according to an embodiment of the present invention. The second plate 920 may be made of various materials, but preferably includes polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (COC) Polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM), polyetherimide (POM) , Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutyleneterephthalate , PBT), fluorinated ethylenepropylene (FEP), perfluoralkoxyalkane (PFA), and combinations thereof. It is chosen or a thermoplastic resin may be a thermosetting resin material. The thickness of the second plate 920 may vary, but may be selected from 100 μm to 200 μm. In addition, the width and length of the reaction channel 921 may vary, but preferably the width of the reaction channel 921 is selected from 0.5 mm to 3 mm, the length of the reaction channel 921 is 20 mm To 40 mm. The inner wall of the second plate 920 may be coated with a material such as silane series or bovine serum albumin (BSA) to prevent adsorption of DNA and protein, Can be carried out according to methods known in the art.

The lower surface of the third plate 930 is disposed on the upper surface of the second plate 920. The third plate 930 has a fixed layer 940, an inlet 931, and an outlet 932 formed on the reaction channel 921 formed in the second plate 920. The inlet 931 is a portion into which the PCR sample and the reagent are introduced. The outflow portion 932 is a portion where the PCR product flows out after the completion of the PCR. The third plate 930 covers the reaction channel 921 formed in the second plate 920 and the inlet 931 and the outlet 932 are connected to the inlet and outlet of the reaction channel 921, And serves as an outlet. The third plate 930 may be made of various materials, but it is preferably made of polydimethylsiloxane (PDMS), cycle olefin copolymer (COC), polymethylmethacrylate , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET) And the like. In addition, the inlet portion 931 may have various sizes, but preferably may be selected from a diameter of 1.0 mm to 3.0 mm. In addition, the outlet 932 may have various sizes, but may preferably be selected from 1.0 mm to 1.5 mm in diameter. The inlet 931 and the outlet 932 are provided with separate cover means (not shown) to prevent the solution from leaking when the PCR for the PCR sample and the reagent proceeds in the reaction channel 921 Can be prevented. The cover means may be embodied in various shapes, sizes or materials. In addition, the thickness of the third plate may vary, but may be selected preferably from 0.1 mm to 2.0 mm. In addition, there may be two or more of the inlet 931 and the outlet 932.

Meanwhile, the PCR reaction unit 900 may include an inlet 931 and an outlet 932 through mechanical processing to provide a third plate 930; A portion of the third plate 930 corresponding to the inlet 931 of the third plate 930 is connected to the outlet 932 of the third plate 930, Forming a reaction channel (921) through mechanical processing to a corresponding portion to provide a second plate (920); Providing a first plate (910) by forming a surface of a hydrophilic material (922) on the upper surface of the plate material having a size corresponding to the lower surface of the second plate (920) through surface treatment; And the lower surface of the third plate 930 are joined to the upper surface of the second plate 920 through a joining process and the lower surface of the second plate 920 is joined to the upper surface of the first plate 910 And a step of joining to the surface through a bonding step. The inlet 931 and outlet 932 of the third plate 930 and the reaction channel 921 of the second plate 920 are injection molded, hot-embossing and casting. ), And laser ablation. In addition, the hydrophilic material 922 on the surface of the first plate 910 can be treated by a method selected from the group consisting of oxygen and argon plasma treatment, corona discharge treatment, and surfactant application, . &Lt; / RTI > In addition, the lower surface of the third plate 930 and the upper surface of the second plate 920, the lower surface of the second plate 920 and the upper surface of the first plate 910 may be thermally bonded, It can be adhered by ultrasonic fusion, solvent bonding processes and can be carried out according to methods known in the art. A double-sided adhesive, a thermoplastic resin or a thermosetting resin 500 may be applied between the third plate 930 and the second plate 920 and between the second plate 920 and the third plate 910.

Meanwhile, according to FIGS. 13 to 14 in which the portion “a” of FIG. 10 is enlarged, the detection electrode 950 may be implemented in various ways. For example, a two-electrode module having a working electrode 950a in which an oxidation or reduction reaction occurs and a reference electrode 950b in which no oxidation or reduction reaction occurs, as shown in FIG. 13, or FIG. As illustrated in FIG. 14, the three-electrode module may include the indicator electrode 950a, the reference electrode 950b, and a counter electrode 950c for adjusting the electronic balance generated from the indicator electrode. . As such, when the structure of the detection electrode 950 is implemented in the multi-electrode module method as illustrated in FIGS. 13 to 14, the sensitivity of the electrochemical signal generated inside the reaction channel 921 may be increased, and the generation may be performed. Detection and measurement of signals can be performed easily.

15 shows a chip holder of a real-time PCR device according to a second embodiment of the present invention.

According to FIG. 15, the chip holder 300 includes a connection port 310 mounted to the PCR chip, and electrically connected to an end of the detection electrode 950 of the PCR reaction unit 900 of the PCR chip. . The chip holder 300 is a portion in which the PCR chip is mounted on the real-time PCR device. An inner wall of the chip holder 300 may have a shape and structure for fixedly mounting with the outer wall of the PCR chip so that the PCR chip having a plate shape does not leave the chip holder 300. That is, when the PCR chip is mounted on the chip holder 300, the end of the detection electrode 950 of the PCR reaction unit 900 is electrically connected to the connection port 310 of the chip holder 300 to perform the reaction. The electrochemical signal generated inside the channel 921 is transmitted to the electrochemical signal measuring module 800 which will be described later. On the other hand, the PCR chip is removable from the chip holder 300. In addition, the chip holder 300 may be connected to any driving means (not shown) to move up and down or left and right inside the real-time PCR apparatus.

16 shows a real-time PCR device according to a second embodiment of the present invention having a PCR chip, a power supply, and a pump.

According to FIG. 16, the PCR reaction unit 900 is disposed on the thermal block 100, and specifically, the detection electrode 950 is repeatedly disposed on an upper surface of the thermal block 100. It is arrange | positioned repeatedly between 2 heaters. The PCR reaction unit 900 and the components included therein are as described above.

The power supply unit 400 is a module for supplying power to the column electrode unit 200, and may be connected to the first column electrode 210 and the second column electrode 220 of the column electrode unit 200, respectively. have. For example, when the PCR chip is disposed in the real-time PCR device to perform PCR, a first power port (not shown) of the power supply 400 is electrically connected to the first column electrode 210. The second power port (not shown) of the power supply unit 400 is electrically connected to the second column electrode 220. Subsequently, when there is a user instruction for performing a PCR, the power supply unit 400 supplies power to the first column electrode 210 and the second column electrode 220, respectively, so that the first block of the column block 100 is provided. The heater 110 and the second heater 120 can be quickly heated, and when the heaters 110 and 120 reach a predetermined temperature, the power supply is controlled to maintain the predetermined temperature. For example, the predetermined temperature may be a PCR denaturation temperature (85 ° C. to 105 ° C., preferably 95 ° C.) in the first heater 110 and a PCR annealing / extension temperature (in the second heater 120) 50 ° C to 80 ° C, preferably 72 ° C), or the PCR annealing / extension step temperature (50 ° C to 80 ° C, preferably 72 ° C or 60 ° C) in the first heater 110 and the PCR annealing / (85 deg. C to 105 deg. C, preferably 95 deg. C) in the PCR denaturation step (120).

The pump 500 is a module for controlling the flow rate and flow rate of the fluid flowing in the at least one reaction channel 921 of the PCR reaction unit 900 and may be a positive pressure pump or a negative pressure pump, It may be a syringe pump. The pump 500 may be drivably disposed on a portion of the reaction channel 921 but preferably includes an inlet 931 and / or an outlet 932 formed at both ends of the reaction channel 921 ). The pump 500 acts not only as a pump when the pump 500 is connected to the inlet 931 and / or the outlet 932 but also through the inlet 931 and / or the outlet 932, It may also serve as a stopper to prevent reagent solution from escaping. In addition, when it is desired to control the flow rate and flow rate of the fluid flowing in the reaction channel 921, that is, the sample and reagent solutions in one direction, the pump 500 includes the inlet part 931 and the outlet part 932. If only one of the connections, and the remaining one can be a general plug is sealed, the flow of the fluid flowing in the reaction channel 921, that is, if you want to control the flow rate and flow rate of the sample and reagent solution in both directions The pump 500 may be connected to both the inlet part 931 and the outlet part 932.

The nucleic acid amplification reaction of the sample and the reagent in the real-time PCR device may be performed by the following steps as an example.

1.Double-stranded target DNA, oligonucleotide primer having a sequence complementary to the specific nucleotide sequence to be amplified, DNA polymerase, deoxyribonucleotide triphosphates (dNTP), PCR reaction buffer (PCR reaction buffer) Prepare a sample and reagent solution containing.

2. The sample and reagent solutions are introduced into the PCR reaction unit 900. In this case, the sample and reagent solutions are disposed in the reaction channel 921 inside the PCR reaction unit 900 through the inlet 931.

3. The column electrode unit 200, specifically, the first column electrode 210 and the second column electrode 220 are connected to the power supply unit 400, respectively, the inflow of the PCR reaction unit 900 The unit 931 and the outlet 932 are sealingly connected to the pump 500.

4. The power supply unit 400 is instructed to supply power to heat the first heater 110 and the second heater 120 through the first column electrode 210 and the second column electrode 220. And a specific temperature, for example, a PCR denaturation step temperature (95 ° C.) for the first heater 110 and a PCR annealing / extension step temperature (72 ° C.) for the second heater 120.

5. If a positive pressure is provided by the pump 500 connected to the inlet 931 or a negative pressure is provided by the pump 500 connected to the outlet 932, the sample and reagent solutions are transferred to the reaction channel 921. ) Flow in the horizontal direction from the inside. In this case, the flow rate and flow rate of the sample and reagent solutions may be controlled by adjusting the strength of the positive pressure or the negative pressure provided by the pump 500.

By performing the above steps, the sample and reagent solution may be transferred from the inlet 931 end of the reaction channel 921 to the outlet 932 end of the upper corresponding portion 301 and the first heater 110. 2 PCR is performed while moving the upper corresponding portion 302 of the heater 120 in the longitudinal direction. According to FIG. 16, the first sample and reagent solution receives heat from a heat block 100 in which a heater unit including the first heater 110 and the second heater 120 is repeatedly disposed 10 times. 10 PCR cycles are completed while undergoing a PCR denaturation step in the upper counterpart 301 of the heater 110 and a PCR annealing / extension step in the upper counterpart 302 of the second heater 120. Subsequently, optionally, the sample and reagent solution is formed from the upper end of the first heater 110 and the second heater 120 from the outlet 931 end of the reaction channel 921 to the end of the inlet 932. PCR can be performed again by moving the upper corresponding part of the back side in the longitudinal direction.

17 illustrates a nucleic acid amplification process by a real time PCR apparatus according to a second embodiment of the present invention, and a process of detecting and measuring a nucleic acid amplification signal in real time.

According to FIG. 17, the real-time PCR apparatus includes a heat block 100 in which the first heater 110 and the second heater 120 are repeatedly disposed in a horizontal direction, the first heater 110, and the second heater 120. A PCR reaction unit 900 repeatedly arranged so that the fixed layer 940 and the detection electrode 950 correspond to each other, and are electrically connected to a connection port (not shown) of the chip holder (not shown). The electrochemical signal measuring module 800 is implemented to measure in real time the electrochemical signal generated in the reaction channel 921 of the PCR reaction unit 900, and other power supply, pump, etc., although not shown Include. The electrochemical signal measurement module 800 may be electrically connected to the connection port of the chip holder through an electrical connection means 700, for example, a lead wire. Therefore, the electrochemical signals generated repeatedly by the sequential nucleic acid amplification in the reaction channel 921 of the PCR reaction unit 900 are sequentially detected through the detection electrode 950 of the PCR reaction unit 900 , The detected signal may be measured in the electrochemical signal measurement module 800 via the connection port of the chip holder and the electrical connection means 700 and further processed or analyzed. The electrochemical signal measuring module 800 may be various but may be an anodic stripping voltammetry (ASV), a chronoamperometry (CA), a cyclic voltammetry, a square voltage ammeter wave voltammetry (SWV), differential pulse voltammetry (DPV), and impedance (Impedance). Therefore, according to the real-time PCR device according to the second embodiment of the present invention according to Figure 17, it is possible to measure and analyze the nucleic acid amplification process in real time (real-time) during PCR. In this case, unlike the conventional real-time PCR device, the sample and reagent solutions do not need to be added a separate fluorescent material. In addition, the step of measuring the nucleic acid amplification reaction in real-time (real-time) by the real-time PCR device according to a second embodiment of the present invention can be confirmed. For example, the sample and reagent solutions pass through the upper corresponding portion 301 of the first heater 110 and the upper corresponding portion 302 of the second heater 120 in the reaction channel 921. A PCR denaturation step and a PCR annealing / extension step are performed, in which case the sample and reagent solution is between the first heater 110 and the second heater 120 and between the first heater 110 and the second heater. It passes through the detection electrode 950 region repeatedly disposed between the heater unit including the (120). When the sample and reagent solution passes through the upper corresponding portion of the detection electrode 950, the amplification target nucleic acid and the capture probe and the amplification target nucleic acid and the capture probe and the The electrochemical signal (change of current) generated by complementary coupling of the complex with the signal probe may be sequentially detected and measured in real time through the detection electrode 950. Therefore, by monitoring the reaction result by amplification of the nucleic acid (without the fluorescent material and the optical detection system) in the reaction channel 921 during real-time during each cycle of the PCR, and can be detected and measured in real-time.

18 illustrates a series of procedures for detecting and measuring nucleic acid amplification and nucleic acid amplification signals in real time using a real time PCR apparatus according to a second embodiment of the present invention.

According to FIG. 18, a real-time PCR method using a real-time PCR device according to an embodiment of the present invention may include providing the above-described real-time PCR device; Injecting a PCR sample comprising a template nucleic acid and a PCR reagent including the metal nanoparticle-signal probe complex into a reaction channel 921 of the PCR reaction unit 900 of the PCR chip; Mounting a PCR chip in which the PCR sample and the PCR reagent are injected to the chip holder 300 such that an electrode 950 end of the PCR reaction unit 900 is electrically connected to the connection port 310; The PCR sample and the PCR reagent are sequentially moved to the first heater and the second heater to maintain the denaturation stage temperature of the PCR and the annealing and extension (or amplification) stage temperatures of the PCR while moving the reaction channel 921 in the longitudinal direction, respectively. Repeatedly performing thermal contact with PCR; And detecting and measuring an electrochemical signal (change of current) generated by complementary coupling between the amplification target nucleic acid, the capture probe, and the signal probe of the complex in the PCR reaction unit 900 during the PCR. Steps.

The real-time PCR device providing step S1 is a step of preparing the above-described real-time PCR device. Therefore, the real-time PCR method according to an embodiment of the present invention below assumes the operation of the real-time PCR device.

Sample and reagent injection step (S2) is a material capable of generating an electrical signal through a chemical reaction (combination) with the PCR sample and reagent, and the template nucleic acid to be amplified in the PCR reaction unit 900 of the PCR chip, for example For example, a metal nanoparticle-signal probe complex is injected.

PCR chip mounting step (S3) is a step of mounting the PCR chip containing the PCR sample and reagent to the chip holder 300 of the real-time PCR device. In this case, the detection electrode 950 of the PCR reaction unit 900 of the PCR chip should be electrically connected to the connection port 310 of the chip holder 300 to detect the electrochemical signal.

The PCR step S4 maintains the temperature of the first heater 110 and the second heater 120 of the thermal block 100 and heats the reaction channel 921 of the PCR reaction unit 900 of the PCR chip. PCR is performed while the sample and the reagent move in the longitudinal direction. In this case, target nucleic acid sites are sequentially amplified based on template nucleic acids in samples and reagents moving inside the reaction channel 921, and signal probes of the capture probes and the complexes are continuously amplified according to the continuous amplification of the target nucleic acid sites. The complementary combination of causes an electrochemical signal.

Electrochemical signal detection and measurement step (S5) is the electrochemical signal (current value change) generated by the continuous amplification of the nucleic acid in the step S4 to the detection electrode 950 of the PCR reaction unit 900, the chip holder ( Detecting and measuring through the connection port 310 of the 300, the electrical connection means 700, and the electrochemical signal measurement module 800.

Claims

Heater group having at least one heater, at least two of the heater group and the two or more heater group is a repeating arrangement of two or more heater units spaced apart so that mutual heat exchange does not occur, the sample and reagents on at least one side A thermal block having a contact surface of a PCR chip to be received;
A column electrode unit having a column electrode connected to supply electric power to heaters provided in the column block; And
At least one reaction channel having an inlet and an outlet at both ends, and repeatedly disposed across the cross section in the longitudinal direction of the reaction channel, disposed at an upper portion of the thermal block, and located at one region inside the reaction channel. And a capture probe formed on the surface-treated fixed layer and the other region inside the reaction channel, the capture probe being formed and capable of complementarily binding to one region of the amplification target nucleic acid, the detection electrode being configured to detect an electrochemical signal. A plate-like PCR reaction part including a complex having a metal nanoparticle and a signal probe connected to the metal nanoparticle and complementarily binding to another region of the amplification target nucleic acid;
Including, PCR (Polymerase Chain Reaction) chip.

The method of claim 1,
The metal nanoparticles are selected from the group consisting of zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu), gallium (Ga), indium (In), gold (Au), and silver (Ag). PCR chip, characterized in that selected above.

The method of claim 1,
Wherein said electrochemical signal is due to a change in current generated as said amplification target nucleic acid complementarily binds said capture probe and a signal probe of said complex.

The method of claim 1,
The detection electrode is at least one selected from the group consisting of gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes, graphene, and carbon. PCR chip, characterized in that.

The method of claim 1,
Wherein said amplifying target nucleic acid, said capture probe, and said signal probe are single-stranded DNA.

The method of claim 1,
The electrode is a two-electrode module having a working electrode in which an oxidation or reduction reaction occurs and a reference electrode in which no oxidation or reduction reaction occurs, or the indicator electrode, the reference electrode, and the indicator electrode PCR chip, characterized in that implemented as a three-electrode module having a counter electrode (counter electrode) for adjusting the electronic balance generated from.

The method of claim 1,
The thermal block is a PCR chip, characterized in that it comprises two to four heater groups.

The method of claim 1,
The thermal block has two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group is PCR annealing And / or maintaining the extension step temperature and the second heater group maintains the PCR denaturation step temperature.

The method of claim 1,
The thermal block has three heater groups, the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing step temperature, and the third heater group maintains the PCR extension step temperature. Or the first heater group maintains a PCR annealing step temperature and the second heater group maintains a PCR extension step temperature and the third heater group maintains a PCR denaturation step temperature, or the first heater group The PCR chip, characterized in that to maintain the PCR extension step temperature, the second heater group maintains the PCR denaturation step temperature and the third heater group maintains the PCR annealing step temperature.

The method of claim 1,
And said at least one reaction channel is extended so as to pass in a straight longitudinal direction through an upper corresponding part of a heater disposed most optimally and an upper corresponding part of a heater disposed last.

The method of claim 1,
The PCR reaction unit comprises a first plate provided with the detection electrode; A second plate disposed on the first plate and provided with the one or more reaction channels; And a third plate disposed on the second plate and provided with the inlet and the outlet.

A PCR chip according to any one of claims 1 to 11;
A power supply unit for supplying power to the column electrode unit;
A chip holder mounted with the PCR chip, the chip holder having a connection port configured to be electrically connected to the detection electrode end of the PCR chip; And
An electrochemical signal measuring module electrically connected to a connection port of the chip holder and configured to measure in real time an electrochemical signal generated in a reaction channel of the PCR chip;
Time PCR device.

The method of claim 12,
The electrochemical signal measuring module includes an anodic stripping voltammetry (ASV), a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV), and a pulse voltage Real-time PCR device, characterized in that it is selected from the group consisting of differential pulse voltammetry (DPV), and impedance (impedance).

The method of claim 12,
The PCR chip is a real-time PCR device, characterized in that detachable implementation in the chip holder.

The method of claim 12,
And a pump arranged to provide a positive pressure or a negative pressure to control the flow rate and flow rate of the fluid flowing in the one or more reaction channels.