WO2014035163A1

WO2014035163A1 - Real-time pcr device comprising thermal block in which heater units are repeatedly arranged for detecting electrochemical signals and real-time pcr method using same

Info

Publication number: WO2014035163A1
Application number: PCT/KR2013/007783
Authority: WO
Inventors: 김성우; 박현규; 이정환; 이유진; 김덕중; 원병연; 백송이
Original assignee: 나노바이오시스(주); 한국과학기술원
Priority date: 2012-08-29
Filing date: 2013-08-29
Publication date: 2014-03-06
Also published as: KR101946339B1; KR20140028430A

Abstract

One embodiment of the present invention relates to a real-time PCR device comprising a thermal block in which heater units are repeatedly arranged for detecting electrochemical signals, and to a real-time PCR method using same. According to the present invention, a plurality of samples can be analyzed simultaneously at an ultra-high speed through the thermal block in which the heater units are repeatedly arranged and a plate-shaped PCR chip, and a simple module that can easily detect continuous electrochemical signals, which are generated during a nucleic acid amplification process, can significantly contribute to the miniaturization and increased portability of a product.

Description

Real-time PCR device for detecting an electrochemical signal including a heat block in which the heater unit is repeatedly arranged, and a real-time PCR method using the same

One embodiment of the present invention relates to a real-time PCR device and a real-time PCR method using the same that can detect and measure the electrochemical signal according to the amplified nucleic acid in real time.

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 problems of the background art as described above, an embodiment of the present invention is to propose a real-time PCR device and a real-time PCR method using the same that can reasonably improve the PCR time and yield, and further miniaturization and portability of the product.

One embodiment of the present invention is a heater group having at least one heater, the heater group having at least two and the two or more heater groups are at least two heater units spaced apart so that mutual heat exchange does not occur, at least, A thermal block having a contact surface of a PCR chip containing a sample and a reagent on one surface thereof; A column electrode unit having a column electrode connected to supply electric power to heaters provided in the column block; At least one reaction channel having both an inlet and an outlet at both ends, and repeatedly spaced apart across the cross section in the longitudinal direction of the reaction channel, and formed in one region inside the reaction channel to complement one region of the amplification target nucleic acid; The capture probe that can be coupled to each other is provided with a fixed surface-treated fixed layer and a detection electrode formed in another region inside the reaction channel and configured to detect an electrochemical signal. A plate-shaped PCR chip comprising a complex connected to the signal probe capable of complementarily binding to another region of the amplification target nucleic acid; 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. to provide.

In the real-time PCR device according to an 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 may be implemented as a three-electrode module having a counter electrode (counter electrode) for adjusting the electronic balance generated from.

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 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 be to maintain the PCR denaturation step temperature and the third heater group to 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 chip may include 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.

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

The apparatus may further include a power supply unit for supplying power to the column electrode unit.

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 the real-time PCR device according to an embodiment of the present invention, not only can a plurality of samples be analyzed at a high speed at the same time through a heat block and a plate-shaped PCR chip in which a heater unit is repeatedly arranged, Simple module implementations that can easily detect continuous electrochemical signals can contribute significantly to product miniaturization and portability.

1 to 5 show a column block and a column electrode part of a real-time PCR device according to an embodiment of the present invention.

6 to 9 illustrate the coupling between the capture probe and the signal probe and the amplification target nucleic acid, and the electrochemical signal generation process, which occur in the reaction chamber of the PCR chip of the real-time PCR device according to an embodiment of the present invention.

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

13 to 14 are enlarged views of one horizontal section 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.

Figure 16 shows a real-time PCR device according to an 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 an 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 an embodiment of the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. The following description is only for easily understanding the embodiments according to the present invention, but 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, to amplify deoxyribonucleic acid (DNA) having a specific base sequence, a PCR device may prepare a solution containing a PCR sample and a reagent comprising double stranded DNA as a template nucleic acid at a specific temperature, for example about 95 ° C. A denaturing step of separating the double-stranded DNA into a single-stranded DNA by heating, to provide an oligonucleotide primer having a sequence complementary to the base sequence to be amplified, and the isolated single-stranded DNA An annealing step in which the primer is coupled to a specific base sequence of the single strand of DNA by cooling to a specific temperature, for example, 55 ° C., to form a partial DNA-primer complex, and after the annealing step, The solution was kept at an appropriate temperature, for example 72 ° C., to primers of the partial DNA-primer complexes by DNA polymerase. An extension (or amplification) step of forming a double strand of DNA on the basis is performed, and the above steps are repeated, for example, 20 to 40 times to exponentially amplify the DNA having the specific base sequence. Can be. In some cases, the PCR device may simultaneously perform the annealing step and the extension (or amplification) step, and in this case, the PCR device performs two steps including the extension step and the annealing and extension (or amplification) step. By doing so, the first circulation can be completed. 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.

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 the 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. In addition, the thermal block includes three heater groups, wherein 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 has 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 The group may maintain the PCR extension step temperature, the second heater group may maintain the PCR denaturation step temperature, and the third heater group may 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. The heaters are spaced apart so that mutual heat exchange does not occur, and as a result, in the nucleic acid amplification reaction that can be greatly affected by minute temperature changes, the denaturation step, annealing step and extension step (or the denaturation step and annealing). Accurate temperature control), and it is possible to maintain the desired temperature or temperature range only at the site 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 in half of the predetermined PCR cycle. For example, when the PCR apparatus according to an embodiment of the present invention is to be applied to a PCR having a circulation cycle of 20 cycles, the heater unit may be repeatedly arranged ten 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 is a thermal electrode 210 connected to supply power to the thermal block 100 of the PCR device and the heaters provided in the thermal block 100 according to an embodiment of the present invention in thermal contact with the PCR chip 900. And a column electrode portion 200 having a 220. Specifically, in the thermal block 100 according to an embodiment of the present invention in thermal contact with the PCR chip 900, the upper end of Figure 5 shows a vertical cross-sectional view of the thermal block 100, The bottom shows a top view of the row block 100. 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 chip 900 will be described later.

6 to 9 illustrate a combination between a capture probe and a signal probe and an amplification target nucleic acid, and an electrochemical signal generation process, which occur inside a reaction chamber of a PCR chip of a real-time PCR device according to an embodiment of the present invention.

According to Figure 6, the PCR chip 900 is a nucleic acid, for example, a template nucleic acid double-stranded DNA PCR sample, oligonucleotide primer having a sequence complementary to a specific base sequence to be amplified PCR reagent, DNA polymerase, three A solution containing deoxyribonucleotide triphosphates (dNTP) and PCR reaction buffer can be accommodated. The PCR chip 900 includes an inlet 931 for introducing the sample and a reagent, an outlet 932 for discharging the solution having completed the nucleic acid amplification reaction, and a nucleic acid amplification reaction of the sample and the reagent. Channel 921 is provided. 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 chip 900 is in thermal contact with the thermal block 100, the PCR sample is provided with heat from the thermal block 100 and included in the reaction channel 921 of the PCR chip 900. And reagents may be heated and maintained. In addition, the PCR chip 900 is implemented in a plate shape as a whole to increase the thermal conductivity and to have two or more reaction channels 921. In addition, the outer structure of the PCR chip 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 chip 900 may be implemented as a plastic material of a transparent or opaque material, the thickness of the plastic material is easy to adjust the thickness can increase the heat transfer efficiency only by adjusting the thickness, manufacturing process is simple chip manufacturing You can save money.

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 chip 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 chip 900 of the real-time PCR device according to an 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.

The PCR chip 900 may be repeatedly spaced across the cross section in one or more reaction channels 921 having the inlet part 931 and the outlet part 932 at both ends, and the length of the reaction channel 921. A fixed layer 940 disposed in a region within the reaction channel 921 and surface-treated with a capture probe capable of complementarily binding to a region of the amplification target nucleic acid, and the other inside the reaction channel 921. A detection electrode 950 is formed in one region and implemented 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 chip 900, the fixed layer 940 and the detection electrode 950 are located in the reaction channel 921 region from the inlet portion 931 to the outlet portion 932. It is repeatedly spaced at regular intervals, through this 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 chip 900, it is confirmed that the fixed layer 940 and the detection electrode 950 are disposed to face each other in the cross section of the reaction channel 921. The positions of the pinned layer 940 and the detection electrode 950 may be changed up and down.

11 to 12, the PCR chip 900 may be divided into three layers based on a vertical cross-sectional view. 11 to 12, the PCR chip 900 includes: a first plate 910 provided with the detection electrode 950; A second plate 920 disposed on the first plate 910 and provided with the one or more reaction channels 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.

An upper surface of the first plate 910 provided with the detection electrode 950 is adhesively disposed on a lower surface of the second plate 920. The first plate 910 is adhered to the second plate 920 having the reaction channel 921 to secure a space with respect to the reaction channel 921, and further, at least the reaction channel 921. The detection electrode 950 is disposed in one region (surface). On the other hand, the first plate 910 may be implemented in a variety of materials, preferably polydimethylsiloxane (PDMS), cycloolefin copolymer (cycle olefin copolymer, COC), polymethyl methacrylate (polymethylmetharcylate) , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof It may be a material selected from. In addition, a hydrophilic material (not shown) may be processed on the upper surface of the first plate 910 to smoothly perform PCR. By treating the hydrophilic material, a single layer including a hydrophilic material may be formed on the first plate 910. The hydrophilic material may be a variety of materials, but preferably may be selected from the group consisting of carboxyl group (-COOH), amine group (-NH2), hydroxy group (-OH), and sulfone group (-SH), Treatment of the hydrophilic material can be carried out according to methods known in the art.

An upper surface of the second plate 920 is disposed in contact with a 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 portion 931 and the outlet portion 932 formed on the third plate 910 so that the inlet portion 931 and the outlet portion 932 are implemented at both ends. One or more reaction channels (921). Therefore, PCR is performed after the PCR sample and the reagent are introduced into the reaction channel 921. In addition, the reaction channel 921 may be present in two or more according to the purpose and range of use of the PCR device according to an embodiment of the present invention. In addition, the second plate 920 may be formed of various materials, but preferably, polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (cycloolefin copolymer, COC) , Polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM) Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (polybutylene terephthalate) , PBT), fluorinated ethylenepropylene (FEP), perfluoroalkoxyalkane (PFA), and combinations thereof It is chosen or a thermoplastic resin may be a thermosetting resin material. In addition, 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. In addition, the inner wall of the second plate 920 may be coated with a material such as silane-based and Bovine Serum Albumin (BSA) to prevent DNA and protein adsorption. The treatment of 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 portion 931 is a portion into which the PCR sample and the reagent are introduced. The outlet 932 is a portion where the PCR product flows out after the PCR is completed. Accordingly, the third plate 930 covers the reaction channel 921 formed in the second plate 920, but the inlet part 931 and the outlet part 932 are the inlet part of the reaction channel 921 and the same. It will act as an outlet. In addition, the third plate 930 may be made of various materials, but preferably, polydimethylsiloxane (PDMS), cycloolefin copolymer (CCO), polymethylmethacrylate (polymethylmetharcylate) , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof It may be a material selected from. 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 portion 932 may have various sizes, but preferably may be selected from a diameter of 1.0 mm to 1.5 mm. In addition, the inlet part 931 and the outlet part 932 are provided with separate cover means (not shown), so that the solution leaks when the PCR sample and the reagent in the reaction channel 921 proceed with the PCR. Can be prevented. The cover means may be implemented in various shapes, sizes or materials. In addition, the thickness of the third plate may vary, but preferably may be selected from 0.1 mm to 2.0 mm. In addition, the inlet part 931 and the outlet part 932 may exist at least two.

On the other hand, the PCR chip 900 to form an inlet (931) and outlet 932 through mechanical processing to provide a third plate (930); The plate having a size corresponding to the bottom surface of the third plate 930 from the portion corresponding to the inlet portion 931 of the third plate 930 to the outlet portion 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 made of a hydrophilic material 922 through surface treatment on an upper surface of a plate having a size corresponding to a lower surface of the second plate 920; And bonding a lower surface of the third plate 930 to an upper surface of the second plate 920 through a bonding process, and attaching a lower surface of the second plate 920 to an upper portion of the first plate 910. It can be easily produced by a method comprising the step of bonding to the surface through a bonding process. 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 may be treated by a method selected from the group consisting of oxygen and argon plasma treatment, corona discharge treatment, and surfactant application and are known in the art. Can be performed according to. 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 processed 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.

According to FIG. 15, the chip holder 300 includes a connection port 310 mounted on the PCR chip 900 and electrically connected to an end of the detection electrode 950 of the PCR chip 900. The chip holder 300 is a portion in which the PCR chip 900 is mounted to the PCR device. The inner wall of the chip holder 300 may have a shape and structure for fixed mounting with the outer wall of the PCR chip 900 so that the PCR chip 900 having a plate shape does not leave the chip holder 300. That is, when the PCR chip 900 is mounted on the chip holder 300, an end of the detection electrode 950 of the PCR chip 900 is electrically connected to the connection port 310 of the chip holder 300. The electrochemical signal generated inside the reaction channel 921 of the PCR chip 900 is transferred to the electrochemical signal measuring module 800 which will be described later. On the other hand, the PCR chip 900 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 illustrates a real-time PCR device according to an embodiment of the present invention having a PCR chip 900, a power supply 400, and a pump 500.

According to FIG. 16, the PCR chip 900 is disposed on and in contact with the heat block, and specifically, the detection electrode 950 is disposed between the repeatedly arranged first and second heaters on the heat block 100. It is arranged repeatedly. The PCR chip 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 900 is disposed in contact with the column block 100 to perform PCR, a first power port (not shown) of the power supply 400 may be connected to the first column electrode (not shown). Electrically connected to 210, a second power port (not shown) of the power supply 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 step temperature (85 ° C. to 105 ° C., preferably 95 ° C.) in the first heater 110 and a PCR annealing / extension step temperature (in the second heater 120). 50 ° C. to 80 ° C., preferably 72 ° C.), or in the first heater 110, a PCR annealing / extension step temperature (50 ° C. to 80 ° C., preferably 72 ° C. or 60 ° C.) and the second heater At 120, the PCR denaturation step temperature (85 ℃ to 105 ℃, preferably 95 ℃) may be.

The pump 500 is a module for controlling the flow rate and the flow rate of the fluid flowing in one or more reaction channels 921 of the PCR chip 900, may be a positive pressure pump or a negative pressure pump, for example a syringe It may be a syringe pump. The pump 500 may be operably disposed in a portion of the reaction channel 921, but preferably the inlet 931 and / or outlet 932 formed at both ends of the reaction channel 921. Is placed in the connection. When the pump 500 is connected to the inlet 931 and / or the outlet 932, the pump 500 may not only serve as a pump but also provide a sample and the like through the inlet 931 and / or the outlet 932. It may also serve as a stopper to prevent leakage of reagent solution. 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.

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

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 chip 100. In this case, the sample and reagent solutions are disposed in the reaction channel 921 inside the PCR chip 900 through the inlet portion 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 400, respectively, and the inlet of the PCR chip 900 931 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.

According to FIG. 17, a PCR device according to an embodiment of the present invention may include a heat block 100, the first heater 110, and the first heater 110 and the second heater 120 repeatedly arranged in a horizontal direction. A PCR chip 900 repeatedly arranged such that the fixed layer 940 and the detection electrode 950 correspond to a space between the second heaters 120, and a connection port (not shown) of the chip holder (not shown). ) Is electrically connected to the electrochemical signal measuring module 800, which is implemented to measure in real time the electrochemical signal generated inside the reaction channel 921 of the PCR chip 900, other power supply unit, although not shown , Pumps and the like. The electrochemical signal measuring 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, electrochemical signals repeatedly generated by sequential nucleic acid amplification inside the reaction channel 921 of the PCR chip 900 are sequentially detected through the detection electrode 950 of the PCR chip 900. The detected signal may be measured and further processed or analyzed by the electrochemical signal measuring module 800 via the connection port of the chip holder and the electrical connection means 700. The electrochemical signal measuring module 800 may vary, but an anode stripping voltammetry (ASV), a chronoamperometry (CA), a cyclic voltammetry, a square wave voltmeter (square) wave voltammetry (SWV), differential pulse voltammetry (DPV), and impedance. Therefore, according to the PCR device according to an embodiment of the present invention according to FIG. 17, the nucleic acid amplification process may be measured and analyzed in 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 an 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. Accordingly, the amount of target nucleic acid is monitored in real time by monitoring the result of the reaction by amplification of the nucleic acid in the reaction channel 921 (without the fluorescent substance and the light detection system) in real time during each cycle of PCR. Can be detected and measured in real-time

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 real-time PCR device described above; Injecting a PCR sample comprising a template nucleic acid and a PCR reagent comprising the metal nanoparticle-signal probe complex into a reaction channel (921) of the PCR chip (900); Mounting a PCR chip (900) into which the PCR sample and the PCR reagent are injected to the chip holder (300) such that an electrode (950) end of the PCR chip (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, in real time, an electrochemical signal (current change) generated by complementary coupling between the amplification target nucleic acid, the capture probe, and the signal probe of the complex in the PCR chip 900 during the PCR. It includes.

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 that can generate an electrical signal, for example, metal nanoparticles by chemical reaction (combination) with the PCR sample and reagent, the template nucleic acid to be amplified in the PCR chip 900 Injecting the 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 to detect the electrochemical signal.

In the PCR step S4, the temperature of the first heater 110 and the second heater 120 of the thermal block 100 is maintained while heating, and the sample and the reagent are removed from the reaction channel 921 of the PCR chip 900. PCR is performed while moving in the longitudinal direction. In this case, target nucleic acid sites are sequentially amplified on the basis of template nucleic acids in samples and reagents moving in the reaction channel 921, and the capture probes and the signal probes of the complex are sequentially amplified according to the continuous amplification of the target nucleic acid sites. Complementary coupling results in electrochemical signals.

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 the electrode 950 of the PCR chip 900, the chip holder 300 Detecting and measuring through the connection port 310 of the, the electrical connection means 700, and the electrochemical signal measuring 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;

At least one reaction channel having both an inlet and an outlet at both ends, and repeatedly spaced apart across the cross section in the longitudinal direction of the reaction channel, and formed in one region inside the reaction channel to complement one region of the amplification target nucleic acid; The capture probe that can be coupled to each other is provided with a fixed surface-treated fixed layer and a detection electrode formed in another region inside the reaction channel and configured to detect an electrochemical signal. A plate-shaped PCR chip comprising a complex connected to the signal probe capable of complementarily binding to another region of the amplification target nucleic acid;

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;

Real-time PCR (Polymerase Chain Reaction) apparatus comprising a.

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). The real-time PCR device characterized in that the above selection.

The method of claim 1,

The electrochemical signal is a real-time PCR device, characterized in that due to the change in current generated as the amplification target nucleic acid complementarily binds to the capture probe and the signal probe of the 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. Real time PCR device, characterized in that.

The method of claim 1,

The amplification target nucleic acid, the capture probe, and the 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 Real-time PCR device, 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 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 1,

The thermal block is a real-time PCR device, 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 Real-time PCR device, characterized in that to maintain 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 Real-time PCR device, 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 length direction through an upper corresponding portion of the heater disposed most optimally and an upper corresponding portion of the heater disposed last.

The method of claim 1,

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

The method of claim 1,

Real-time PCR device further comprises a power supply for supplying power to the column electrode.

The method of claim 1,

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.