KR20140028430A

KR20140028430A - Real-time pcr device for detecting electrochemcial signal comprising heating block of repetitively disposed heater unit, and real-time pcr using the same

Info

Publication number: KR20140028430A
Application number: KR1020120094676A
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: 2014-03-10
Also published as: KR101946339B1; WO2014035163A1

Abstract

One embodiment of the present invention relates to a real-time PCR apparatus for detecting an electrochemical signal including a heat block in which a heater unit is repeatedly arranged, and a real-time PCR method using the same. The plate-shaped PCR chip enables simultaneous analysis of a large number of samples at a very high speed, as well as simple module implementation that enables continuous detection of electrochemical signals generated during the nucleic acid amplification process. Can greatly contribute to anger.

Description

TECHNICAL FIELD The present invention relates to a real-time PCR apparatus for detecting an electrochemical signal including a heat block in which a heater unit is repeatedly arranged, and a real-time PCR method using the same, time PCR using the same}

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

Polymerase Chain Reaction (PCR) is a technique for repetitively heating and cooling a specific region of a template nucleic acid to successively replicate the specific region and amplify a nucleic acid having the specific region exponentially, Science, genetic engineering, and medical fields. Recently, a variety of PCR apparatuses for performing the PCR have been developed. One example of a conventional PCR device is one in which a container containing a sample solution containing a template nucleic acid is mounted in one reaction chamber, and the container is repeatedly heated and cooled to perform a PCR reaction. However, since the PCR apparatus has a single reaction chamber, the overall structure is not complicated. However, it is necessary to provide a complicated circuit for accurate temperature control, and the total PCR execution time There is a problem that this becomes longer. Another example of a conventional PCR apparatus is a PCR system in which a plurality of reaction chambers having a PCR progress temperature are mounted and a sample solution containing a nucleic acid is flowed through one channel passing through the 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 channel for passing through a reaction chamber of a high temperature and a low temperature is necessarily required, A separate control device for controlling the flow rate of the sample solution containing the nucleic acid flowing in the channel passing through the chamber is required. Meanwhile, recent PCR apparatuses are being developed in order to open an efficient method for grasping not only an effort to improve the PCR yield, but also a real time PCR process. Real-time PCR is a so-called " real-time PCR "technique in which a PCR process can be grasped in real time. A real-time PCR apparatus includes a fluorescence substance injected into a PCR chamber, A measurement technique is adopted. However, in this case, the real-time PCR apparatus may have a complex structure such as a separate light source module for activating an optical signal from a fluorescent substance, a light detecting module for detecting an optical signal obtained from an amplified nucleic acid, It is difficult to miniaturize the device and it is difficult to use the device as a portable device.

Accordingly, there is a need for a real-time PCR device capable of obtaining a reliable PCR yield while reducing the PCR time, and further miniaturizing and porting the product.

In order to solve the problems of the background art as described above, one embodiment of the present invention is to propose a real-time PCR apparatus capable of reasonably improving the PCR time and yield, further miniaturizing and porting the product, and real-

One embodiment of the present invention is a heater group having at least one heater, at least two heater groups, and two or more heater groups are repeatedly arranged at least two heater units spaced apart from each other so that mutual heat exchange does not occur, A thermal block having a contact surface of the PCR chip on one side containing the sample and the reagent; A column electrode unit having column electrodes connected to supply electric power to the heaters provided in the column block; At least one reaction channel in which an inlet and an outlet are formed at both ends, and a plurality of reaction channels formed in one region inside the reaction channel, the reaction channel being repeatedly disposed across the cross section in the longitudinal direction of the reaction channel, And a detection electrode formed on the other surface of the reaction channel and adapted to detect an electrochemical signal, wherein the metal nanoparticle and the metal nanoparticle A plate-like PCR chip comprising a complex comprising a signal probe connected and complementary to another region of the amplification target nucleic acid; A chip holder having a connection port on which the PCR chip is mounted and is electrically connected to a detection electrode end of the PCR chip; And a electrochemical signal measuring module electrically connected to the connection port of the chip holder to measure an electrochemical signal generated in a reaction channel of the PCR chip in real time, to provide.

In the real-time PCR apparatus according to an embodiment of the present invention,

The metal nanoparticles may be selected from the group consisting of zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu), gallium (Ga), indium (In), gold (Au) Or more.

The electrochemical signal may be due to a current change that occurs as the amplification target nucleic acid is complementary to the capture probe and the signal probe of the complex.

The detection electrode may be at least one selected from the group consisting of Au, Co, Pt, Ag, carbon nanotube, graphene, and carbon. .

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

The electrode includes a working electrode having an oxidation or reduction reaction and a reference electrode having no oxidation or reduction reaction, or a two-electrode module having a reference electrode, a reference electrode, Electrode module having a counter electrode for adjusting the electronic balance generated from the electrode assembly.

The electrochemical signal measuring module may be an anodic stripping voltammetry (ASV), a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV) A differential pulse voltammetry (DPV), and an impedance system.

The thermal block may include two to four heater groups.

Wherein the thermal block comprises two heater groups, wherein the first heater group maintains the PCR denaturation temperature and the second heater group maintains the PCR annealing / extension temperature, or the first heater group is PCR annealed / Extension stage temperature and the second heater group may be to maintain the PCR denaturation step temperature.

Wherein the thermal block includes three heater groups, wherein 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 The PCR extension step temperature may be maintained 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 at least one reaction channel may be extended so as to pass the upper corresponding portion of the heater disposed at the best position among the heater units and the upper corresponding portion of the heater disposed at the end disposed in the straight length direction.

Wherein the PCR chip comprises: a first plate provided with the detection electrode; A second plate disposed on the first plate and having the at least one reaction channel; And a third plate disposed on the second plate and having the inlet and the outlet.

The PCR chip may be detachably mounted on the chip holder.

And a power supply unit for supplying power to the column electrode unit.

The pump may further include a pump arranged to provide a positive or negative pressure to control the flow rate and flow rate of the fluid flowing in the at least one reaction channel.

According to the real-time PCR apparatus according to an embodiment of the present invention, a plurality of samples can be simultaneously analyzed at a very high speed through a thermal block and a plate-shaped PCR chip in which heater units are repeatedly arranged, Simple module implementation that can easily detect continuous electrochemical signals can contribute significantly to miniaturization and portability of the product.

Figures 1 to 5 illustrate a column block and a column electrode portion of a real-time PCR device according to an embodiment of the present invention.
FIGS. 6 to 9 show the binding between the capture probe and the amplified target nucleic acid in the reaction chamber of the PCR chip of the real-time PCR device according to an embodiment of the present invention, and the electrochemical signal generation process.
10 to 12 show detailed components of a PCR chip of a real-time PCR device according to an embodiment of the present invention.
13 to 14 are enlarged horizontal cross-sections of a real-time PCR apparatus according to an embodiment of the present invention.
15 shows a chip holder of a real-time PCR device according to an embodiment of the present invention.
16 shows a real-time PCR apparatus according to an embodiment of the present invention, which includes a PCR chip, a power supply, and a pump.
17 shows a nucleic acid amplification process by a real-time PCR device according to an embodiment of the present invention, and a process of detecting and measuring nucleic acid amplification signals in real time in accordance with the present invention.
FIG. 18 shows a series of procedures for real-time detection and measurement of nucleic acid amplification and nucleic acid amplification signals using a real-time PCR apparatus according to an 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.

A PCR device according to an embodiment of the present invention refers to a device used in 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 perform the annealing step and the extension (or amplification) step simultaneously, wherein the PCR device performs two steps of the extension step and the annealing and extension (or amplification) step , Thereby completing the first circulation. Accordingly, a real-time PCR apparatus 1 according to an embodiment of the present invention refers to an apparatus including modules for performing the above steps, and detailed modules not described in the present specification are disclosed in the prior art It is presumed that it is included in the scope of the invention.

Figures 1 to 5 illustrate a column block and a column electrode portion of a real-time PCR device according to an 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 in order to perform PCR. The thermal block 100 has a contact surface of a PCR chip in which a sample and a reagent are contained on at least one surface thereof, In contact with one side of the PCR chip to be described, PCR is performed by supplying heat to a sample and a reagent present in at least one reaction channel. The thermal block 100 may be implemented using a substrate as a body. The substrate is implemented with any material that does not change its physical and / or chemical properties due to heating and temperature maintenance of the heater disposed within the substrate and does not cause mutual heat exchange between two or more heaters spaced apart within the substrate . For example, the substrate may be made of plastic, glass, silicon, or the like, and may be transparent or translucent. The thermal block 100 may be implemented in a plate shape as a whole, but is not limited thereto. The heat block 100 includes at least two heaters, at least two heaters, and at least two heaters, which are spaced apart from each other so as not to exchange heat with each other. In addition, the contact surface of the PCR chip is formed on at least one surface of the thermal block 100, and various shapes for efficiently supplying heat to the PCR chip containing the sample and the reagent, for example, And may be implemented in a planar shape or a pillar shape.

The heaters 111, 112, 121, 122, 131, and 132 are heat generating elements, and heat waves (not shown) may be disposed therein. The hot wire may be drivably connected to various heat sources to maintain a predetermined temperature, and may be drivably connected to various temperature sensors for monitoring the temperature of the hot wire. The heating line may be arranged to be symmetrical in the up and down and / or the left and right directions with respect to the surface center point of the heater in order to maintain the internal temperature of the heater as a whole. Also, a thin film heater (not shown) may be disposed inside the heater. The thin film heater may be disposed at regular intervals in the up and down and / or the left and right directions with respect to the center point of the surface of the heater in order to maintain the internal temperature of the heater as a whole. The heater may also be a heating element, a metal material itself, such as chromium, aluminum, copper, iron, silver, etc., for even heat distribution and rapid heat transfer to the same area. The heater may be a light-transmitting heat generating element, for example, an oxide semiconductor material or a conductive nanoparticle including a substance to which an impurity selected from the group consisting of In, Sb, Al, Ga, C and Sn is added to the oxide semiconductor material, And at least one selected from the group consisting of indium tin oxide, conductive high molecular materials, carbon nanotubes, and graphene.

The heaters 110, 120, and 130 are units that include the one or more heaters, and maintain the temperatures for performing the denaturation step, the annealing step, and / or the extension step for performing the PCR. At least two heaters are disposed in the heat block 100, and the two or more heaters are spaced apart from each other such that mutual heat exchange does not occur. The heater group may include two to four heaters in the heat block 100. That is, the heat block includes two heater groups, the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing / extension step temperature, or the first heater group The PCR annealing / extension step temperature can be maintained and the second heater group can 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 is disposed three times in the thermal block 100 to maintain the temperature for performing PCR in three stages, i.e., the denaturation step, the annealing step and the extension step, The heater group may be disposed twice in the thermal block 100 to maintain the temperature for performing PCR in two stages, i.e., the denaturation step and the annealing / extension step, but is not limited thereto. When the heater group is disposed in the thermal block 100 twice and performs two steps for performing the PCR, that is, the denaturation step and the annealing / extending step, there are three steps for performing the PCR, namely, the denaturation step, the annealing step, The reaction time can be reduced and the number of heaters can be reduced to simplify the structure. In this case, in the third step for carrying out PCR, the temperature for carrying out the denaturation step is 85 ° C to 105 ° C, preferably 95 ° C, and the temperature for carrying out the annealing step is 40 ° C to 60 ° C, preferably 50 ° C., and the temperature for carrying out the extension step is 50 ° C. to 80 ° C., preferably 72 ° C. In the second step for carrying out the PCR, the temperature for carrying out the denaturation step is 85 ° C. to 105 ° C., 95 < 0 > C, and the temperature for carrying out the annealing / lengthening step is from 50 [deg.] C to 80 [deg.] C, preferably 72 [deg.] C. However, the specified temperature and temperature range for performing the PCR can be adjusted within a range that can be realized in performing PCR. The heater group may further include a heater for performing a temperature buffering function.

Wherein the heater unit (10, 20) is a unit including the at least two heater groups including the at least one heater, wherein the first cycle including the denaturation step, the annealing step and / Area. The heater unit is repeatedly disposed in the heat block 100 at least two times. Preferably, the heater unit may be repeatedly disposed in the heat block 100 10 times, 20 times, 30 times, or 40 times, but the present invention is not limited thereto.

1, the thermal block 100 includes heater units 10 and 20 which are repeatedly arranged, two heater groups 110 and 120 respectively included therein, and one heater 111 and 121, respectively, Thereby sequentially providing a two-step temperature for performing the PCR, that is, one temperature of the denaturation step and one temperature of the annealing / extending step. For example, the first heater 111 maintains a temperature of 85 ° C to 105 ° C, preferably at 95 ° C, so that the first heater group 110 provides a temperature for performing the denaturation step, 2 heaters 121 are maintained at a temperature of 50 ° C to 80 ° C, preferably at 72 ° C, so that the second heater group 120 provides a temperature for performing the annealing / 100 sequentially provides a second-stage temperature for PCR execution in the first heater unit 10 and the second heater unit 20 in sequence.

2, the thermal block 100 includes heater units 10 and 20 which are repeatedly arranged, two heater groups 110 and 120 respectively included therein, and two heaters 111, 112, 121, and 122 to sequentially provide two temperatures for performing PCR, that is, two temperatures of the denaturation step and two annealing / lengthening steps. For example, the temperature of the first heater 111 is set to a temperature of 85 ° C to 105 ° C, and the temperature of the second heater 112 is set to be equal to or different from the temperature of the first heater 111 in the range of 85 ° C to 105 ° C. The third heater 121 provides a temperature of 1 to 50 ° C to 80 ° C, the fourth heater 122 provides a temperature of 50 ° C to 80 ° C, The second heater group 120 maintains a temperature equal to or different from the temperature of the third heater 121 in the range of 80 ° C to provide the temperature for performing the annealing / The temperature of the second heater unit 10 and the temperature of the second heater unit 20 are sequentially repeated.

3, the thermal block 100 includes heater units 10 and 20 which are repeatedly arranged, three heater groups 110, 120 and 130 respectively included therein, and one heater 111, 121, and 131, thereby providing a three-step temperature for PCR execution, 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 a temperature of 85 ° C to 105 ° C, preferably at 95 ° C, so that the first heater group 110 provides a temperature for performing the denaturation step, The second heater 121 maintains a temperature of 40 ° C to 60 ° C at 1 temperature, preferably 50 ° C, so that the second heater group 120 provides a temperature for performing the annealing step, The heat block 100 is maintained at a temperature of 50 ° C to 80 ° C, preferably at 72 ° C, so that the third heater group 130 provides a temperature for performing the extension step, And the three-stage temperature for performing the PCR in the first heater unit 10 and the second heater unit 20 in sequence.

4, heater units 10 and 20 are repeatedly arranged, three heater groups 110, 120 and 130 respectively included in the heater units 10 and 20, and two heaters 111, 112, 121, 122 and 131 , 132), thereby providing a three-step temperature for PCR execution, that is, two temperatures of the denaturation step, two temperatures of the annealing step, and two temperatures of the extension step, in sequence. For example, the temperature of the first heater 111 is set to a temperature of 85 ° C to 105 ° C, and the temperature of the second heater 112 is set to be equal to or different from the temperature of the first heater 111 in the range of 85 ° C to 105 ° C. The third heater 121 provides a temperature of 1 to 40 deg. C to 60 deg. C, the fourth heater 122 provides a temperature of 40 deg. C to 60 deg. C, The second heater group 120 provides a temperature for performing the annealing step while maintaining a temperature equal to or different from the temperature of the third heater 121 in the range of 60 ° C, The third heater group 130 is maintained at a temperature of 1 to 80 ° C, and the sixth heater 132 maintains the same temperature as the temperature of the fifth heater 131 in the range of 50 ° C to 80 ° C, The thermal block 100 can be operated in three stages for PCR in the first heater unit 10 and the second heater unit 20, And the system temperature is repeatedly provided in sequence.

As shown in Figs. 1 to 4, by repeatedly arranging two or more heaters that maintain a constant temperature, the rate of temperature change can be significantly improved. For example, according to the conventional single heater method adopting only one heater, the rate of temperature change is within the range of 3 ° C. to 7 ° C. per second, whereas according to the repetition heater arrangement method according to the embodiment of the present invention, The temperature change rate between the electrodes is within the range of 20 ° C to 40 ° C per second, so that the reaction time can be greatly shortened. 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. The number of repetitive arrangements of the heater units 10 and 20 may vary depending on the type of the user or the sample and the reagent to be subjected to the PCR. have. For example, in a case where the PCR apparatus according to an embodiment of the present invention is applied to PCR in which the circulation cycle is 10 cycles, the heater unit can be repeatedly arranged 10 times. That is, the heater unit can be repeatedly arranged 10 times, 20 times, 30 times, 40 times, 50 times, or the like depending on the type of user or sample and reagent to perform PCR, But is not limited to. 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 solution is subjected to a PCR cycle of 10 times in the direction from the inlet to the outlet in one or more reaction channels, which will be described in detail below, followed by 10 cycles of the PCR cycle from the outlet to the inlet Can be repeatedly executed.

5 illustrates a thermal block 100 of a PCR device according to an embodiment of the present invention in thermal contact with a PCR chip 900 and a column electrode 210 connected to supply power to the heaters provided in the thermal block 100 And 220, respectively. 5 is a vertical cross-sectional view of the thermal block 100, and FIG. 2 (b) is a vertical cross-sectional view of the thermal block 100. In the thermal block 100, The bottom shows a top view of the thermal block 100. Referring to FIG. 5, the thermal block 100 includes a heater unit disposed repeatedly 10 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 I.e., a first heater 110 and a second heater 120, respectively. The heater, the heater group, the heater unit and the heat block according to Fig. 5 are as described above. The column electrode unit 200 is a module for heating the thermal block 100 by supplying electric power to the thermal block 100 from a power supply unit And column electrodes 210 and 220 connected to supply power. 5, the first column electrode 210 of the thermal block 100 is connected to supply power to the first heater 110, and the second column electrode 220 is connected to the second heater 120, but is not limited thereto. If the temperature of the first heater 110 is maintained at a PCR denaturation temperature, for example, 85 ° C to 105 ° C, and the temperature of the second heater 120 is maintained at a PCR annealing / The first column electrode 210 is supplied with power for maintaining the PCR denaturation step temperature from the power supply unit and the second column electrode 220 supplies power for maintaining the PCR annealing / Can receive. 5, the first column electrode 210 and the second column electrode 220 are connected to the first heater 110 and the second heater 120 repeatedly disposed in the column block 100, Can be connected. The first column electrode 210 and the second column electrode 220 may be made of a conductive material such as gold, silver, or copper, and are not particularly limited. The PCR chip 900 will be described later.

FIGS. 6 to 9 show the binding between the capture probe and the amplified target nucleic acid within the reaction chamber of the PCR chip of the real-time PCR device according to an embodiment of the present invention, and the electrochemical signal generation process.

6, the PCR chip 900 may include a nucleic acid, for example, a template nucleic acid double-stranded DNA as a PCR sample, an oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified as a PCR reagent, a DNA polymerase, A solution containing deoxyribonucleotide triphosphates (dNTP), a PCR reaction buffer, and the like. The PCR chip 900 includes an inlet 931 for introducing the sample and the reagent, an outlet 932 for discharging the nucleic acid amplification-completed solution, and a reaction in which the nucleic acid amplification reaction of the sample and the reagent is performed And a channel 921. According to FIG. 6, the reaction channel 921 is extended so as to pass through the upper side corresponding portion of the first heater and the upper side corresponding portion of the second heater in the longitudinal direction. When the outer surface of the PCR chip 900 is thermally contacted with the thermal block 100, the thermal block 100 receives heat from the thermal block 100 and the PCR sample 900 included in the reaction channel 921 of the PCR chip 900, And reagents can be heated and maintained. In addition, the PCR chip 900 is implemented in a plate shape so as to increase the thermal conductivity and to have two or more reaction channels 921. The outer structure of the PCR chip 900 is 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. The PCR chip 900 may be made of a transparent or opaque plastic material. Since the thickness of the PCR chip 900 can be easily controlled by the characteristics of the plastic material, heat transfer efficiency can be increased only by controlling the thickness. The cost can be reduced.

Referring to FIG. 7, the reaction chamber 921 is formed in the PCR chip 900 as a space in which PCR is performed by a PCR sample and a reagent. The reaction chamber 921 may be embodied in various shapes and structures such as an empty columnar shape, a bar shape, and a square columnar shape. 7 is a detailed view of the reaction chamber 921 of the PCR chip 900 of the real-time PCR apparatus and the complex 29 accommodated therein according to an embodiment of the present invention. The reaction chamber 921 includes a fixed layer 940 disposed on one surface of the reaction chamber 921 and surface-treated with a capture probe 24 capable of complementarily binding with one region of the amplified target nucleic acid, And a detection electrode 950 disposed in the reaction chamber 921 and 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 shown in Fig. 7, are preferably arranged so as to face each other up and down or right and left. The reaction chamber 921 includes metal nanoparticles 27 and a signal probe 28 connected to the metal nanoparticles 27 and capable of complementarily binding with another region of the amplification target nucleic acid, (29). In this case, the complex 29 may be accommodated in the reaction chamber 921 before the introduction of the PCR sample including the template nucleic acid, and may be contained in the reaction chamber (PCR) containing the PCR reagent including the primer, 23). The fixed layer 940 may be formed of various materials such as silicon, plastic, glass, and metal so that the capture probes 24 may be deposited on one surface thereof. In this case, the surface of the fixed layer 940 may be surface-treated with a substance such as an amine NH ₃ ⁺ , an aldehyde COH, a carboxyl group COOH, or the like before the capture probe 24 is deposited. The capture probe 24 is designed to be complementary to a site (region) of the amplification target nucleic acid and binds to the metal nanoparticles 27 to form a complex 29. The metal nanoparticles 27 may be various but may be selected from the group consisting of Zn, Cd, Pb, Cu, Ga, In, Au, (Ag). In this case, in the amplification target nucleic acid, the complementary binding region of the signal probe 28 is connected to the capture probe 28 24). &Lt; / RTI > Thus, the capture probe 24 and the signal probe 28 may be complementarily coupled to an amplification target nucleic acid (see FIG. 8). Referring to FIG. 8, the left side shows the case where the amplified target nucleic acid is not present before the PCR, and the right side shows the case where the amplified target nucleic acid is present after the PCR, The amplified target nucleic acid 2 is complementarily bound to the capture probe 24 surface-treated on the immobilization layer 940, and the amplified target nucleic acid 2 is immobilized on the metal nanoparticles 27 And combines with the connected signal probe 28 to concentrate the metal nanoparticles 27 in a region close to the fixed layer 940. As a result, the metal nanoparticles 27 do not reach the detection electrode 26, causing a change (reduction) in current between the metal nanoparticles 27 and the detection electrode 26, A detectable electrochemical signal due to the amplification is generated. 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 configured to detect an electrochemical signal generated inside the reaction chamber 921. The detection electrode 950 may be formed of various materials to perform the functions as described above. For example, the detection electrode 950 may be formed of gold (Au), cobalt (Co), platinum (Pt), silver (Ag) nanotube, graphene, and carbon. The detection electrode 950 may be formed in various shapes and structures to efficiently detect an electrochemical signal generated in the reaction chamber 921. For example, as shown in FIG. 7, the detection electrode 950 may be formed in the reaction chamber 921 Or a metal plate disposed along the inner surface. The electrochemical signals may be measured by an electrochemical signal measuring module to be described later. The electrochemical signal measuring module may be various, but an anodic stripping voltammetry (ASV), a chronoamperometry (CA) And may be selected from the group consisting of a pulse voltage meter, a cyclic voltammetry, a square wave voltammetry (SWV), a differential pulse voltammetry (DPV), and an impedance. The electrochemical signal may be due to a current change that occurs as the amplification target nucleic acid is complementarily coupled with the capture probe 24 and the signal probe 28. FIG. 9 shows a process of generating an electrochemical signal in a real-time PCR device according to an embodiment of the present invention. According to Fig. 9, the step S1 is a step in which, before the start of the PCR, a complex comprising the capturing probe (24), the signal probe 28 and the metal nanoparticles 27 surface-treated to the fixed layer 25 (Signal, signal) generated by the reduction or oxidation between the detection electrode 950 (GC electrode) and the metal nanoparticles 27 (yellow particles) Amplification target nucleic acid 2 (H1N1 DNA) is added to the capture probe 24 and the signaling probe 28 (Signaling probe-AuNP) ) To cause a decrease in current change (signal decrease). Specifically, when a reduction voltage is applied to the metal nano-particles 27 (AuNP) of the complex 29, the metal nano-particles 27 (AuNP) (NiNP) is oxidized (Stripping) when a voltage is applied to the sensing electrode 950 (Accumulation of AuNP) while being accumulated on the surface of the sensing electrode 950 to form an accumulation layer. That is, a signal is generated (Signal), and the current change can be easily measured as a voltage value represented by the oxidation current peak (S2). In this case, the current change value in the reaction chamber 921, that is, the electrochemical signal, shows the maximum value. Also, since the current change is different for each kind of metal nanoparticles (27, AuNP), simultaneous signal detection for two or more samples is possible when two or more metal nanoparticles (27, AuNP) are used. (2, H1N1 DNA) is amplified from the template nucleic acid (2, H1N1 DNA) by the capture probe (24, Signaling probe (AuNP) of the complex 29 as described above by complementarily binding (Hybridized target DNA) with the signal probe 28 of the probe 29 (AuNP) of the capture probe 24 and the complex 29, while increasing the amount of amplified target nucleic acid (2, H1N1 DNA) as the PCR cycle progresses, The hybridized target DNA is also increased with the signaling probe 28 of the signaling probe-AuNP to further reduce the current value (signal). Therefore, real-time PCR can be realized by detecting and measuring the above-described current reduction phenomenon, that is, an electrochemical signal.

10 to 12 show detailed components of a PCR chip of a real-time PCR device according to an embodiment of the present invention.

The PCR chip 900 is provided with at least one reaction channel 921 at both ends of which an inlet 931 and an outlet 932 are implemented and a plurality of reaction channels 921 which are repeatedly spaced across the cross section in the longitudinal direction of the reaction channel 921 A fixed layer 940 formed on one surface of the reaction channel 921 and capable of complementarily binding with one region of the amplification target nucleic acid, And a detection electrode 950 formed in one region and configured to detect an electrochemical signal.

10-12, the fixed layer 940 and the detection electrode 950 are repeatedly disposed across the cross section in the longitudinal direction of the reaction channel 921, and the thermal contact with the thermal block 100 The fixed layer 940 and the detection electrode 950 are arranged to be disposed between the at least two heater groups 110, 10 showing a plan view of the PCR chip 900, the fixed layer 940 and the detection electrode 950 are formed in the reaction channel 921 region from the inflow portion 931 to the outflow portion 932 And thus the electrochemical signal can be repeatedly detected from nucleic acids sequentially amplified while passing through the reaction channel 921 in the longitudinal direction through such a structure. 11 to 12 showing vertical cross-sectional views of the PCR chip 900, it is confirmed that the fixed layer 940 and the detection electrode 950 are disposed opposite to each other in the cross section of the reaction channel 921 The position of the fixed layer 940 and the detection electrode 950 may be varied up and down.

11 to 12, the PCR chip 900 may be divided into three layers based on vertical cross-sectional views. 11 and 12, the PCR chip 900 includes a first plate 910 having 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 having the fixed 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 fixed 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. 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 and 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 includes a fixed layer 940 formed on a reaction channel 921 formed in the second plate 920, an inlet 931 and an outlet 932. 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 931 may have various sizes, but may preferably be selected from 1.0 mm to 3.0 mm in diameter. 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 chip 900 may be mechanically processed to form an inlet 931 and an outlet 932 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 the outlet 932 of the third plate 930 and the reaction channel 921 of the second plate 920 are formed by injection molding, hot-embossing, casting ), And laser ablation. &Lt; IMAGE > 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 > The lower surface of the third plate 930 and the upper surface of the second plate 920 and the lower surface of the second plate 920 and the upper surface of the first plate 910 are thermally bonded, Ultrasonic welding, solvent bonding, and may be performed 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.

On the other hand, according to Figs. 13 to 14 in which the portion "a" of Fig. 10 is enlarged, the detection electrode 950 can be variously implemented. For example, a two-electrode module having a working electrode 950a where an oxidation or reduction reaction takes place as shown in FIG. 13 and a reference electrode 950b where no oxidation or reduction reaction takes place, Electrode module including a counter electrode 950c for adjusting the electronic balance generated from the indicating electrode 950a, the reference electrode 950b, and the indicating electrode as shown in FIG. 14 . When the structure of the detection electrode 950 is realized in the multi-electrode module type as shown in FIGS. 13 to 14, the sensitivity of the electrochemical signal generated in the reaction channel 921 can be increased, The detection and measurement of the signal can be easily performed.

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

15, the chip holder 300 includes a connection port 310 to which the PCR chip 900 is mounted and is electrically connected to the end of the detection electrode 950 of the PCR chip 900. The chip holder 300 is a part where the PCR chip 900 is mounted in the PCR device. The inner wall of the chip holder 300 may have a shape and a structure to be fixedly mounted on the outer wall of the PCR chip 900 so that the plate-shaped PCR chip 900 does not separate from the chip holder 300. That is, when the PCR chip 900 is mounted on the chip holder 300, the end of the detection electrode 950 of the PCR chip 900 is electrically connected to the connection port 310 of the chip holder 300 An electrochemical signal generated in the reaction channel 921 of the PCR chip 900 is transmitted to an electrochemical signal measurement module 800 to be described later. Meanwhile, the PCR chip 900 is detachable from the chip holder 300. It should be noted that the chip holder 300 may be connected to any driving means (not shown) to move vertically or horizontally within the real-time PCR device.

16 shows a real-time PCR apparatus according to an embodiment of the present invention, which includes a PCR chip 900, a power supply unit 400, and a pump 500.

16, the PCR chip 900 is disposed in contact with the thermal block. Specifically, the detection electrode 950 is disposed between the first heater and the second heater which are repeatedly disposed on the thermal block 100 Are repeatedly arranged. The PCR chip 900 and its constituent elements 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, have. For example, when the PCR chip 900 is placed on the thermal block 100 for PCR, a first power port (not shown) of the power supply 400 is connected to the first column electrode And a second power port (not shown) of the power supply 400 is electrically connected to the second column electrode 220. The second power port (not shown) When there is a user instruction to perform the PCR, the power supply unit 400 supplies power to the first column electrode 210 and the second column electrode 220, respectively, The heater 110 and the second heater 120 can be rapidly heated and when the respective heaters 110 and 120 reach the predetermined temperature, the power supply amount 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 fluid flowing in at least one reaction channel 921 of the PCR chip 900 and may be a positive pressure pump or a negative pressure pump, 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 order to control the flow rate and the flow rate of the fluid flowing in the reaction channel 921 in one direction, the pump 500 is connected to the inlet 931 and the outlet 932, And a common stopper may be hermetically connected to the remaining one, and when it is desired to control the flow rate and the flow rate of the fluid flowing in the reaction channel 921, that is, the sample and reagent solution, in both directions, The pump 500 may be connected to both the inlet 931 and the outlet 932.

The nucleic acid amplification reaction of the sample and the reagent in the PCR apparatus including the PCR chip 900, the power supply unit 400 and the pump 500 may be performed through the following steps as an embodiment.

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

2. The sample and reagent solution is introduced into the PCR chip 100. In this case, the sample and the reagent solution are placed in the reaction channel 921 inside the PCR chip 900 through the inlet 931.

3. The column electrode unit 200 and the first column electrode 210 and the second column electrode 220 are connected to the power supply unit 400, (931) and the outlet (932) to the pump (500).

4. An electric power supply unit 400 for supplying power to the first heater 110 and the second heater 120 through the first column electrode 210 and the second column electrode 220, And maintains the PCR denaturation step temperature (95 캜) for a specific temperature, for example, the first heater 110, and the PCR annealing / elongating step temperature 72 캜 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 solution may flow into the reaction channel 921 In the horizontal direction. In this case, the flow rate and the flow rate of the sample and the reagent solution can be controlled by adjusting the intensity of the positive pressure or the negative pressure provided by the pump 500.

By performing the above steps, the sample and reagent solution is supplied to the upper portion 301 of the first heater 110 from the end of the inlet 931 of the reaction channel 921 to the end of the outlet 932, 2 heater while moving the upper corresponding portion 302 of the heater 120 in the longitudinal direction. Referring to FIG. 16, the sample and reagent solution is supplied with heat from the heat block 100 in which the heater unit including the first heater 110 and the second heater 120 is repeatedly arranged ten times, The PCR cycle is completed through the PCR denaturation step at the upper part 301 of the heater 110 and the PCR annealing / extension step at the upper part 302 corresponding to the second heater 120. Optionally, the sample and reagent solution is supplied to the upper portion of the first heater 110 from the end of the outlet 931 of the reaction channel 921 to the end of the inlet 932, ) Can be re-executed while shifting the corresponding portion on the upper side in the longitudinal direction.

17 shows a nucleic acid amplification process by a real-time PCR device according to an embodiment of the present invention, and a process of detecting and measuring nucleic acid amplification signals in real time in accordance with the present invention.

17, a PCR apparatus according to an embodiment of the present invention includes a thermal block 100 in which a first heater 110 and a second heater 120 are repeatedly arranged in a horizontal direction, a first heater 110, And a PCR chip 900 repeatedly arranged so that a fixed layer 940 and a detection electrode 950 correspond to a space between the first and second heaters 120. The connection port of the chip holder (not shown) An electrochemical signal measuring module 800 electrically connected to the reaction channel 921 of the PCR chip 900 to measure an electrochemical signal generated in the reaction channel 921 of the PCR chip 900 in real time, , A pump, and the like. 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, an electrochemical signal repeatedly generated by the sequential nucleic acid amplification in the reaction channel 921 of the PCR chip 900 is sequentially detected through the detection electrode 950 of the PCR chip 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 PCR apparatus of the embodiment of FIG. 17, the nucleic acid amplification process can be measured and analyzed in real time during PCR. In this case, unlike the conventional real-time PCR apparatus, the sample and the reagent solution do not need separate fluorescent materials. In addition, it can be confirmed that the nucleic acid amplification reaction is measured in real-time by the real-time PCR apparatus according to an embodiment of the present invention. For example, the sample and reagent solution may be passed through the reaction channel 921 in succession to the upper corresponding portion 301 of the first heater 110 and the upper corresponding portion 302 of the second heater 120 PCR denaturation step and PCR annealing / lengthening step. In this case, the sample and reagent solution is transferred between the first heater 110 and the second heater 120, and between the first heater 110 and the second heater 120, Passes through the detection electrode 950 region repeatedly disposed between the heater unit including the heater 120. When the sample and reagent solution passes through the corresponding portion of the detection electrode 950 on the upper side, the flow rate of the sample and the reagent solution is slowed or temporarily held in a stopped state through fluid control, and then the amplified target nucleic acid, The electrochemical signal (current change) due to the complementary combination of the signal probe of the complex can be sequentially detected and measured through the detection electrode 950 in real time. 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.

FIG. 18 shows a series of procedures for real-time detection and measurement of nucleic acid amplification and nucleic acid amplification signals using a real-time PCR apparatus according to an embodiment of the present invention.

18, a real-time PCR method using a real-time PCR apparatus according to an embodiment of the present invention includes the steps of: providing the real-time PCR apparatus; Injecting a PCR sample containing a template nucleic acid and a PCR reagent including the metal nanoparticle-signal probe complex into a reaction channel 921 of the PCR chip 900; Mounting the PCR chip 900 into which the PCR sample and the PCR reagent are injected to the chip holder 300 so that the terminal of the electrode 950 of the PCR chip 900 is electrically connected to the connection port 310; The PCR and the PCR reagent are sequentially transferred to the first heater and the second heater which maintain the denaturation temperature of the PCR and the annealing and the extension (or amplification) of the PCR, respectively, while moving the reaction channel 921 in the longitudinal direction And performing PCR by repeated thermal contact; And detecting and measuring in real time an electrochemical signal (current change) due to complementary binding between the amplified target nucleic acid and the capture probe and the signal probe of the complex in the PCR chip 900 during the PCR .

The real-time PCR device providing step S1 is a step of preparing the above-mentioned real-time PCR device. Therefore, the real-time PCR method according to an embodiment of the present invention is based on the premise of driving the real-time PCR apparatus.

The sample and reagent injecting step S2 is a step of injecting a PCR sample and a reagent into the PCR chip 900 and a substance capable of generating an electrical signal through a chemical reaction (binding) with the template nucleic acid to be amplified, for example, a metal nanoparticle - < / RTI > signal probe complex.

The PCR chip mounting step (S3) is a step of mounting the PCR chip (900) containing the PCR sample and the reagent to the chip holder (300) of the real time PCR device (1). In this case, the electrode 950 of the PCR chip 900 should be electrically connected to the connection port 310 of the chip holder 300 for electrochemical signal detection.

The PCR step S4 heats and maintains the temperatures of the first heater 110 and the second heater 120 of the thermal block 100 and the sample and the reagent are supplied to the reaction channel 921 of the PCR chip 900 And the PCR is performed while moving in the longitudinal direction. In this case, the target nucleic acid sites are sequentially amplified on the basis of the template nucleic acid in the sample and the reagent moving in the reaction channel 921, and the target nucleic acid sequence is amplified by successive amplification of the target nucleic acid site and the capture probe and the signal probe of the complex Electrochemical signals are generated due to complementary coupling.

The electrochemical signal detection and measurement step S5 may be performed in the same manner as the electrochemical signal detection and measurement step S5 in which the electrochemical signal (change in current value) generated by the continuous amplification of the nucleic acid in the step S4 is inputted to the electrode 950 of the PCR chip 900, And the electrochemical signal measuring module 800. The electrochemical signal measuring module 800 is connected to the electrochemical signal measuring module 800,

Claims

A heater group having at least one heater and at least two heater groups, wherein the at least two heater groups are repeatedly arranged at least two heater units spaced apart from each other so that mutual heat exchange does not occur, A thermal block having a contact surface of the received PCR chip;
A column electrode unit having column electrodes connected to supply electric power to the heaters provided in the column block;
At least one reaction channel in which an inlet and an outlet are formed at both ends, and a plurality of reaction channels formed in one region inside the reaction channel, the reaction channel being repeatedly disposed across the cross section in the longitudinal direction of the reaction channel, And a detection electrode formed on the other surface of the reaction channel and adapted to detect an electrochemical signal, wherein the metal nanoparticle and the metal nanoparticle A plate-like PCR chip comprising a complex comprising a signal probe connected and complementary to another region of the amplification target nucleic acid;
A chip holder having a connection port on which the PCR chip is mounted and is electrically connected to a 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 an electrochemical signal generated in a reaction channel of the PCR chip in real time;
(Polymerase Chain Reaction) device.

The method according to claim 1,
The metal nanoparticles may be selected from the group consisting of zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu), gallium (Ga), indium (In), gold (Au) Or more is selected.

The method according to claim 1,
Wherein the electrochemical signal is due to a change in current that occurs as the amplification target nucleic acid is complementarily bound to the capture probe and the signal probe of the complex.

The method according to claim 1,
The detection electrode may be at least one selected from the group consisting of Au, Co, Pt, Ag, carbon nanotube, graphene, and carbon. Time PCR device.

The method according to claim 1,
Wherein the amplification target nucleic acid, the capture probe, and the signal probe are single stranded DNA.

The method according to claim 1,
The electrode includes a working electrode having an oxidation or reduction reaction and a reference electrode having no oxidation or reduction reaction, or a two-electrode module having a reference electrode, a reference electrode, Electrode module having a counter electrode for adjusting the electron balance generated from the electrode.

The method according to claim 1,
The electrochemical signal measuring module may be an anodic stripping voltammetry (ASV), a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV) A differential pulse voltammetry (DPV), and an impedance system.

The method according to claim 1,
Wherein the thermal block comprises two to four heater groups.

The method according to claim 1,
Wherein the thermal block comprises two heater groups, wherein the first heater group maintains the PCR denaturation temperature and the second heater group maintains the PCR annealing / extension temperature, or the first heater group is PCR annealed / Extension step temperature and the second heater group maintains PCR denaturation step temperature.

The method according to claim 1,
Wherein the thermal block includes three heater groups, wherein 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 Wherein the PCR extension step temperature is maintained and the second heater group maintains the PCR denaturation step temperature and the third heater group maintains the PCR annealing step temperature.

The method according to claim 1,
Wherein the at least one reaction channel is extended so as to pass the upper corresponding portion of the heater disposed at the best position among the heater units and the upper corresponding portion of the heater disposed at the last position in the straight length direction.

The method according to claim 1,
Wherein the PCR chip comprises: a first plate provided with the detection electrode; A second plate disposed on the first plate and having the at least one reaction channel; And a third plate disposed on the second plate, the third plate having the inlet and the outlet.

The method according to claim 1,
Wherein the PCR chip is detachably mounted on the chip holder.

The method according to claim 1,
And a power supply unit for supplying power to the column electrode unit.

The method according to claim 1,
Further comprising a pump arranged to provide a positive or negative pressure to control the flow rate and flow rate of the fluid flowing in the at least one reaction channel.