WO2014007557A1 - Real-time pcr device for detecting electrochemical signals, and real-time pcr method using same - Google Patents

Real-time pcr device for detecting electrochemical signals, and real-time pcr method using same Download PDF

Info

Publication number
WO2014007557A1
WO2014007557A1 PCT/KR2013/005939 KR2013005939W WO2014007557A1 WO 2014007557 A1 WO2014007557 A1 WO 2014007557A1 KR 2013005939 W KR2013005939 W KR 2013005939W WO 2014007557 A1 WO2014007557 A1 WO 2014007557A1
Authority
WO
WIPO (PCT)
Prior art keywords
pcr
real
chip
electrode
time pcr
Prior art date
Application number
PCT/KR2013/005939
Other languages
French (fr)
Korean (ko)
Inventor
김성우
이정환
이유진
김덕중
Original Assignee
나노바이오시스(주)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 나노바이오시스(주) filed Critical 나노바이오시스(주)
Publication of WO2014007557A1 publication Critical patent/WO2014007557A1/en

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/028—Modular arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept

Definitions

  • 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.
  • PCR Polymerase Chain Reaction
  • PCR Polymerase Chain Reaction
  • 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.
  • 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.
  • 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.
  • 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.
  • real-time PCR 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.
  • 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.
  • 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 includes a first column block and a second column block spaced apart on the substrate; At least one reaction channel having both an inlet and an outlet at both ends, and disposed in at least one region of the reaction channel, and configured to detect an electrochemical signal generated by the binding of the amplifying nucleic acid and the active material within the reaction channel.
  • a plate-shaped PCR chip having an electrode;
  • Drive means implemented to move the chip holder on which the PCR chip is mounted vertically or horizontally so that the PCR chip is in thermal contact with the first row block or the second row block;
  • an electrochemical signal measuring module electrically connected to a connection port of the chip holder to measure in real time an electrochemical signal generated in a reaction channel of the PCR chip.
  • the active material may be a cationic material in the ionization product of the ionic binding material.
  • the ion-bonding material may be methylene blue.
  • the electrochemical signal may be due to the change in the total current value due to the combination of the negative charge of the amplified nucleic acid and the positive charge of the active material.
  • the electrode is at least one from the group consisting of gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotube (carbon nanotube), graphene (graphene) and carbon (Carbon). Can be selected.
  • the electrode is a two-electrode module having a working electrode (combination of the amplification nucleic acid and the active material) and a reference electrode (combination) of the amplification nucleic acid and the active material does not occur, or the indicator It may be implemented as a three-electrode module having a counter electrode for adjusting the electronic balance generated from the electrode, the reference electrode, and the indicator electrode.
  • the electrochemical signal measuring module is an anode stripping voltammetry (ASV), a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV), It may be selected from the group consisting of differential pulse voltammetry (DPV), and impedance (impedance).
  • ASV anode stripping voltammetry
  • CA chronoamperometry
  • SWV square wave voltammetry
  • DPV differential pulse voltammetry
  • impedance impedance
  • first and second heat blocks may be symmetrical in the up, down, and / or left and right directions with respect to the center point of each of the heat blocks in order to maintain a constant temperature of the first and second heat blocks.
  • the heating wire may be arranged at.
  • any one of the first row block and the second row block may be implemented to maintain the denaturation step temperature of the PCR, and the other may maintain the annealing and extension (or amplification) step temperature of the PCR.
  • the denaturation step temperature may be 90 ° C to 100 ° C
  • the extension (or amplification) step temperature may be 55 ° C to 75 ° C.
  • first and second heat blocks may be spaced apart at a predetermined distance such that mutual heat exchange does not occur.
  • the driving means includes a rail extending in the left and right direction, and a connecting member slidably movable in the left and right direction through the rail and slidable in the vertical direction, wherein one end of the connecting member is the chip holder Can be arranged.
  • the PCR chip may be implemented detachably to the chip holder.
  • the PCR chip comprises a first plate provided with the 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.
  • Another embodiment of the present invention comprises the steps of providing a real-time PCR device; Injecting a PCR sample comprising a template nucleic acid and a PCR reagent comprising the active material into a reaction channel of the PCR chip; Mounting a PCR chip in which the PCR sample and the PCR reagent are injected into the chip holder such that an electrode end of the PCR chip is electrically connected to the connection port; By operating the driving means, the PCR chip mounted on the chip holder is repeatedly applied to the first row block and the second row block to maintain the denaturation step temperature of PCR and the annealing and extension (or amplification) step temperature of PCR, respectively. Performing PCR in thermal contact; And it provides a real-time PCR method comprising the step of detecting in real time the electrochemical signal generated by the combination of the amplified nucleic acid and the active material in the PCR chip during the PCR.
  • the active material may be a cationic material in the ionization product of the ionic binding material.
  • the ion-bonding material may be methylene blue.
  • the electrochemical signal may be due to the change in the total current value due to the combination of the negative charge of the amplified nucleic acid and the positive charge of the active material.
  • FIG. 1 shows a real-time PCR device according to an embodiment of the present invention.
  • Figure 2 shows the driving principle of the real-time PCR device according to an embodiment of the present invention.
  • 3 to 6 show a PCR chip according to an embodiment of the present invention.
  • FIG. 7 illustrates a chip holder in accordance with one embodiment of the present invention.
  • FIG. 8 is a layout view of an electrochemical signal measurement module of a real-time PCR device according to an embodiment of the present invention.
  • FIG. 9 is a flowchart illustrating a real-time PCR method according to an embodiment of the present invention.
  • FIG. 10 is a graph showing the results of performing a real-time PCR method according to an embodiment of the present invention.
  • 11 is an electrophoretic picture showing the result of performing a real-time PCR method according to an embodiment of the present invention.
  • FIG. 1 shows a real-time PCR device according to an embodiment of the present invention.
  • the real-time PCR device 1 comprises a first column block 100 and a second column block 200 spaced apart on the substrate 400; At least one reaction channel 921 having both an inlet portion 931 and an outlet portion 932 formed at both ends, and disposed in at least one region of the reaction channel 921, wherein the amplification nucleic acid is formed within the reaction channel 921.
  • a plate-shaped PCR chip 900 having an electrode 950 implemented to detect an electrochemical signal generated by the binding of an active material;
  • a chip holder 300 mounted with the PCR chip 900 and having a connection port 310 configured to be electrically connected to an end of the electrode 950 of the PCR chip 900;
  • the PCR chip 900 may be in thermal contact with the first row block 100 or the second row block 200 by moving the chip holder 300 on which the PCR chip 900 is mounted vertically or horizontally.
  • Drive means 500, 510, 520 implemented such that;
  • an electrochemical signal measuring module electrically connected to the connection port 310 of the chip holder 300 to measure in real time an electrochemical signal generated in the reaction channel 921 of the PCR chip 900. 800).
  • FIG. 1 illustrates a first column block 100 and a second column block 200, a plate-shaped PCR chip 900, and a chip holder on which the PCR chip 900 is mounted.
  • 300, the chip holder 300 on which the PCR chip 900 is mounted is moved up and down or left and right so that the PCR chip 900 is opened in the first row block 100 or the second row block 200.
  • the PCR device refers to a device used for PCR (Polymerase Chain Reaction) for amplifying a nucleic acid having a specific nucleotide sequence.
  • PCR Polymerase Chain Reaction
  • 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.
  • the real-time PCR device 1 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 On the premise that all are provided in the obvious range.
  • the real-time PCR apparatus 1 may include a first row block 100 disposed on a substrate 400 and the first row block 100 on the substrate 400. ) And a second row block 200 spaced apart from each other.
  • the substrate 400 does not change its physical or chemical properties due to heating of the first thermal block 100 and the second thermal block 200, and the first thermal block 100 and the second thermal block 200 do not change. It may be implemented as a material having a material so that heat exchange does not occur between).
  • the first row block 100 and the second row block 200 may maintain a temperature for performing a denaturation step, annealing step and extension (or amplification) step for amplifying the nucleic acid.
  • the first thermal block 100 and the second thermal block 200 may include or be operably connected with various modules for providing and maintaining the temperature required for the respective steps. Therefore, when the PCR chip 900 mounted on the chip holder 300 contacts one surface of each of the row blocks 100 and 200, the first row block 100 and the second row block 200 may be moved. The contact surface with the PCR chip 900 can be heated as a whole, so that the solution contained in the PCR chip 900 can be uniformly heated or maintained at temperature.
  • the real time PCR apparatus employing a conventional single row block has a rate of temperature change in a single row block within a range of 3 to 7 ° C.
  • the rate of temperature change in each of the thermal blocks 100 and 200 is within a range of 20 to 40 ° C. per second, which can significantly shorten the PCR execution time.
  • hot wires may be disposed in the first row block 100 and the second row block 200.
  • the hot wire can be operably connected with various heat sources to maintain a temperature for performing the denaturing, annealing and extending (or amplifying) steps, and can be operably connected with various temperature sensors for monitoring the temperature of the hot wire. Can be.
  • the heating wires may be moved up and down and / or left and right with respect to the center point of the surface of each of the heat blocks 100 and 200 in order to maintain a constant internal temperature of the first and second heat blocks 100 and 200. It may be arranged to be symmetrical.
  • a thin film heater (not shown) may be disposed in the first thermal block 100 and the second thermal block 200. The thin-film heater is vertically and / or horizontally based on a center point of each of the heat block 100 and 200 in order to maintain a constant internal temperature of the first and second heat blocks 100 and 200. May be spaced apart at regular intervals.
  • the first thermal block 100 and the second thermal block 200 are embodied in a plate shape for even heat distribution and rapid heat transfer of the same area to the PCR chip 900, and a metal material, for example, aluminum material It may comprise or be made of aluminum.
  • the first thermal block 100 may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) steps.
  • the first row block 100 of the real-time PCR device 1 can maintain 50 °C to 100 °C, preferably in the first thermal block 100
  • the denaturation step may be maintained at 90 °C to 100 °C, preferably 95 °C, 55 °C to 75 when performing the annealing and extension (or amplification) step in the first heat block.
  • °C can be maintained, preferably 72 °C.
  • the specific temperature and range are not limited as long as the denaturation step or the temperature at which the annealing and extension (or amplification) steps can be performed.
  • the second thermal block 200 may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) steps.
  • the second row block 200 of the real-time PCR apparatus 1 may maintain 90 ° C to 100 ° C when performing the denaturation step in the second row block,
  • the temperature may be maintained at 95 ° C., and may be maintained at 55 ° C. to 75 ° C., preferably at 72 ° C., when the annealing and extension (or amplification) steps are performed in the second heat block.
  • the specific temperature and range are not limited as long as the denaturation step or the temperature at which the annealing and extension (or amplification) steps can be performed.
  • the first heat block 100 may maintain the denaturing temperature of the PCR (denaturing temperature), the denaturation of the template nucleic acid occurs when the denaturation step temperature is lower than 90 °C yield If the denaturation step temperature is higher than 100 °C may decrease or disappear the activity of the enzyme used in the PCR, the denaturation step temperature may be 90 °C to 100 °C, preferably 95 °C Can be.
  • the second row block 200 may maintain annealing / extension temperature of annealing and extension (or amplification) of a PCR reaction.
  • extension (or amplification) step temperature is lower than 55 ° C, the specificity of the PCR reaction product may be lowered, and if the annealing and extension (or amplification) step temperature is higher than 74 ° C, extension by primers may not occur. Since the PCR efficiency is lowered, the annealing and extension (or amplification) step temperature may be 55 ° C to 75 ° C, preferably 72 ° C.
  • the first thermal block 100 and the second thermal block 200 are spaced apart at a predetermined distance such that mutual heat exchange does not occur.
  • the heat exchange does not occur between the first heat block 100 and the second heat block 200, in the nucleic acid amplification reaction that can be significantly affected by minute temperature changes, the denaturation step and the Accurate temperature control of the annealing and extension (or amplification) steps is possible.
  • Real-time PCR device 1 includes a driving means (500).
  • the driving means 500 moves the chip holder 300 on which the PCR chip 900 is mounted up, down, left, or right so that the PCR chip 900 has the first row block 100 or the second row block ( Each of which is in thermal contact.
  • the chip holder 300 on which the PCR chip 900 is mounted is capable of reciprocating left and right between the first row block 100 and the second row block 200,
  • the driving means 500 the chip holder 300 on which the PCR chip 900 is mounted may be contacted or separated up and down with the first row block 100 and the second row block 200. According to FIG.
  • the driving means 500 is arranged to be slidably movable in a left and right direction through a rail 510 extending in a left and right direction, and the rail 510, and a connecting member 520 that is slidably movable in an up and down direction.
  • the chip holder 300 is connected to one end of the connection member 520.
  • the left and right and / or vertical movement of the driving means 500 may be controlled by control means (not shown) which is operably disposed inside or outside the real-time PCR apparatus 1.
  • the real-time PCR device 1 includes a plate-shaped PCR chip 900 and a chip holder 300 for receiving PCR samples and reagents, the PCR chip 900 is the chip holder Removably implemented at 300, the details of the PCR chip 900 and the chip holder 300 will be described later.
  • Figure 2 shows the driving principle of the real-time PCR device according to an embodiment of the present invention.
  • the nucleic acid amplification reaction using the real-time PCR device 1 is implemented as follows. First, for example, a template nucleic acid (eg, double-stranded DNA), an oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified, a DNA polymerase, and a triphosphate deoxyribo After introducing a PCR sample including a nucleotide (deoxyribonucleotide triphosphates (dNTP), PCR buffer) and a solution containing a reagent, the PCR chip 900 is mounted on the chip holder 300.
  • dNTP deoxyribonucleotide triphosphates
  • the first heat block 100 is heated and maintained at a denaturation step temperature, for example 90 ° C. to 100 ° C., preferably 95 ° C.
  • the second heat block 200 is heated and maintained at a temperature for an annealing and extension (or amplification) step, for example 55 ° C. to 75 ° C., preferably 72 ° C.
  • the PCR chip 900 is moved downward by controlling the connection member 520 of the driving means 500 to move the PCR chip 900 mounted on the chip holder 300 to the first row block ( 100) to perform the PCR first denaturation step (step x).
  • the PCR chip 900 is moved upward by controlling the connecting member 520 of the driving means 500 to move the PCR chip 900 mounted on the chip holder 300 to the first row block 100. End the PCR first denaturation step and control the rail 510 and the connection member 520 of the driving means 500 to move the PCR chip 900 above the second row block 200. (Step y). Thereafter, the PCR chip 900 is moved downward by controlling the connection member 520 of the driving means 500 to move the PCR chip 900 mounted on the chip holder 300 to the second row block ( 100) to perform PCR first annealing and extension (or amplification) steps (step z).
  • the PCR chip 900 is moved upward by controlling the connecting member 520 of the driving means 500 to move the PCR chip 900 mounted on the chip holder 300 to the second row block 100. End the first annealing and extension (or amplification) step of the PCR, and control the rail 510 and the connecting member 520 of the driving means 500 to control the PCR chip 900 in a first row. After moving up block 200, the steps x, y, and z are repeated to perform nucleic acid amplification reactions at predetermined cycles (circulation step).
  • 3 to 6 are detailed views of the PCR chip 900 according to an embodiment of the present invention.
  • PCR chip 900 is one or more reaction channels 921, the inlet 931 and the outlet 932 is implemented at both ends, and at least one region of the reaction channel 921
  • the electrode 950 is disposed in the reaction channel 921 and is configured to detect an electrochemical signal generated by the binding of the amplifying nucleic acid and the active material in the reaction channel 921.
  • the PCR chip 900 is 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, DNA polymerase, and triphosphate deoxyribonucleotide (deoxyribonucleotide triphosphates (dNTP), a solution containing a PCR reaction buffer can be accommodated.
  • dNTP triphosphate deoxyribonucleotide triphosphates
  • 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.
  • 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.
  • the external 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.
  • 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.
  • the active material is defined as a substance that chemically reacts (couples) with the amplifying nucleic acid to generate an electrochemical signal
  • the electrochemical signal can be continuously detected and measured according to the continuous amplification of the nucleic acid Say a signal.
  • a double stranded nucleic acid DNA
  • the amplified nucleic acid reacts with the active material as a result of continuous amplification of the nucleic acid, thereby detecting a change in total charge amount. Can be derived.
  • the electrochemical signal may be due to the change in the total current value due to the combination of the negative charge of the amplified nucleic acid and the positive charge of the active material
  • the active material may be a cationic material in the ionization product of the ion-binding material have.
  • the ionizable material may be methylene blue
  • the active material may be a cationic material in the ionization product of methylene blue.
  • the methylene blue (C 16 H 18 N 3 SCl.3H 2 O) is ionized when dissolved in a solvent and ionized with C 16 H 18 N 3 S + and Cl ⁇ , in the case of the former is positively charged by a sulfur atom (S).
  • Double-stranded nucleic acid is composed of sugar, base and phosphoric acid, of which the phosphate group is negatively charged, double-stranded nucleic acid (DNA) is negatively charged as a whole.
  • the cation of methylene blue binds to the phosphate group of DNA, reducing the apparent diffusion of methylene blue bound to the double-stranded nucleic acid rather than the apparent diffusion of methylene blue, thus reducing the peak value of the current. Therefore, as the PCR cycle proceeds, the double-stranded nucleic acid (DNA) is amplified and the amount of methylene blue bound to the double-stranded nucleic acid (DNA) increases, resulting in a decrease in the peak value of the current. Real-time quantification of amplified nucleic acids is possible through an electrical signal due to chemical bonding of.
  • the electrode 950 is disposed in at least one region of the reaction channel 921, and is configured to detect an electrochemical signal generated due to the combination of an amplifying nucleic acid and an active material inside the reaction channel 921.
  • the electrode 950 may be formed of various materials to perform the above functions, but for example, gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes (carbon nanotubes) ), Graphene, and carbon may be selected from one or more selected from the group consisting of.
  • the electrode 950 may be implemented in various shapes or structures to perform the above function, but is disposed at the bottom of the central region of the reaction channel 921, for example, as shown in FIG.
  • the working electrode (950a) and the amplification of the nucleic acid and the active material that the binding of the amplifying nucleic acid and the active material occurs A two-electrode module (right side of FIG. 4) having a reference electrode 950b where no coupling occurs, or electrons generated from the indicator electrode 950a, the reference electrode 950b, and the indicator electrode It may be implemented as a three-electrode module (left side of FIG. 4) having a counter electrode 950c for adjusting the balance.
  • the structure of the electrode 950 is implemented in the multi-electrode module method as illustrated in FIG. 4, not only the sensitivity of the electrochemical signal generated in the reaction channel 921 may be increased, but also the detection of the generated signal may be performed. And measurement can be easily performed.
  • the upper surface of the first plate 910 with the electrode 950 is adhesively disposed on the 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 electrode 950 is disposed in one region (surface).
  • 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.
  • a hydrophilic material (not shown) may be processed on the upper surface of the first plate 910 to smoothly perform PCR.
  • hydrophilic material 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.
  • 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.
  • the reaction channel 921 may be present in two or more depending on the purpose and range of use of the PCR device 1 according to an embodiment of the present invention, according to Figure 3, two reaction channels 921 are illustrated have.
  • 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.
  • the thickness of the second plate 920 may vary, but may be selected from 100 ⁇ m to 200 ⁇ m.
  • the width and length of the reaction channel 921 may vary, but preferably the width of the reaction channel 921 is selected from 0.5 mm to 3 mm, the length of the reaction channel 921 is 20 mm To 40 mm.
  • the inner wall of the second plate 920 may be coated with a material such as silane-based and Bovine Serum Albumin (BSA) to prevent DNA and protein adsorption.
  • BSA Bovine Serum Albumin
  • the lower surface of the third plate 930 is disposed on the upper surface of the second plate 920.
  • the third plate 930 includes an inlet portion 931 formed in one region on the reaction channel 921 formed in the second plate 920 and an outlet portion 932 formed in the other region.
  • 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.
  • 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.
  • the inlet portion 931 may have various sizes, but preferably may be selected from a diameter of 1.0 mm to 3.0 mm.
  • the outlet portion 932 may have various sizes, but preferably may be selected from a diameter of 1.0 mm to 1.5 mm.
  • 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.
  • the thickness of the third plate may vary, but preferably may be selected from 0.1 mm to 2.0 mm.
  • the inlet part 931 and the outlet part 932 may exist at least two.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 7 illustrates a chip holder in accordance with one embodiment of the present invention.
  • the chip holder 300 includes a connection port 310 on which the PCR chip 900 is mounted but is electrically connected to an end of the 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 1.
  • 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, the end of the electrode 950 of the PCR chip 900 is electrically connected to the connection port 310 of the chip holder 300.
  • the electrochemical signal generated by the binding of the amplifying nucleic acid and the active material in the reaction channel 921 of the PCR chip 900 is transferred to the electrochemical signal measuring module 800 which will be described later.
  • the PCR chip 900 is removable from the chip holder 300.
  • the chip holder 300 is connected to the driving means 500, specifically, the end of the connecting member 520 may be moved up and down or left and right inside the real-time PCR device (1).
  • FIG. 8 is a layout view of an electrochemical signal measurement module of a real-time PCR device according to an embodiment of the present invention.
  • the real-time PCR device 1 is electrically connected to the connection port 310 of the chip holder 300 so as to be inside the reaction channel 921 of the PCR chip 900.
  • An electrochemical signal measurement module 800 is implemented to measure in real time the electrochemical signal generated in the.
  • the electrochemical signal measuring module 800 may be electrically connected to the connection port 310 of the chip holder 300 through an electrical connection means 700, for example, a lead wire. Therefore, an electrochemical signal generated in the reaction channel 921 of the PCR chip 900 is detected through the electrode 950 of the PCR chip 900, and the detected signal is detected by the chip holder 300.
  • 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.
  • ASV anode stripping voltammetry
  • CA chronoamperometry
  • SWV square wave voltmeter
  • DPV differential pulse voltammetry
  • FIG. 9 is a flowchart illustrating a real-time PCR method according to an embodiment of the present invention.
  • a real-time PCR method comprises the steps of providing the above-described real-time PCR device (1); Injecting a PCR sample containing a template nucleic acid and a PCR reagent containing the active material into the 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 first row block for operating the driving means 500 to maintain the denaturation step temperature of the PCR and the annealing and extension (or amplification) step temperature of the PCR, respectively, mounted on the chip holder 300.
  • the real time PCR device providing step S1 is a step of preparing the above-described real time PCR device 1. Therefore, the real-time PCR method according to an embodiment of the present invention below assumes the driving of the real-time PCR device (1).
  • Sample and reagent injection step (S2) is a material that can generate an electrical signal, such as methylene blue to the PCR chip 900 through the chemical reaction (combination) with the PCR sample and reagents, and the template nucleic acid to be amplified Injecting.
  • 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.
  • 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.
  • the first heat block 100 and the second heat block 200 are heated and maintained, and the driving means 500 is operated to perform PCR in the reaction channel 921 of the PCR chip 900.
  • This is the step to be performed.
  • the target nucleic acid site is amplified based on the template nucleic acid in the reaction channel 921, and the electrochemical signal is generated due to the continuous reaction (binding) with the active material according to the continuous amplification of the target nucleic acid site. .
  • 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.
  • the time point for detecting and measuring the electrochemical signal may vary, but the PCR chip 900 mounted on the chip holder 300 maintains the temperature of the extension (or amplification) step of the PCR according to the time when the nucleic acid is amplified. The point of time in thermal contact with the heat block (first heat block or second heat block) or immediately after the heat contact is preferred.
  • the point of time immediately after the thermal contact refers to the driving means 500 after the PCR chip 900 mounted on the chip holder 300 is in thermal contact with a thermal block that maintains the temperature of the PCR (or amplification) step.
  • the PCR chip 900 includes three reaction channels 921 and electrodes 950 connected to the ends thereof, respectively, in a plate shape made of plastic,
  • the electrode 950 was fabricated using carbon nanotubes and silver (Ag), and the electrochemical signal measuring module 800 adopted an anode stripping voltammetry (ASV). 100 ⁇ l of methylene blue was added to the PCR reagent as a substance to provide the active substance.
  • ASV anode stripping voltammetry
  • 100 ⁇ l of methylene blue was added to the PCR reagent as a substance to provide the active substance.
  • the detection conditions of the electrochemical signal of the anode peeling voltammeter were assumed as follows.
  • 0.1 ng / ⁇ l of double stranded template DNA (template ds-DNA) is prepared as a PCR sample, and a pair of primers complementarily binding to a specific nucleotide sequence as a PCR reagent, specifically, a forward primer (0.125 ⁇ l, 1 pmole), Reverse primer (0.125 ⁇ l, 1pmole), 0.2 mL of dNTP, 0.2 mL of polymerase (i-starmax II polymerase, iNtRON Biotechnology), PCR buffer (pH 9, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl) 2 , 30 mM salt), and the like, the PCR sample and reagent solution are introduced into the PCR chip 900 according to an embodiment of the present invention, and the PCR chip 900 is mounted on the chip holder 300.
  • a forward primer (0.125 ⁇ l, 1 pmole
  • Reverse primer (0.125 ⁇ l
  • the first heat block 100 is heated and maintained at 95 ° C., that is, the allowable temperature range is 90 ° C. to 100 ° C.
  • the second heat block 200 is 72 ° C., that is, the allowable temperature range is 55 ° C. Heated to and maintained at 75 ° C.
  • the PCR device 1 was operated to perform 40 cycles of PCR (Pre-denaturation, 95 ° C., 30 sec, once; Denaturation 95 ° C., 4 sec, 40 times; Annealing & Extension 72 ° C.). , 30 sec, 40 times).
  • ASV anodizing stripping voltammetry
  • FIG. 10 is a graph showing a result of performing a real-time PCR method according to an embodiment of the present invention
  • Figure 11 is an electrophoresis picture showing the result of performing a real-time PCR method according to an embodiment of the present invention.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Clinical Laboratory Science (AREA)
  • Dispersion Chemistry (AREA)
  • Hematology (AREA)
  • Electrochemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

An embodiment of the present invention relates to a real-time PCR device and method, whereby not only is it possible to perform simultaneous ultra-high-speed analysis of a plurality of samples via a dual heat block and plate-shaped PCR chip but it is also possible to significantly contribute to the microminiaturization and increased portability of products through simple modularization allowing the easy detection of continuous electrochemical signals generated in the process of nucleic acid amplification.

Description

전기화학적 신호를 검출하기 위한 실시간 PCR 장치, 및 이를 이용한 실시간 PCR 방법Real time PCR apparatus for detecting electrochemical signal, and real time PCR method using same
본 발명의 일 실시예는 증폭 핵산에 따른 전기화학적 신호를 실시간으로 검출 및 측정할 수 있는 실시간 PCR 장치 및 이를 이용한 실시간 PCR 방법에 관한 것이다.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.
중합효소 연쇄 반응, 즉 PCR(Polymerase Chain Reaction)은 주형 핵산의 특정 부위를 반복적으로 가열 및 냉각하여 상기 특정 부위를 연쇄적으로 복제하여 그 특정 부위를 갖는 핵산을 기하급수적으로 증폭하는 기술로써, 생명과학, 유전공학 및 의료 분야 등에서 분석 및 진단 목적으로 널리 사용되고 있다. 최근 상기 PCR을 수행하기 위한 PCR 장치가 다양하게 개발되고 있다. 종래 PCR 장치의 일 예는 하나의 반응 챔버에 주형 핵산을 포함하는 샘플 용액을 포함하는 용기를 장착하고, 상기 용기를 반복적으로 가열 및 냉각하여 PCR 반응을 수행한다. 그러나, 상기 PCR 장치는 하나의 반응 챔버를 구비하기 때문에 전체적인 구조가 복잡하진 않지만, 정확한 온도 제어를 위해 복잡한 회로를 구비해야 하고, 하나의 반응 챔버에 대한 반복적인 가열 및 냉각으로 인해 전체 PCR 수행 시간이 길어지는 문제점이 있다. 또한, 종래 PCR 장치의 다른 예는 PCR 진행 온도를 갖는 복수 개의 반응 챔버를 장착하고, 이들 반응 챔버를 통과하는 하나의 채널을 통해 핵산을 포함하는 샘플 용액을 흐르게 하여 PCR을 수행한다. 그러나, 상기 PCR 장치는 복수 개의 반응 챔버를 이용하기 때문에 정확한 온도 제어를 위한 복잡한 회로가 요구되진 않지만, 고온 및 저온의 반응 챔버를 통과하기 위한 긴 유로가 반드시 필요하므로 전체 구조가 복잡하고, 상기 반응 챔버를 통과하는 채널에 흐르는 핵산을 포함하는 샘플 용액의 유속을 제어하기 위한 별도의 제어 장치가 요구된다. 한편, 최근 PCR 장치는 PCR 수율을 개선하기 위한 노력뿐만 아니라 PCR 진행 과정을 실시간으로 파악하기 위한 효율적인 방법을 개방하기 위한 방향으로 개발되고 있다. 이와 같이 PCR 진행 과정을 실시간으로 파악할 수 있는 기술을 소위 "실시간 PCR(real-time PCR)"이라고 하는데, 실시간 PCR 장치는 PCR 챔버에 형광물질을 투입하여 증폭 산물과의 결합으로 발생하는 광신호를 측정하는 기술이 채용된다. 그러나, 이 경우 상기 실시간 PCR 장치는 형광물질로부터 광신호를 활성화하기 위한 별도의 광원 모듈, 증폭 핵산으로부터 획득된 광신호를 검출하기 위한 광검출 모듈, 및 기타 광 경로를 조절하기 위한 반사경 등 복잡한 구조를 반드시 채용해야 하는 바, 기기의 소형화가 어렵고, 휴대용으로 활용하기 어려운 문제점이 있다.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.
따라서, PCR 시간을 줄임과 동시에 신뢰할 수 있는 PCR 수율을 얻을 수 있고, 더 나아가 제품의 소형화 및 휴대화가 가능한 실시간 PCR 장치의 필요성이 부각되고 있는 실정이다.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.
위와 같은 배경기술의 문제점을 해결하고자, 본 발명의 일 실시예는 PCR 시간 및 수율을 합리적으로 개선하고, 더 나아가 제품의 소형화 및 휴대화가 가능한 실시간 PCR 장치 및 이를 이용한 실시간 PCR 방법을 제안하고자 한다.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.
본 발명의 일 실시예는 기판 상부에 이격 배치된 제1 열 블록 및 제2 열 블록; 양 말단에 유입부 및 유출부가 구현된 1 이상의 반응 채널, 및 상기 반응 채널의 적어도 일 영역에 배치되되 상기 반응 채널 내부에서 증폭 핵산과 활성물질의 결합으로 인해 발생하는 전기화학적 신호를 검출하도록 구현된 전극을 구비하는, 판 형상의 PCR 칩; 상기 PCR 칩이 장착되되 상기 PCR 칩의 전극 말단과 전기적으로 연결되도록 구현된 연결 포트를 구비하는 칩 홀더; 상기 PCR 칩이 장착된 칩 홀더를 상하 또는 좌우로 이동하여 상기 PCR 칩이 상기 제1 열 블록 또는 상기 제2 열 블록에 열 접촉할 수 있도록 구현된 구동 수단; 및 상기 칩 홀더의 연결 포트와 전기적으로 연결되어 상기 PCR 칩의 반응 채널 내부에서 발생하는 전기화학적 신호를 실시간으로 측정하도록 구현된 전기화학적 신호 측정 모듈을 포함하는 실시간 PCR 장치를 제공한다.One embodiment of the present invention includes a first column block and a second column block spaced apart on the substrate; At least one reaction channel having both an inlet and an outlet at both ends, and disposed in at least one region of the reaction channel, and configured to detect an electrochemical signal generated by the binding of the amplifying nucleic acid and the active material within the reaction channel. A plate-shaped PCR chip having an electrode; A chip holder mounted with the PCR chip, the chip holder having a connection port configured to be electrically connected to an electrode end of the PCR chip; Drive means implemented to move the chip holder on which the PCR chip is mounted vertically or horizontally so that the PCR chip is in thermal contact with the first row block or the second row block; And an electrochemical signal measuring module electrically connected to a connection port of the chip holder to measure in real time an electrochemical signal generated in a reaction channel of the PCR chip.
본 발명의 일 실시예에 있어서,In one embodiment of the invention,
상기 활성물질은 이온결합성 물질의 이온화 산물 중 양이온 물질일 수 있다. 이 경우 상기 이온결합성 물질은 메틸렌 블루(methylene blue)일 수 있다.The active material may be a cationic material in the ionization product of the ionic binding material. In this case, the ion-bonding material may be methylene blue.
또한, 상기 전기화학적 신호는 상기 증폭 핵산의 음 전하와 상기 활성물질의 양 전하의 결합에 인한 총 전류값 변화에 기인하는 것일 수 있다.In addition, the electrochemical signal may be due to the change in the total current value due to the combination of the negative charge of the amplified nucleic acid and the positive charge of the active material.
또한, 상기 전극은 금(Au), 코발트(Co), 백금(Pt), 은(Ag), 탄소나노튜브(carbon nanotube), 그래핀(graphene), 및 탄소(Carbon)로 구성된 군으로부터 1 이상 선택될 수 있다.In addition, the electrode is at least one from the group consisting of gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotube (carbon nanotube), graphene (graphene) and carbon (Carbon). Can be selected.
또한, 상기 전극은 상기 증폭 핵산과 활성물질의 결합이 일어나는 지시 전극(working electrode) 및 상기 증폭 핵산과 활성물질의 결합이 일어나지 않는 기준 전극(reference electrode)을 구비하는 2-전극 모듈, 또는 상기 지시 전극, 상기 기준 전극, 및 상기 지시 전극으로부터 발생하는 전자 밸런스를 조절하는 카운터 전극(counter electrode)을 구비하는 3-전극 모듈로 구현될 수 있다.In addition, the electrode is a two-electrode module having a working electrode (combination of the amplification nucleic acid and the active material) and a reference electrode (combination) of the amplification nucleic acid and the active material does not occur, or the indicator It may be implemented as a three-electrode module having a counter electrode for adjusting the electronic balance generated from the electrode, the reference electrode, and the indicator electrode.
또한, 상기 전기화학적 신호 측정 모듈은 양극 벗김 전압전류계(anodic stripping voltammetry, ASV), 대시간 전류계 (chronoamperometry, CA), 순환 전압전류계(cyclic voltammetry), 네모파 전압전류계(square wave voltammetry, SWV), 펄스 전압전류계(differential pulse voltammetry, DPV), 및 임피던스계(impedance)로 구성된 군으로부터 선택될 수 있다.In addition, the electrochemical signal measuring module is an anode stripping voltammetry (ASV), a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV), It may be selected from the group consisting of differential pulse voltammetry (DPV), and impedance (impedance).
또한, 상기 제1 열 블록 및 제2 열 블록은 상기 제1 열 블록 및 제2 열 블록의 온도를 전체적으로 일정하게 유지하기 위해 각 열 블록의 중심점을 기준으로 상하 및/또는 좌우 방향으로 대칭되도록 내부에 열선이 배치될 수 있다.In addition, the first and second heat blocks may be symmetrical in the up, down, and / or left and right directions with respect to the center point of each of the heat blocks in order to maintain a constant temperature of the first and second heat blocks. The heating wire may be arranged at.
또한, 상기 제1 열 블록 및 상기 제2 열 블록 중 어느 하나는 PCR의 변성 단계 온도를 유지하고, 다른 하나는 PCR의 어닐링 및 연장 (혹은 증폭) 단계 온도를 유지하도록 구현될 수 있다. 이 경우 상기 변성 단계 온도는 90℃ 내지 100℃이고, 상기 연장 (혹은 증폭) 단계 온도는 55℃ 내지 75℃일 수 있다.In addition, any one of the first row block and the second row block may be implemented to maintain the denaturation step temperature of the PCR, and the other may maintain the annealing and extension (or amplification) step temperature of the PCR. In this case, the denaturation step temperature may be 90 ° C to 100 ° C, and the extension (or amplification) step temperature may be 55 ° C to 75 ° C.
또한, 상기 제1 열 블록과 제2 열 블록은 상호 열 교환이 일어나지 않도록 미리 결정된 거리로 이격 배치될 수 있다.In addition, the first and second heat blocks may be spaced apart at a predetermined distance such that mutual heat exchange does not occur.
또한, 상기 구동 수단은 좌우 방향으로 연장된 레일, 및 상기 레일을 통해 좌우 방향으로 슬라이딩 이동가능하게 배치되고 상하 방향으로 슬라이딩 이동 가능한 연결 부재를 포함하고, 상기 연결 부재의 일 말단에는 상기 칩 홀더가 배치될 수 있다.In addition, the driving means includes a rail extending in the left and right direction, and a connecting member slidably movable in the left and right direction through the rail and slidable in the vertical direction, wherein one end of the connecting member is the chip holder Can be arranged.
또한, 상기 PCR 칩은 상기 칩 홀더에 탈착 가능하게 구현될 수 있다.In addition, the PCR chip may be implemented detachably to the chip holder.
또한, 상기 PCR 칩은 상기 전극이 구비된 제1 판; 상기 제1 판 상에 배치되되 상기 1 이상의 반응 채널이 구비된 제2 판; 및 상기 제2 판 상에 배치되되 상기 유입부 및 유출부가 구비된 제3 판을 포함할 수 있다.In addition, the PCR chip comprises a first plate provided with the 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.
본 발명의 다른 일 실시예는 상기 실시간 PCR 장치를 제공하는 단계; 주형 핵산을 포함하는 PCR 시료 및 상기 활성물질을 포함하는 PCR 시약을 상기 PCR 칩의 반응 채널에 주입하는 단계; 상기 PCR 칩의 전극 말단이 상기 연결 포트에 전기적으로 연결되도록 상기 PCR 시료 및 PCR 시약이 주입된 PCR 칩을 상기 칩 홀더에 장착하는 단계; 상기 구동 수단을 가동하여 상기 칩 홀더에 장착된 PCR 칩이 PCR의 변성 단계 온도 및 PCR의 어닐링 및 연장(혹은 증폭) 단계 온도를 각각 유지하는 상기 제1 열 블록 및 상기 제2 열 블록에 반복적으로 열 접촉하여 PCR을 수행하는 단계; 및 상기 PCR 수행 중 상기 PCR 칩 내부에서 증폭 핵산과 상기 활성물질의 결합으로 인해 발생하는 전기화학적 신호를 실시간으로 검출 및 측정하는 단계를 포함하는, 실시간 PCR 방법을 제공한다.Another embodiment of the present invention comprises the steps of providing a real-time PCR device; Injecting a PCR sample comprising a template nucleic acid and a PCR reagent comprising the active material into a reaction channel of the PCR chip; Mounting a PCR chip in which the PCR sample and the PCR reagent are injected into the chip holder such that an electrode end of the PCR chip is electrically connected to the connection port; By operating the driving means, the PCR chip mounted on the chip holder is repeatedly applied to the first row block and the second row block to maintain the denaturation step temperature of PCR and the annealing and extension (or amplification) step temperature of PCR, respectively. Performing PCR in thermal contact; And it provides a real-time PCR method comprising the step of detecting in real time the electrochemical signal generated by the combination of the amplified nucleic acid and the active material in the PCR chip during the PCR.
본 발명의 다른 일 실시예에 있어서,In another embodiment of the present invention,
상기 활성물질은 이온결합성 물질의 이온화 산물 중 양이온 물질일 수 있다. 이 경우 상기 이온결합성 물질은 메틸렌 블루(methylene blue)일 수 있다.The active material may be a cationic material in the ionization product of the ionic binding material. In this case, the ion-bonding material may be methylene blue.
또한, 상기 전기화학적 신호는 상기 증폭 핵산의 음 전하와 상기 활성물질의 양 전하의 결합에 인한 총 전류값 변화에 기인하는 것일 수 있다.In addition, the electrochemical signal may be due to the change in the total current value due to the combination of the negative charge of the amplified nucleic acid and the positive charge of the active material.
본 발명의 일 실시예에 따른 실시간 PCR 장치 및 방법에 따르면, 듀얼(dual) 열 블록 및 판 형상의 PCR 칩을 통해 다수의 샘플을 동시에 초고속으로 분석할 수 있을 뿐만 아니라, 핵산 증폭 과정에서 발생하는 연속적인 전기화학적 신호를 쉽게 검출할 수 있는 단순한 모듈 구현을 통해 제품의 극-소형화 및 휴대화에 상당히 기여할 수 있다.According to the real-time PCR apparatus and method 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 dual column block and a plate-shaped PCR chip, Simple module implementations that can easily detect continuous electrochemical signals can contribute significantly to product miniaturization and portability.
도 1은 본 발명의 일 실시예에 따른 실시간 PCR 장치를 도시한다.1 shows a real-time PCR device according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 실시간 PCR 장치의 구동원리를 도시한다.Figure 2 shows the driving principle of the real-time PCR device according to an embodiment of the present invention.
도 3 내지 6은 본 발명의 일 실시예에 따른 PCR 칩을 도시한다.3 to 6 show a PCR chip according to an embodiment of the present invention.
도 7은 본 발명의 일 실시예에 따른 칩 홀더를 도시한다.7 illustrates a chip holder in accordance with one embodiment of the present invention.
도 8은 본 발명의 일 실시예에 따른 실시간 PCR 장치의 전기화학적 신호 측정 모듈의 배치도이다.8 is a layout view of an electrochemical signal measurement module of a real-time PCR device according to an embodiment of the present invention.
도 9는 본 발명의 일시예에 따른 실시간 PCR 방법을 나타내는 순서도이다.9 is a flowchart illustrating a real-time PCR method according to an embodiment of the present invention.
도 10은 본 발명의 일시예에 따른 실시간 PCR 방법을 수행한 결과를 나타내는 그래프이다.10 is a graph showing the results of performing a real-time PCR method according to an embodiment of the present invention.
도 11은 본 발명의 일 실시예에 따른 실시간 PCR 방법을 수행한 결과를 나타내는 전기영동 사진이다.11 is an electrophoretic picture showing the result of performing a real-time PCR method according to an embodiment of the present invention.
이하, 첨부 도면을 참조하여 본 발명에 따른 실시예들을 상세하게 설명한다. 이하 설명은 본 발명에 따른 일 실시예들을 용이하게 이해하기 위한 것일 뿐이며, 보호범위를 제한하기 위한 것은 아니다.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.
도 1은 본 발명의 일 실시예에 따른 실시간 PCR 장치를 도시한다. 1 shows a real-time PCR device according to an embodiment of the present invention.
본 발명의 일 실시예에 따른 실시간 PCR 장치(1)는 기판(400) 상부에 이격 배치된 제1 열 블록(100) 및 제2 열 블록(200); 양 말단에 유입부(931) 및 유출부(932)가 구현된 1 이상의 반응 채널(921), 및 상기 반응 채널(921)의 적어도 일 영역에 배치되되 상기 반응 채널(921) 내부에서 증폭 핵산과 활성물질의 결합으로 인해 발생하는 전기화학적 신호를 검출하도록 구현된 전극(950)을 구비하는, 판 형상의 PCR 칩(900); 상기 PCR 칩(900)이 장착되되 상기 PCR 칩(900)의 전극(950) 말단과 전기적으로 연결되도록 구현된 연결 포트(310)를 구비하는 칩 홀더(300); 상기 PCR 칩(900)이 장착된 칩 홀더(300)를 상하 또는 좌우로 이동하여 상기 PCR 칩(900)이 상기 제1 열 블록(100) 또는 상기 제2 열 블록(200)에 열 접촉할 수 있도록 구현된 구동 수단(500, 510, 520); 및 상기 칩 홀더(300)의 연결 포트(310)와 전기적으로 연결되어 상기 PCR 칩(900)의 반응 채널(921) 내부에서 발생하는 전기화학적 신호를 실시간으로 측정하도록 구현된 전기화학적 신호 측정 모듈(800)을 포함한다. The real-time PCR device 1 according to an embodiment of the present invention comprises a first column block 100 and a second column block 200 spaced apart on the substrate 400; At least one reaction channel 921 having both an inlet portion 931 and an outlet portion 932 formed at both ends, and disposed in at least one region of the reaction channel 921, wherein the amplification nucleic acid is formed within the reaction channel 921. A plate-shaped PCR chip 900 having an electrode 950 implemented to detect an electrochemical signal generated by the binding of an active material; A chip holder 300 mounted with the PCR chip 900 and having a connection port 310 configured to be electrically connected to an end of the electrode 950 of the PCR chip 900; The PCR chip 900 may be in thermal contact with the first row block 100 or the second row block 200 by moving the chip holder 300 on which the PCR chip 900 is mounted vertically or horizontally. Drive means 500, 510, 520 implemented such that; And an electrochemical signal measuring module electrically connected to the connection port 310 of the chip holder 300 to measure in real time an electrochemical signal generated in the reaction channel 921 of the PCR chip 900. 800).
도 1은 기판(400) 상부에 이격 배치된 제1 열 블록(100) 및 제2 열 블록(200), 판 형상의 PCR 칩(900), 및 상기 PCR 칩(900)이 장착되는 칩 홀더(300), 상기 PCR 칩(900)이 장착된 칩 홀더(300)를 상하 또는 좌우로 이동하여 상기 PCR 칩(900)이 상기 제1 열 블록(100) 또는 상기 제2 열 블록(200)에 열 접촉할 수 있도록 구현된 구동 수단(500, 510, 520)을 도시한다.1 illustrates a first column block 100 and a second column block 200, a plate-shaped PCR chip 900, and a chip holder on which the PCR chip 900 is mounted. 300, the chip holder 300 on which the PCR chip 900 is mounted is moved up and down or left and right so that the PCR chip 900 is opened in the first row block 100 or the second row block 200. It shows a drive means 500, 510, 520 implemented to be in contact.
PCR 장치란 특정 염기 서열을 갖는 핵산을 증폭하는 PCR(Polymerase Chain Reaction)에 사용하는 장치를 말한다. 예를 들어, 특정 염기 서열을 갖는 DNA(deoxyribonucleic acid)를 증폭하기 위해 PCR 장치는 주형 핵산인 이중 가닥의 DNA를 포함하는 PCR 시료 및 시약을 포함하는 용액을 특정 온도, 예를 들어 약 95℃로 가열하여 상기 이중 가닥의 DNA를 단일 가닥의 DNA로 분리하는 변성 단계(denaturing step), 증폭하고자 하는 염기 서열과 상보적인 서열을 갖는 올리고뉴클레오티드(oligonucleotide) 프라이머를 제공하고, 상기 분리된 단일 가닥의 DNA와 함께 특정 온도, 예를 들어 55℃로 냉각하여 상기 단일 가닥의 DNA의 특정 염기 서열에 상기 프라이머를 결합시켜 부분적인 DNA-프라이머 복합체를 형성하는 어닐링 단계(annealing step), 및 상기 어닐링 단계 이후 상기 용액을 적정 온도, 예를 들어 72℃로 유지하여 DNA 중합효소(polymerase)에 의해 상기 부분적인 DNA-프라이머 복합체의 프라이머를 기초로 이중 가닥의 DNA를 형성하는 연장 (혹은 증폭) 단계(extension step)를 수행하고, 상기 3 단계를 예를 들어 20회 내지 40회로 반복함으로써 상기 특정 염기 서열을 갖는 DNA를 기하급수적으로 증폭할 수 있다. 경우에 따라, 상기 PCR 장치는 상기 어닐링 단계와 상기 연장(혹은 증폭) 단계를 동시에 수행할 수 있고, 이 경우 PCR 장치는 상기 연장 단계와 상기 어닐링 및 연장(혹은 증폭) 단계로 구성된 2 단계를 수행함으로써, 제1 순환을 완성할 수도 있다. 따라서, 본 발명의 일 구체예에 따른 실시간 PCR 장치(1)는 상기 단계들을 수행하기 위한 모듈들을 포함하는 장치를 말하며, 본 명세서에 기재되지 아니한 세부적인 모듈들은 PCR을 수행하기 위한 종래 기술 중 개시되고 자명한 범위에서 모두 구비하고 있는 것을 전제로 한다.The PCR device refers to a device used for PCR (Polymerase Chain Reaction) for amplifying a nucleic acid having a specific nucleotide 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 On the premise that all are provided in the obvious range.
도 1에 따르면, 본 발명의 일 실시예에 따른 실시간 PCR 장치(1)는 기판(400) 상부에 배치된 제1 열 블록(100) 및 상기 기판(400) 상부에 상기 제1 열 블록(100)과 이격 배치된 제2 열 블록(200)을 포함한다. 상기 기판(400)은 상기 제1 열 블록(100) 및 제2 열 블록(200)의 가열로 인해 그 물리적 또는 화학적 성질이 변하지 않고, 상기 제1 열 블록(100) 및 제2 열 블록(200) 간에 열 교환이 일어나지 않도록 하는 재질을 갖는 물질로 구현될 수 있다. 상기 제1 열 블록(100) 및 제2 열 블록(200)은 핵산을 증폭하기 위한 변성 단계, 어닐링 단계 및 연장 (혹은 증폭) 단계를 수행하기 위한 온도를 유지할 수 있다. 따라서 상기 제1 열 블록(100) 및 제2 열 블록(200)은 상기 각 단계들에 요구되는 온도를 제공하고, 이를 유지하기 위한 다양한 모듈을 포함하거나 또는 그러한 모듈과 구동가능하게 연결될 수 있다. 따라서, 상기 칩 홀더(300)에 장착된 PCR 칩(900)이 상기 각 열 블록(100, 200)의 일 면에 접촉하면 상기 제1 열 블록(100) 및 제2 열 블록(200)은 상기 PCR 칩(900)과의 접촉 면을 전체적으로 가열할 수 있어서, 상기 PCR 칩(900) 내부에 수용된 용액을 균일하게 가열 또는 온도 유지할 수 있다. 기존의 단일 열 블록을 채용했던 실시간 PCR 장치는 단일 열 블록에서의 온도 변화율이 초당 3 내지 7℃ 범위 내에서 이루어지는데 반해, 본 발명의 일 실시예에 따른 2개의 열 블록을 포함하는 실시간 PCR 장치(1)는 각각의 열 블록(100, 200)에서의 온도 변화율이 초당 20 내지 40℃ 범위 내에서 이루어져 PCR 수행 시간을 상당히 단축할 수 있다. 또한, 상기 제1 열 블록(100) 및 제2 열 블록(200)은 그 내부에 열선(도시되지 않음)이 배치될 수 있다. 상기 열선은 상기 변성 단계, 어닐링 단계 및 연장 (혹은 증폭) 단계를 수행하기 위한 온도를 유지하도록 다양한 열원과 구동가능하게 연결될 수 있고, 상기 열선의 온도를 모니터하기 위한 다양한 온도 센서와 구동가능하게 연결될 수 있다. 상기 열선은 상기 제1 열 블록(100) 및 제2 열 블록(200) 내부 온도를 전체적으로 일정하게 유지하기 위해 각각의 열 블록(100, 200) 면의 중심점을 기준으로 상하 및/또는 좌우 방향으로 대칭되도록 배치될 수 있다. 또한, 상기 제1 열 블록(100) 및 제2 열 블록(200)은 그 내부에 박막 히터(thin film heater, 도시되지 않음)가 배치될 수 있다. 상기 박막 히터는 상기 제1 열 블록(100) 및 제2 열 블록(200) 내부 온도를 전체적으로 일정하게 유지하기 위해 각각의 열 블록(100, 200) 면의 중심점을 기준으로 상하 및/또는 좌우 방향으로 일정한 간격으로 이격 배치될 수 있다. 상기 제1 열 블록(100) 및 제2 열 블록(200)은 상기 PCR 칩(900)에 동일 면적의 고른 열 분포 및 신속한 열 전달을 위해 판 형상으로 구현되고, 금속 재질, 예를 들어 알루미늄 재질을 포함하거나 또는 알루미늄 재질로 구성될 수 있다. 상기 제1 열 블록(100)은 상기 변성 단계, 또는 어닐링 및 연장 (혹은 증폭) 단계를 수행하기 위한 적정 온도를 유지하도록 구현될 수 있다. 예를 들어, 본 발명의 일 실시예에 따른 실시간 PCR 장치(1)의 제1 열 블록(100)은 50℃ 내지 100℃를 유지할 수 있고, 바람직하게는 상기 제1 열 블록(100)에서 상기 변성 단계를 수행하는 경우 90℃ 내지 100℃를 유지할 수 있고, 바람직하게는 95℃를 유지할 수 있으며, 상기 제1 열 블록에서 상기 어닐링 및 연장 (혹은 증폭) 단계를 수행하는 경우에는 55℃ 내지 75℃를 유지할 수 있고, 바람직하게는 72℃를 유지할 수 있다. 다만, 상기 특정 온도 및 범위는 상기 변성 단계, 또는 어닐링 및 연장 (혹은 증폭) 단계를 수행할 수 있는 온도라면 제한되는 것은 아니다. 상기 제2 열 블록(200)은 상기 변성 단계, 또는 어닐링 및 연장 (혹은 증폭) 단계를 수행하기 위한 적정 온도를 유지하도록 구현될 수 있다. 예를 들어, 본 발명의 일 실시예에 따른 실시간 PCR 장치(1)의 제2 열 블록(200)은 상기 제2 열 블록에서 상기 변성 단계를 수행하는 경우 90℃ 내지 100℃를 유지할 수 있고, 바람직하게는 95℃를 유지할 수 있으며, 상기 제2 열 블록에서 상기 어닐링 및 연장 (혹은 증폭) 단계를 수행하는 경우에는 55℃ 내지 75℃를 유지할 수 있고, 바람직하게는 72℃를 유지할 수 있다. 다만, 상기 특정 온도 및 범위는 상기 변성 단계, 또는 어닐링 및 연장 (혹은 증폭) 단계를 수행할 수 있는 온도라면 제한되는 것은 아니다. 따라서, 본 발명의 일 실시예에 따르면, 상기 제1 열 블록(100)은 PCR의 변성 단계 온도 (denaturing temperature)를 유지할 수 있으며, 변성 단계 온도가 90℃보다 낮으면 주형 핵산의 변성이 일어나 수율이 떨어지거나 반응이 일어나지 않을 수도 있고, 변성 단계 온도가 100℃보다 높아지면 PCR에 사용되는 효소의 활성이 감소하거나 사라져서, 상기 변성 단계 온도는 90℃ 내지 100℃일 수 있고, 바람직하게는 95℃일 수 있다. 또한, 본 발명의 일 실시예에 따르면, 상기 제2 열 블록(200)은 PCR 반응의 어닐링 및 연장 (혹은 증폭) 단계 온도(annealing/extension temperature)를 유지할 수 있다. 연장 (혹은 증폭) 단계 온도가 55℃보다 낮으면 PCR 반응 산물의 특이성(specificity)이 떨어질 수 있고, 어닐링 및 연장 (혹은 증폭) 단계 온도가 74℃보다 높으면 프라이머에 의한 연장이 일어나지 않을 수 있기 때문에 PCR 효율이 떨어지게 되므로 상기 어닐링 및 연장 (혹은 증폭) 단계 온도는 55℃ 내지 75℃일 수 있고, 바람직하게는 72℃일 수 있다. 상기 제1 열 블록(100)과 제2 열 블록(200)은 상호 열 교환이 일어나지 않도록 미리 결정된 거리로 이격 배치된다. 이에 따라, 상기 제1 열 블록(100)과 제2 열 블록(200) 사이에서 열 교환이 일어나지 않기 때문에, 미세한 온도 변화에 의해서도 중대한 영향을 받을 수 있는 핵산 증폭 반응에 있어서, 상기 변성 단계와 상기 어닐링 및 연장 (혹은 증폭) 단계의 정확한 온도 제어가 가능하다.According to FIG. 1, the real-time PCR apparatus 1 according to an exemplary embodiment of the present invention may include a first row block 100 disposed on a substrate 400 and the first row block 100 on the substrate 400. ) And a second row block 200 spaced apart from each other. The substrate 400 does not change its physical or chemical properties due to heating of the first thermal block 100 and the second thermal block 200, and the first thermal block 100 and the second thermal block 200 do not change. It may be implemented as a material having a material so that heat exchange does not occur between). The first row block 100 and the second row block 200 may maintain a temperature for performing a denaturation step, annealing step and extension (or amplification) step for amplifying the nucleic acid. Thus, the first thermal block 100 and the second thermal block 200 may include or be operably connected with various modules for providing and maintaining the temperature required for the respective steps. Therefore, when the PCR chip 900 mounted on the chip holder 300 contacts one surface of each of the row blocks 100 and 200, the first row block 100 and the second row block 200 may be moved. The contact surface with the PCR chip 900 can be heated as a whole, so that the solution contained in the PCR chip 900 can be uniformly heated or maintained at temperature. The real time PCR apparatus employing a conventional single row block has a rate of temperature change in a single row block within a range of 3 to 7 ° C. per second, whereas a real time PCR device including two row blocks according to an embodiment of the present invention (1), the rate of temperature change in each of the thermal blocks 100 and 200 is within a range of 20 to 40 ° C. per second, which can significantly shorten the PCR execution time. In addition, hot wires (not shown) may be disposed in the first row block 100 and the second row block 200. The hot wire can be operably connected with various heat sources to maintain a temperature for performing the denaturing, annealing and extending (or amplifying) steps, and can be operably connected with various temperature sensors for monitoring the temperature of the hot wire. Can be. The heating wires may be moved up and down and / or left and right with respect to the center point of the surface of each of the heat blocks 100 and 200 in order to maintain a constant internal temperature of the first and second heat blocks 100 and 200. It may be arranged to be symmetrical. In addition, a thin film heater (not shown) may be disposed in the first thermal block 100 and the second thermal block 200. The thin-film heater is vertically and / or horizontally based on a center point of each of the heat block 100 and 200 in order to maintain a constant internal temperature of the first and second heat blocks 100 and 200. May be spaced apart at regular intervals. The first thermal block 100 and the second thermal block 200 are embodied in a plate shape for even heat distribution and rapid heat transfer of the same area to the PCR chip 900, and a metal material, for example, aluminum material It may comprise or be made of aluminum. The first thermal block 100 may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) steps. For example, the first row block 100 of the real-time PCR device 1 according to an embodiment of the present invention can maintain 50 ℃ to 100 ℃, preferably in the first thermal block 100 When performing the denaturation step may be maintained at 90 ℃ to 100 ℃, preferably 95 ℃, 55 ℃ to 75 when performing the annealing and extension (or amplification) step in the first heat block. ℃ can be maintained, preferably 72 ℃. However, the specific temperature and range are not limited as long as the denaturation step or the temperature at which the annealing and extension (or amplification) steps can be performed. The second thermal block 200 may be implemented to maintain an appropriate temperature for performing the denaturation step, or the annealing and extension (or amplification) steps. For example, the second row block 200 of the real-time PCR apparatus 1 according to an embodiment of the present invention may maintain 90 ° C to 100 ° C when performing the denaturation step in the second row block, Preferably, the temperature may be maintained at 95 ° C., and may be maintained at 55 ° C. to 75 ° C., preferably at 72 ° C., when the annealing and extension (or amplification) steps are performed in the second heat block. However, the specific temperature and range are not limited as long as the denaturation step or the temperature at which the annealing and extension (or amplification) steps can be performed. Therefore, according to one embodiment of the present invention, the first heat block 100 may maintain the denaturing temperature of the PCR (denaturing temperature), the denaturation of the template nucleic acid occurs when the denaturation step temperature is lower than 90 ℃ yield If the denaturation step temperature is higher than 100 ℃ may decrease or disappear the activity of the enzyme used in the PCR, the denaturation step temperature may be 90 ℃ to 100 ℃, preferably 95 ℃ Can be. In addition, according to an embodiment of the present invention, the second row block 200 may maintain annealing / extension temperature of annealing and extension (or amplification) of a PCR reaction. If the extension (or amplification) step temperature is lower than 55 ° C, the specificity of the PCR reaction product may be lowered, and if the annealing and extension (or amplification) step temperature is higher than 74 ° C, extension by primers may not occur. Since the PCR efficiency is lowered, the annealing and extension (or amplification) step temperature may be 55 ° C to 75 ° C, preferably 72 ° C. The first thermal block 100 and the second thermal block 200 are spaced apart at a predetermined distance such that mutual heat exchange does not occur. Accordingly, since the heat exchange does not occur between the first heat block 100 and the second heat block 200, in the nucleic acid amplification reaction that can be significantly affected by minute temperature changes, the denaturation step and the Accurate temperature control of the annealing and extension (or amplification) steps is possible.
본 발명의 일 실시예에 따른 실시간 PCR 장치(1)는 구동 수단(500)을 포함한다. 상기 구동 수단(500)은 상기 PCR 칩(900)이 장착된 칩 홀더(300)를 상하 또는 좌우로 이동하여 상기 PCR 칩(900)이 상기 제1 열 블록(100) 또는 상기 제2 열 블록(200)에 각각 열 접촉할 수 있도록 구현된다. 상기 구동 수단(500)에 의해, 상기 PCR 칩(900)이 장착된 칩 홀더(300)는 상기 제1 열 블록(100)과 제2 열 블록(200) 사이에서 좌우로 왕복 운동이 가능하고, 상기 구동 수단(500)에 의해, 상기 PCR 칩(900)이 장착된 칩 홀더(300)는 상기 제1 열 블록(100)과 제2 열 블록(200)에 상하로 접촉 또는 분리될 수 있다. 도 1에 따르면, 상기 구동 수단(500)은 좌우 방향으로 연장된 레일(510), 및 상기 레일(510)을 통해 좌우 방향으로 슬라이딩 이동 가능하게 배치되고, 상하 방향으로 슬라이딩 이동 가능한 연결 부재(520)를 포함하고, 상기 연결 부재(520)의 일 말단에 상기 칩 홀더(300)가 연결 배치된다. 상기 구동 수단(500)의 좌우 및/또는 상하 이동은 상기 실시간 PCR 장치(1)의 내부 또는 외부에 구동가능하게 배치된 제어 수단(도시되지 않음)에 의해 제어될 수 있다.Real-time PCR device 1 according to an embodiment of the present invention includes a driving means (500). The driving means 500 moves the chip holder 300 on which the PCR chip 900 is mounted up, down, left, or right so that the PCR chip 900 has the first row block 100 or the second row block ( Each of which is in thermal contact. By the driving means 500, the chip holder 300 on which the PCR chip 900 is mounted is capable of reciprocating left and right between the first row block 100 and the second row block 200, By the driving means 500, the chip holder 300 on which the PCR chip 900 is mounted may be contacted or separated up and down with the first row block 100 and the second row block 200. According to FIG. 1, the driving means 500 is arranged to be slidably movable in a left and right direction through a rail 510 extending in a left and right direction, and the rail 510, and a connecting member 520 that is slidably movable in an up and down direction. The chip holder 300 is connected to one end of the connection member 520. The left and right and / or vertical movement of the driving means 500 may be controlled by control means (not shown) which is operably disposed inside or outside the real-time PCR apparatus 1.
본 발명의 일 실시예에 따른 실시간 PCR 장치(1)는 PCR 시료 및 시약을 수용하는 판 형상의 PCR 칩(900) 및 칩 홀더(300)를 포함하고, 상기 PCR 칩(900)은 상기 칩 홀더(300)에 탈착 가능하게 구현된다, 상기 PCR 칩(900) 및 칩 홀더(300)에 관한 상세사항은 후술한다.The real-time PCR device 1 according to an embodiment of the present invention includes a plate-shaped PCR chip 900 and a chip holder 300 for receiving PCR samples and reagents, the PCR chip 900 is the chip holder Removably implemented at 300, the details of the PCR chip 900 and the chip holder 300 will be described later.
도 2는 본 발명의 일 실시예에 따른 실시간 PCR 장치의 구동원리를 도시한다.Figure 2 shows the driving principle of the real-time PCR device according to an embodiment of the present invention.
도 2에 따르면, 본 발명에 따른 실시간 PCR 장치(1)를 이용한 핵산 증폭 반응은 아래와 같이 구현된다. 먼저, 상기 PCR 칩(900)에 예를 들어, 주형 핵산(예를 들어, 이중 가닥 DNA), 증폭하고자 하는 특정 염기 서열과 상보적인 서열을 갖는 올리고뉴클레오티드 프라이머, DNA 중합효소, 삼인산화데옥시리보뉴클레오티드(deoxyribonucleotide triphosphates, dNTP), PCR 완충액(PCR buffer)를 포함하는 PCR 시료 및 시약을 포함하는 용액을 도입한 후, 상기 PCR 칩(900)을 상기 칩 홀더(300)에 장착한다. 뒤이어, 상기 제1 열 블록(100)을 변성 단계 온도, 예를 들어, 90℃ 내지 100℃, 바람직하게는 95℃로 가열 및 유지한다. 뒤이어 또는 이와 동시에, 상기 제2 열 블록(200)을 어닐링 및 연장 (혹은 증폭) 단계를 위한 온도, 예를 들어, 55℃ 내지 75℃, 바람직하게는 72℃로 가열 및 유지한다. 그 후, 상기 구동 수단(500)의 연결 부재(520)를 제어하여 상기 PCR 칩(900)을 하향 이동시켜, 상기 칩 홀더(300)에 장착된 PCR 칩(900)을 상기 제1 열 블록(100)에 접촉시켜 PCR 제1 변성 단계를 수행한다(x 단계). 뒤이어, 상기 구동 수단(500)의 연결 부재(520)를 제어하여 상기 PCR 칩(900)을 상향 이동시켜, 상기 칩 홀더(300)에 장착된 PCR 칩(900)을 상기 제1 열 블록(100)으로부터 분리시켜 PCR 제1 변성 단계를 종료하고, 상기 구동 수단(500)의 레일(510) 및 연결 부재(520)를 제어하여 상기 PCR 칩(900)을 제2 열 블록(200)의 위로 이동시킨다(y 단계). 그 후, 상기 구동 수단(500)의 연결 부재(520)를 제어하여 상기 PCR 칩(900)을 하향 이동시켜, 상기 칩 홀더(300)에 장착된 PCR 칩(900)을 상기 제2 열 블록(100)에 접촉시켜 PCR 제1 어닐링 및 연장 (혹은 증폭) 단계를 수행한다(z 단계). 뒤이어, 상기 구동 수단(500)의 연결 부재(520)를 제어하여 상기 PCR 칩(900)을 상향 이동시켜, 상기 칩 홀더(300)에 장착된 PCR 칩(900)을 상기 제2 열 블록(100)으로부터 분리시켜 PCR의 제1 어닐링 및 연장 (혹은 증폭) 단계를 종료하고, 상기 구동 수단(500)의 레일(510) 및 연결 부재(520)를 제어하여 상기 PCR 칩(900)을 제1 열 블록(200)의 위로 이동시킨 후 상기 x, y, z 단계를 반복함으로써, 일정 주기로 핵산 증폭 반응을 수행한다(순환 단계).According to Figure 2, the nucleic acid amplification reaction using the real-time PCR device 1 according to the present invention is implemented as follows. First, for example, a template nucleic acid (eg, double-stranded DNA), an oligonucleotide primer having a sequence complementary to a specific nucleotide sequence to be amplified, a DNA polymerase, and a triphosphate deoxyribo After introducing a PCR sample including a nucleotide (deoxyribonucleotide triphosphates (dNTP), PCR buffer) and a solution containing a reagent, the PCR chip 900 is mounted on the chip holder 300. Subsequently, the first heat block 100 is heated and maintained at a denaturation step temperature, for example 90 ° C. to 100 ° C., preferably 95 ° C. Subsequently or at the same time, the second heat block 200 is heated and maintained at a temperature for an annealing and extension (or amplification) step, for example 55 ° C. to 75 ° C., preferably 72 ° C. Thereafter, the PCR chip 900 is moved downward by controlling the connection member 520 of the driving means 500 to move the PCR chip 900 mounted on the chip holder 300 to the first row block ( 100) to perform the PCR first denaturation step (step x). Subsequently, the PCR chip 900 is moved upward by controlling the connecting member 520 of the driving means 500 to move the PCR chip 900 mounted on the chip holder 300 to the first row block 100. End the PCR first denaturation step and control the rail 510 and the connection member 520 of the driving means 500 to move the PCR chip 900 above the second row block 200. (Step y). Thereafter, the PCR chip 900 is moved downward by controlling the connection member 520 of the driving means 500 to move the PCR chip 900 mounted on the chip holder 300 to the second row block ( 100) to perform PCR first annealing and extension (or amplification) steps (step z). Subsequently, the PCR chip 900 is moved upward by controlling the connecting member 520 of the driving means 500 to move the PCR chip 900 mounted on the chip holder 300 to the second row block 100. End the first annealing and extension (or amplification) step of the PCR, and control the rail 510 and the connecting member 520 of the driving means 500 to control the PCR chip 900 in a first row. After moving up block 200, the steps x, y, and z are repeated to perform nucleic acid amplification reactions at predetermined cycles (circulation step).
도 3 내지 6은 본 발명의 일 실시예에 따른 PCR 칩(900)의 상세도이다.3 to 6 are detailed views of the PCR chip 900 according to an embodiment of the present invention.
본 발명의 일 실시예에 따른 PCR 칩(900)은 양 말단에 유입부(931) 및 유출부(932)가 구현된 1 이상의 반응 채널(921), 및 상기 반응 채널(921)의 적어도 일 영역에 배치되되 상기 반응 채널(921) 내부에서 증폭 핵산과 활성물질의 결합으로 인해 발생하는 전기화학적 신호를 검출하도록 구현된 전극(950)을 구비한다. PCR chip 900 according to an embodiment of the present invention is one or more reaction channels 921, the inlet 931 and the outlet 932 is implemented at both ends, and at least one region of the reaction channel 921 The electrode 950 is disposed in the reaction channel 921 and is configured to detect an electrochemical signal generated by the binding of the amplifying nucleic acid and the active material in the reaction channel 921.
상기 PCR 칩(900)은 핵산, 예를 들어 PCR 시료인 주형 핵산 이중 가닥 DNA, PCR 시약인 증폭하고자 하는 특정 염기 서열과 상보적인 서열을 갖는 올리고뉴클레오티드 프라이머, DNA 중합효소, 삼인산화데옥시리보뉴클레오티드(deoxyribonucleotide triphosphates, dNTP), PCR 완충액 (PCR reaction buffer)을 포함하는 용액을 수용할 수 있다. 상기 PCR 칩(900)은 상기 시료 및 시약을 도입하기 위한 유입부(931), 핵산 증폭 반응을 완료한 용액을 배출하기 위한 유출부(932) 및 상기 시료 및 시약의 핵산 증폭 반응이 수행되는 반응 채널(921)을 구비한다. 상기 PCR 칩(900)의 일 표면이 상기 제1 열 블록(100) 및 제2 열 블록(200)에 열 접촉하면 상기 제1 열 블록(100) 및 제2 열 블록(200)으로부터 열을 제공받고, 상기 PCR 칩(900)의 반응 채널(921)에 포함된 PCR 시료 및 시약은 가열 및 유지될 수 있다. 또한, 상기 PCR 칩(900)은 열 전도율을 높이고 2 이상의 반응 채널(921)을 구비할 수 있도록 전체적으로 판 형상으로 구현된다. 또한, 상기 PCR 칩(900)의 외부 구조는 상기 칩 홀더(300)로부터 이탈되지 않도록 상기 칩 홀더(300)의 내부 공간에 고정 장착되도록 구현된다. 또한, 상기 PCR 칩(900)은 투명 또는 불투명 재질의 플라스틱 재질로 구현될 수 있는데, 플라스틱 재질의 특성상 두께 조절이 용이하여 두께 조절만으로 열 전달 효율을 증대시킬 수 있고, 제작 공정이 단순하여 칩 제조 비용을 절감할 수 있다. The PCR chip 900 is 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, DNA polymerase, and triphosphate deoxyribonucleotide (deoxyribonucleotide triphosphates (dNTP), a solution containing a 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. When one surface of the PCR chip 900 is in thermal contact with the first row block 100 and the second row block 200, heat is provided from the first row block 100 and the second row block 200. The PCR samples and reagents included in the reaction channel 921 of the PCR chip 900 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 external 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. 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.
한편, 상기 활성물질(redox indicator)은 증폭 핵산과 화학적으로 반응(결합)하여 전기화학적 신호를 일으키는 물질로 정의되고, 상기 전기화학적 신호는 핵산의 연속적인 증폭에 따라 연속적으로 검출 및 측정될 수 있는 신호를 말한다. 예를 들어, 이중 가닥 핵산(DNA)의 경우 전체적으로 음전하를 띠는데, 활성물질이 양전하를 띠는 경우 핵산의 연속적인 증폭에 따라 증폭 핵산과 상기 활성물질이 반응하여 총 전하량 변화에 의해 검출가능한 신호가 도출될 수 있다. 따라서, 상기 전기화학적 신호는 상기 증폭 핵산의 음 전하와 상기 활성물질의 양 전하의 결합에 인한 총 전류값 변화에 기인할 수 있고, 상기 활성물질은 이온결합성 물질의 이온화 산물 중 양이온 물질일 수 있다. 더 구체적으로, 상기 이온결합성 물질은 메틸렌 블루(methylene blue)이고, 상기 활성물질은 메틸렌 블루의 이온화 산물 중 양이온 물질일 수 있다. 상기 메틸렌 블루(C16H18N3SCl·3H2O)는 용매에 녹이면 이온화 되어 C16H18N3S+와 Cl-로 이온화되고, 전자의 경우 황원자(S)에 의하여 양전하를 띤다. 이중 가닥 핵산(DNA)은 당과 염기와 인산으로 이루어져 있는데, 이 중 인산기가 음전하를 띠고 있어 이중 가닥 핵산(DNA)은 전체적으로 음전하를 띤다. 메틸렌 블루의 양이온이 DNA의 인산기와 결합하여, 메틸렌 블루의 겉보기 확산율보다 이중가닥 핵산과 결합한 메틸렌블루의 겉보기 확산율이 감소하고, 이에 따라 전류의 피크 값을 감소시킨다. 따라서 PCR 주기가 진행됨에 따라 이중 가닥 핵산(DNA)이 증폭되고 이중가닥 핵산(DNA)에 결합되는 메틸렌 블루의 양이 늘어나 전류의 피크 값이 감소하게 되고, 결과적으로 실시간 PCR의 증폭 산물과 메틸렌 블루의 화학적 결합으로 인한 전기적 신호를 통해 증폭 핵산의 실시간 정량이 가능하다.On the other hand, the active material (redox indicator) is defined as a substance that chemically reacts (couples) with the amplifying nucleic acid to generate an electrochemical signal, the electrochemical signal can be continuously detected and measured according to the continuous amplification of the nucleic acid Say a signal. For example, a double stranded nucleic acid (DNA) is generally negatively charged, but when the active material is positively charged, the amplified nucleic acid reacts with the active material as a result of continuous amplification of the nucleic acid, thereby detecting a change in total charge amount. Can be derived. Thus, the electrochemical signal may be due to the change in the total current value due to the combination of the negative charge of the amplified nucleic acid and the positive charge of the active material, the active material may be a cationic material in the ionization product of the ion-binding material have. More specifically, the ionizable material may be methylene blue, and the active material may be a cationic material in the ionization product of methylene blue. The methylene blue (C 16 H 18 N 3 SCl.3H 2 O) is ionized when dissolved in a solvent and ionized with C 16 H 18 N 3 S + and Cl − , in the case of the former is positively charged by a sulfur atom (S). Double-stranded nucleic acid (DNA) is composed of sugar, base and phosphoric acid, of which the phosphate group is negatively charged, double-stranded nucleic acid (DNA) is negatively charged as a whole. The cation of methylene blue binds to the phosphate group of DNA, reducing the apparent diffusion of methylene blue bound to the double-stranded nucleic acid rather than the apparent diffusion of methylene blue, thus reducing the peak value of the current. Therefore, as the PCR cycle proceeds, the double-stranded nucleic acid (DNA) is amplified and the amount of methylene blue bound to the double-stranded nucleic acid (DNA) increases, resulting in a decrease in the peak value of the current. Real-time quantification of amplified nucleic acids is possible through an electrical signal due to chemical bonding of.
상기 전극(950)은 상기 반응 채널(921)의 적어도 일 영역에 배치되되 상기 반응 채널(921) 내부에서 증폭 핵산과 활성물질의 결합으로 인해 발생하는 전기화학적 신호를 검출하도록 구현된다. 상기 전극(950)은 위와 같은 기능을 수행하기 위하여 다양한 재질로 구현될 수 있지만, 예를 들어 금(Au), 코발트(Co), 백금(Pt), 은(Ag), 탄소나노튜브(carbon nanotube), 그래핀(graphene), 및 탄소(Carbon)로 구성된 군으로부터 1 이상 선택될 수 있다. 또한, 상기 전극(950)은 위와 같은 기능을 수행하기 위하여 다양한 형상 또는 구조로 구현될 수 있지만, 예를 들어 도 3과 같이 상기 반응 채널(921)의 중심 영역의 바닥에 배치되고, 그 말단이 상기 PCR 칩의 테두리 경계까지 연장되는 선 형상으로 구현될 수 있고, 더 나아가 도 4와 같이 상기 증폭 핵산과 활성물질의 결합이 일어나는 지시 전극(working electrode)(950a) 및 상기 증폭 핵산과 활성물질의 결합이 일어나지 않는 기준 전극(reference electrode)(950b)을 구비하는 2-전극 모듈(도 4의 우측), 또는 상기 지시 전극(950a), 상기 기준 전극(950b), 및 상기 지시 전극으로부터 발생하는 전자 밸런스를 조절하는 카운터 전극(counter electrode)(950c)을 구비하는 3-전극 모듈(도 4의 좌측)로 구현될 수 있다. 이와 같이, 상기 전극(950)의 구조가 도 4와 같이 다-전극 모듈 방식으로 구현되면, 상기 반응 채널(921) 내부에서 발생하는 전기화학적 신호의 감도를 높일 수 있을 뿐만 아니라, 발생 신호의 검출 및 측정을 용이하게 수행할 수 있다.The electrode 950 is disposed in at least one region of the reaction channel 921, and is configured to detect an electrochemical signal generated due to the combination of an amplifying nucleic acid and an active material inside the reaction channel 921. The electrode 950 may be formed of various materials to perform the above functions, but for example, gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes (carbon nanotubes) ), Graphene, and carbon may be selected from one or more selected from the group consisting of. In addition, the electrode 950 may be implemented in various shapes or structures to perform the above function, but is disposed at the bottom of the central region of the reaction channel 921, for example, as shown in FIG. It may be implemented in a linear shape extending to the border of the PCR chip, and further, as shown in Figure 4 and the working electrode (950a) and the amplification of the nucleic acid and the active material that the binding of the amplifying nucleic acid and the active material occurs A two-electrode module (right side of FIG. 4) having a reference electrode 950b where no coupling occurs, or electrons generated from the indicator electrode 950a, the reference electrode 950b, and the indicator electrode It may be implemented as a three-electrode module (left side of FIG. 4) having a counter electrode 950c for adjusting the balance. As such, when the structure of the electrode 950 is implemented in the multi-electrode module method as illustrated in FIG. 4, not only the sensitivity of the electrochemical signal generated in the reaction channel 921 may be increased, but also the detection of the generated signal may be performed. And measurement can be easily performed.
한편, 도 5 내지 6에 따르면, 상기 PCR 칩(900)은 수직 단면도를 기준으로 크게 3개의 층(layer)으로 구분될 수 있다. 도 5 내지 6에 따르면, 상기 PCR 칩(900)은 상기 전극(950)이 구비된 제1 판(910); 상기 제1 판(910) 상에 배치되되 상기 1 이상의 반응 채널(921)이 구비된 제2 판(920); 및 상기 제2 판(920) 상에 배치되되 상기 유입부(931) 및 유출부(932)가 구비된 제3 판(930)을 포함할 수 있다.Meanwhile, according to FIGS. 5 to 6, the PCR chip 900 may be largely divided into three layers based on a vertical cross-sectional view. 5 to 6, the PCR chip 900 includes: a first plate 910 provided with the 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 inlet part 931 and the outlet part 932.
상기 전극(950)이 구비된 제1 판(910)의 상부 면은 상기 제2 판(920)의 하부 면에 접착 배치된다. 상기 제1 판(910)이 상기 반응 채널(921)을 구비하는 제2 판(920)에 접착 배치됨으로써 상기 반응 채널(921)에 관한 공간이 확보되고, 더 나아가 상기 반응 채널(921)의 적어도 일 영역(표면)에 상기 전극(950)이 배치된다. 한편, 상기 제1 판(910)은 다양한 재질로 구현될 수 있으나, 바람직하게는 폴리디메틸실옥산(polydimethylsiloxane, PDMS), 사이클로올레핀코폴리머(cycle olefin copolymer, COC), 폴리메틸메타크릴레이트(polymethylmetharcylate, PMMA), 폴리카보네이트(polycarbonate, PC), 폴리프로필렌카보네이트(polypropylene carbonate, PPC), 폴리에테르설폰(polyether sulfone, PES), 및 폴리에틸렌텔레프탈레이트(polyethylene terephthalate, PET), 및 그의 조합물로 구성된 군으로부터 선택되는 재질일 수 있다. 또한, 상기 제1 판(910)의 상부 면은 친수성 물질(도시되지 않음)이 처리되어 PCR을 원활하게 수행할 수 있다. 상기 친수성 물질 처리에 의해 상기 제1 판(910) 상에 친수성 물질을 포함하는 단일 층이 형성될 수 있다. 상기 친수성 물질은 다양한 물질일 수 있으나, 바람직하게는 카르복시기(-COOH), 아민기(-NH2), 히드록시기(-OH), 및 술폰기(-SH)로 구성된 군으로부터 선택되는 것일 수 있고, 상기 친수성 물질의 처리는 당 업계에 공지된 방법에 따라 수행할 수 있다. The upper surface of the first plate 910 with the electrode 950 is adhesively disposed on the 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 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.
상기 제2 판(920)의 상부 면은 상기 제3 판(930)의 하부 면과 접촉 배치된다. 상기 제2 판(920)은 상기 반응 채널(921)을 포함한다. 상기 반응 채널(921)은 상기 제3 판(910)에 형성된 유입부(931)와 유출부(932)에 대응되는 부분과 연결되어 양 말단에 유입부(931) 및 유출부(932)가 구현된 1 이상의 반응 채널(921)을 완성한다. 따라서, 상기 반응 채널(921)에 PCR 시료 및 시약이 도입된 후 PCR이 진행된다. 또한, 상기 반응 채널(921)은 본 발명의 일 실시예에 따른 PCR 장치(1)의 사용 목적 및 범위에 따라 2 이상 존재할 수 있고, 도 3에 따르면, 2개의 반응 채널(921)이 예시되고 있다. 또한, 상기 제2 판(920)은 다양한 재질로 구현될 수 있으나, 바람직하게는 폴리메틸메타크릴레이트(polymethylmethacrylate, PMMA), 폴리카보네이트(polycarbonate, PC), 사이클로올레핀 코폴리머(cycloolefin copolymer, COC), 폴리아미드(polyamide, PA), 폴리에틸렌(polyethylene, PE), 폴리프로필렌(polypropylene, PP), 폴리페닐렌 에테르(polyphenylene ether, PPE), 폴리스티렌(polystyrene, PS), 폴리옥시메틸렌(polyoxymethylene, POM), 폴리에테르에테르케톤(polyetheretherketone, PEEK), 폴리테트라프로오르에틸렌(polytetrafluoroethylene, PTFE), 폴리비닐클로라이드(polyvinylchloride, PVC), 폴리비닐리덴 플로라이드(polyvinylidene fluoride, PVDF), 폴리부틸렌테레프탈레이트(polybutyleneterephthalate, PBT), 불소화에틸렌프로필렌(fluorinated ethylenepropylene, FEP), 퍼플로로알콕시알칸(perfluoralkoxyalkane, PFA), 및 그의 조합물로 구성된 군으로부터 선택되는 열 가소성 수지 또는 열 경화성 수지 재질일 수 있다. 또한, 상기 제2 판(920)의 두께는 다양할 수 있으나, 100 ㎛ 내지 200 ㎛에서 선택될 수 있다. 또한, 상기 반응 채널(921)의 폭과 길이는 다양할 수 있으나, 바람직하게는 상기 반응 채널(921)의 폭은 0.5 mm 내지 3 mm에서 선택되고, 상기 반응 채널(921)의 길이는 20 mm 내지 40 mm에서 선택될 수 있다. 또한, 상기 제2 판(920) 내벽은 DNA, 단백질(protein) 흡착을 방지하기 위해 실란(silane) 계열, 보바인 시럼 알부민(Bovine Serum Albumin, BSA) 등의 물질로 코팅할 수 있고, 상기 물질의 처리는 당 업계에 공지된 방법에 따라 수행될 수 있다.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 depending on the purpose and range of use of the PCR device 1 according to an embodiment of the present invention, according to Figure 3, two reaction channels 921 are illustrated have. 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.
상기 제3 판(930)의 하부 면은 상기 제2 판(920)의 상부 면에 배치된다. 상기 제3 판(930)은 상기 제2 판(920)에 형성된 반응 채널(921) 상의 일 영역에 형성된 유입부(931) 및 다른 일 영역에 형성된 유출부(932)를 구비한다. 상기 유입부(931)는 PCR 시료 및 시약이 유입되는 부분이다. 상기 유출부(932)는 PCR이 종료된 후 PCR 산물이 유출되는 부분이다. 따라서, 상기 제3 판(930)은 상기 제2 판(920)에 형성된 반응 채널(921)을 커버하되 상기 유입부(931) 및 유출부(932)는 상기 반응 채널(921)의 유입부 및 유출부 역할을 수행하게 된다. 또한, 상기 제3 판(930)은 다양한 재질로 구현될 수 있지만, 바람직하게는 폴리디메틸실옥산(polydimethylsiloxane, PDMS), 사이클로올레핀코폴리머(cycle olefin copolymer, COC), 폴리메틸메타크릴레이트(polymethylmetharcylate, PMMA), 폴리카보네이트(polycarbonate, PC), 폴리프로필렌카보네이트(polypropylene carbonate, PPC), 폴리에테르설폰(polyether sulfone, PES), 및 폴리에틸렌텔레프탈레이트(polyethylene terephthalate, PET), 및 그의 조합물로 구성된 군으로부터 선택되는 재질일 수 있다. 또한, 상기 유입부(931)은 다양한 크기를 구비할 수 있으나, 바람직하게는 지름 1.0 mm 내지 3.0 mm에서 선택될 수 있다. 또한, 상기 유출부(932)는 다양한 크기를 구비할 수 있으나, 바람직하게는 지름 1.0 mm 내지 1.5 mm에서 선택될 수 있다. 또한, 상기 유입부(931) 및 유출부(932)는 별도의 커버 수단(도시되지 않음)을 구비하여, 상기 반응 채널(921) 내에서 PCR 시료 및 시약에 대한 PCR이 진행될 때 용액이 누출되는 것을 방지할 수 있다. 상기 커버 수단은 다양한 형상, 크기 또는 재질로서 구현될 수 있다. 또한, 상기 제3 판의 두께는 다양할 수 있으나, 바람직하게는 0.1 mm 내지 2.0 mm에서 선택될 수 있다. 또한, 상기 유입부(931) 및 상기 유출부(932)는 2 이상 존재할 수 있다.The lower surface of the third plate 930 is disposed on the upper surface of the second plate 920. The third plate 930 includes an inlet portion 931 formed in one region on the reaction channel 921 formed in the second plate 920 and an outlet portion 932 formed in the other region. 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.
한편, 상기 PCR 칩(900)은 기계적 가공을 통해 유입부(931) 및 유출부(932)를 형성하여 제3 판(930)을 제공하는 단계; 상기 제3 판(930)의 하부 면과 대응되는 크기를 갖는 판재에 상기 제3 판(930)의 유입부(931)와 대응되는 부분으로부터 상기 제3 판(930)의 유출부(932)에 대응되는 부분까지 기계적 가공을 통해 반응 채널(921)을 형성하여 제2 판(920)을 제공하는 단계; 상기 제2 판(920)의 하부 면과 대응되는 크기를 갖는 판재의 상부 면에 표면 처리 가공을 통해 친수성 물질(922)로 구현된 표면을 형성하여 제1 판(910)을 제공하는 단계; 및 상기 제3 판(930)의 하부 면을 상기 제2 판(920)의 상부 면에 접합 공정을 통해 접합하고, 상기 제2 판(920)의 하부 면을 상기 제1 판(910)의 상부 면에 접합 공정을 통해 접합하는 단계를 포함하는 방법에 의해 용이하게 제조될 수 있다. 상기 제3 판(930)의 유입부(931) 및 유출부(932), 및 상기 제2 판(920)의 반응 채널(921)은 사출성형, 핫-엠보싱(hot-embossing), 캐스팅(casting), 및 레이저 어블레이션(laser ablation)으로 구성된 군으로부터 선택되는 가공 방법에 의해 제조될 수 있다. 또한, 상기 제1 판(910) 표면의 친수성 물질(922)은 산소 및 아르곤 플라즈마 처리, 코로나 방전 처리, 및 계면 활성제 도포로 구성된 군으로부터 선택되는 방법에 의해 처리될 수 있고 당 업계에 공지된 방법에 따라 수행될 수 있다. 또한, 상기 제3 판(930)의 하부 면과 상기 제2 판(920)의 상부 면, 및 상기 제2 판(920)의 하부 면과 상기 제1 판(910)의 상부 면은 열 접합, 초음파 융착, 용매 접합 공정에 의해 접착될 수 있고 당 업계에 공지된 방법에 따라 수행될 수 있다. 상기 제3 판(930)과 제2 판(920) 사이 및 상기 제2 판(920)과 제3 판(910) 사이에는 양면 접착제 또는 열가소성 수지 또는 열 경화성 수지(500)가 처리될 수 있다.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.
도 7은 본 발명의 일 실시예에 따른 칩 홀더를 도시한다.7 illustrates a chip holder in accordance with one embodiment of the present invention.
도 7에 따르면, 상기 칩 홀더(300)는 상기 PCR 칩(900)이 장착되되 상기 PCR 칩(900)의 전극(950) 말단과 전기적으로 연결되도록 구현된 연결 포트(310)를 구비한다. 상기 칩 홀더(300)는 상기 PCR 칩(900)이 상기 PCR 장치(1)에 장착되는 부분이다. 상기 칩 홀더(300)의 내벽은 판 형상을 갖는 PCR 칩(900)이 상기 칩 홀더(300)로부터 이탈하지 않도록 상기 PCR 칩(900)의 외벽과 고정 장착되기 위한 형상 및 구조를 가질 수 있다. 즉, 상기 PCR 칩(900)이 상기 칩 홀더(300)에 장착되는 경우 상기 PCR 칩(900)의 전극(950) 말단은 상기 칩 홀더(300)의 연결 포트(310)와 전기적으로 연결되어 상기 PCR 칩(900)의 반응 채널(921) 내부에서 증폭 핵산과 활성물질의 결합으로 인해 발생하는 전기화학적 신호가 후술할 전기화학적 신호 측정 모듈(800)로 전달된다. 한편, 상기 PCR 칩(900)은 상기 칩 홀더(300)와 탈착 가능하다. 또한, 상기 칩 홀더(300)는 상기 구동 수단(500), 구체적으로 상기 연결 부재(520)의 말단에 연결되어 상기 실시간 PCR 장치(1) 내부에서 상하 또는 좌우로 이동할 수 있다.According to FIG. 7, the chip holder 300 includes a connection port 310 on which the PCR chip 900 is mounted but is electrically connected to an end of the 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 1. 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, the end of the electrode 950 of the PCR chip 900 is electrically connected to the connection port 310 of the chip holder 300. The electrochemical signal generated by the binding of the amplifying nucleic acid and the active material in 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 is connected to the driving means 500, specifically, the end of the connecting member 520 may be moved up and down or left and right inside the real-time PCR device (1).
도 8은 본 발명의 일 실시예에 따른 실시간 PCR 장치의 전기화학적 신호 측정 모듈의 배치도이다.8 is a layout view of an electrochemical signal measurement module of a real-time PCR device according to an embodiment of the present invention.
도 8에 따르면, 본 발명의 일 실시예에 따른 실시간 PCR 장치(1)는 상기 칩 홀더(300)의 연결 포트(310)와 전기적으로 연결되어 상기 PCR 칩(900)의 반응 채널(921) 내부에서 발생하는 전기화학적 신호를 실시간으로 측정하도록 구현된 전기화학적 신호 측정 모듈(800)을 포함한다. 상기 전기화학적 신호 측정 모듈(800)은 상기 칩 홀더(300)의 연결 포트(310)와 전기적 연결 수단(700), 예를 들어 리드 전선을 통해 전기 소통가능하게 연결될 수 있다. 따라서, 상기 PCR 칩(900)의 반응 채널(921) 내부에서 발생된 전기화학적 신호는 상기 PCR 칩(900)의 전극(950)을 통해 검출되고, 상기 검출된 신호는 상기 칩 홀더(300)의 연결 포트(310)와 상기 전기적 연결 수단(700)을 경유하여 상기 전기화학적 신호 측정 모듈(800)에서 측정되고 더 나아가 가공 또는 분석될 수 있다. 상기 전기화학적 신호 측정 모듈(800)은 다양할 수 있으나, 양극 벗김 전압전류계(anodic stripping voltammetry, ASV), 대시간 전류계 (chronoamperometry, CA), 순환 전압전류계(cyclic voltammetry), 네모파 전압전류계(square wave voltammetry, SWV), 펄스 전압전류계(differential pulse voltammetry, DPV), 및 임피던스계(impedance)로 구성된 군으로부터 선택될 수 있다.According to FIG. 8, the real-time PCR device 1 according to the exemplary embodiment of the present invention is electrically connected to the connection port 310 of the chip holder 300 so as to be inside the reaction channel 921 of the PCR chip 900. An electrochemical signal measurement module 800 is implemented to measure in real time the electrochemical signal generated in the. The electrochemical signal measuring module 800 may be electrically connected to the connection port 310 of the chip holder 300 through an electrical connection means 700, for example, a lead wire. Therefore, an electrochemical signal generated in the reaction channel 921 of the PCR chip 900 is detected through the electrode 950 of the PCR chip 900, and the detected signal is detected by the chip holder 300. It may be measured and further processed or analyzed in the electrochemical signal measuring module 800 via the connection port 310 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.
도 9는 본 발명의 일시예에 따른 실시간 PCR 방법을 나타내는 순서도이다.9 is a flowchart illustrating a real-time PCR method according to an embodiment of the present invention.
도 9에 따르면, 본 발명의 일 실시예에 따른 실시간 PCR 방법은 상술한 실시간 PCR 장치(1)를 제공하는 단계; 주형 핵산을 포함하는 PCR 시료 및 상기 활성물질을 포함하는 PCR 시약을 상기 PCR 칩(900)의 반응 채널(921)에 주입하는 단계; 상기 PCR 칩(900)의 전극(950) 말단이 상기 연결 포트(310)에 전기적으로 연결되도록 상기 PCR 시료 및 PCR 시약이 주입된 PCR 칩(900)을 상기 칩 홀더(300)에 장착하는 단계; 상기 구동 수단(500)을 가동하여 상기 칩 홀더(300)에 장착된 PCR 칩(900)이 PCR의 변성 단계 온도 및 PCR의 어닐링 및 연장(혹은 증폭) 단계 온도를 각각 유지하는 상기 제1 열 블록(100) 및 상기 제2 열 블록(200)에 반복적으로 열 접촉하여 PCR을 수행하는 단계; 및 상기 PCR 수행 중 상기 PCR 칩(900) 내부에서 증폭 핵산과 상기 활성물질의 결합으로 인해 발생하는 전기화학적 신호를 실시간으로 검출 및 측정하는 단계를 포함한다.9, a real-time PCR method according to an embodiment of the present invention comprises the steps of providing the above-described real-time PCR device (1); Injecting a PCR sample containing a template nucleic acid and a PCR reagent containing the active material into the 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 first row block for operating the driving means 500 to maintain the denaturation step temperature of the PCR and the annealing and extension (or amplification) step temperature of the PCR, respectively, mounted on the chip holder 300. Performing PCR by thermally contacting the thermal block (100) and the second thermal block (200) repeatedly; And detecting and measuring, in real time, an electrochemical signal generated due to the binding of the amplifying nucleic acid and the active material in the PCR chip 900 during the PCR.
실시간 PCR 장치 제공 단계(S1)는 상술한 실시간 PCR 장치(1)를 준비하는 단계이다. 따라서, 이하 본 발명의 일 실시예에 따른 실시간 PCR 방법은 상기 실시간 PCR 장치(1)의 구동을 전제로 한다. The real time PCR device providing step S1 is a step of preparing the above-described real time PCR device 1. Therefore, the real-time PCR method according to an embodiment of the present invention below assumes the driving of the real-time PCR device (1).
시료 및 시약 주입 단계(S2)는 상기 PCR 칩(900)에 PCR 시료 및 시약, 아울러 증폭하고자 하는 주형 핵산과 화학적 반응(결합)을 통해 전기적 신호를 발생시킬 수 있는 물질, 예를 들어 메틸렌 블루를 주입하는 단계이다.Sample and reagent injection step (S2) is a material that can generate an electrical signal, such as methylene blue to the PCR chip 900 through the chemical reaction (combination) with the PCR sample and reagents, and the template nucleic acid to be amplified Injecting.
PCR 칩 장착 단계(S3)는 상기 PCR 시료 및 시약이 수용된 PCR 칩(900)을 상기 실시간 PCR 장치(1)의 칩 홀더(300)에 장착하는 단계이다. 이 경우 전기화학적 신호 검출을 위해 상기 PCR 칩(900)의 전극(950)이 상기 칩 홀더(300)의 연결 포트(310)와 전기적으로 연결되어야 한다.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.
PCR 단계(S4)는 상기 제1 열 블록(100) 및 제2 열 블록(200)을 가열 유지하고, 상기 구동 수단(500)을 가동하여 상기 PCR 칩(900)의 반응 채널(921)에서 PCR이 수행되는 단계이다. 이 경우 상기 반응 채널(921) 내부에서 주형 핵산을 기초로 표적 핵산 부위가 증폭되고, 표적 핵산 부위의 연속적인 증폭에 따라 상기 활성물질과의 연속적인 반응(결합)으로 인해 전기화학적 신호가 발생한다.In the PCR step S4, the first heat block 100 and the second heat block 200 are heated and maintained, and the driving means 500 is operated to perform PCR in the reaction channel 921 of the PCR chip 900. This is the step to be performed. In this case, the target nucleic acid site is amplified based on the template nucleic acid in the reaction channel 921, and the electrochemical signal is generated due to the continuous reaction (binding) with the active material according to the continuous amplification of the target nucleic acid site. .
전기화학적 신호 검출 및 측정 단계(S5)는 상기 S4 단계에서 핵산의 연속적인 증폭에 의해 발생한 전기화학적 신호(전류값 변화)를 상기 PCR 칩(900)의 전극(950), 상기 칩 홀더(300)의 연결 포트(310), 상기 전기적 연결 수단(700), 및 상기 전기화학적 신호 측정 모듈(800)을 통해 검출 및 측정하는 단계이다. 이 경우 상기 전기화학적 신호 검출 및 측정 시점은 다양할 수 있으나, 핵산이 증폭되는 시점에 맞춰 상기 칩 홀더(300)에 장착된 PCR 칩(900)이 PCR의 연장 (혹은 증폭) 단계 온도를 유지하는 열 블록(제1 열 블록 또는 제2 열 블록)과 열 접촉하는 시점 또는 열 접촉 직후 시점이 바람직하다. 이 경우 열 접촉 직후 시점이라 함은 상기 칩 홀더(300)에 장착된 PCR 칩(900)이 PCR의 연장 (혹은 증폭) 단계 온도를 유지하는 열 블록에 열 접촉한 후 상기 구동 수단(500)을 통해 다른 열 블록으로 이동하는 시점뿐만 아니라 상기 칩 홀더(300)에 장착된 PCR 칩(900)이 PCR의 연장 (혹은 증폭) 단계 온도를 유지하는 열 블록에 열 접촉한 상태에서 그 열 블록의 온도가 하강하는 시점, 예를 들어 72℃에서 60℃로 하강하는 시점일 수 있다. 따라서, 본 단계에서 증폭 핵산의 실시간 확인이 가능하게 된다.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. In this case, the time point for detecting and measuring the electrochemical signal may vary, but the PCR chip 900 mounted on the chip holder 300 maintains the temperature of the extension (or amplification) step of the PCR according to the time when the nucleic acid is amplified. The point of time in thermal contact with the heat block (first heat block or second heat block) or immediately after the heat contact is preferred. In this case, the point of time immediately after the thermal contact refers to the driving means 500 after the PCR chip 900 mounted on the chip holder 300 is in thermal contact with a thermal block that maintains the temperature of the PCR (or amplification) step. The temperature of the heat block in a state in which the PCR chip 900 mounted on the chip holder 300 is in thermal contact with the heat block maintaining the extension (or amplification) step temperature of the PCR as well as the time of moving to another heat block through It may be a time when the falling, for example, the time to drop from 72 ℃ to 60 ℃. Therefore, real-time confirmation of the amplified nucleic acid is possible in this step.
실시예Example
본 발명의 일 실시예에 따른 PCR 장치(1)를 사용하여 핵산 증폭 반응을 수행하였다. 본 발명의 일 실시예에 따른 PCR 장치(1)에 있어서, 상기 PCR 칩(900)은 플라스틱 재질의 판 형상으로 3개의 반응 채널(921) 및 이의 말단에 각각 연결된 전극(950)을 구비하되, 상기 전극(950)은 탄소 나노튜브(carbon nanotube) 및 은(Ag)을 이용하여 제작하였고, 상기 전기화학적 신호 측정 모듈(800)은 양극 벗김 전압전류계(anodic stripping voltammetry, ASV)를 채용하였으며, 상기 활성물질을 제공하는 물질로서 메틸렌 블루 100 ㎕를 이하, PCR 시약에 첨가하였다. 한편, 상기 양극 벗김 전압전류계의 전기화학적 신호의 검출 조건을 아래와 같이 전제하였다.The nucleic acid amplification reaction was performed using the PCR device 1 according to the embodiment of the present invention. In the PCR device 1 according to an embodiment of the present invention, the PCR chip 900 includes three reaction channels 921 and electrodes 950 connected to the ends thereof, respectively, in a plate shape made of plastic, The electrode 950 was fabricated using carbon nanotubes and silver (Ag), and the electrochemical signal measuring module 800 adopted an anode stripping voltammetry (ASV). 100 μl of methylene blue was added to the PCR reagent as a substance to provide the active substance. On the other hand, the detection conditions of the electrochemical signal of the anode peeling voltammeter were assumed as follows.
- Preconcentration step : accumulation at -0.5 V 50초-Preconcentration step: accumulation at -0.5 V 50 seconds
- Stripping step : - 0.5 V → 0.5 V, step potential of 2 mV, amplitude of 25 mV, and frequency of 25 HzStripping step-0.5 V → 0.5 V, step potential of 2 mV, amplitude of 25 mV, and frequency of 25 Hz
PCR 시료로서 이중 가닥 주형 DNA(template ds-DNA) 0.1 ng/㎕을 준비하고, PCR 시약으로서 특정 염기 서열에 상보적으로 결합하는 프라이머 쌍, 구체적으로 포워드 프라이머(forward primer, 0.125 ㎕, 1pmole), 리버스 프라이머(reverse primer, 0.125 ㎕, 1pmole), dNTP 0.2 ㎖, 중합효소(i-starmax Ⅱ polymerase, iNtRON Biotechnology) 0.2 ㎖, PCR 버퍼(pH 9, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 30 mM salt) 등을 준비한 후, 상기 PCR 시료 및 시약 용액을 본 발명의 일 실시예에 따른 PCR 칩(900)에 도입하고, 상기 PCR 칩(900)을 상기 칩 홀더(300)에 장착하였다. 한편, 상기 제1 열 블록(100)을 95℃, 즉 허용 온도 범위는 90℃ 내지 100℃로 가열 및 온도 유지하고, 상기 제2 열 블록(200)을 72℃, 즉 허용 온도 범위는 55℃ 내지 75℃로 가열 및 온도 유지하였다. 그 후 40 순환(cycle)의 PCR을 수행하기 위해 PCR 장치(1)를 구동하였다(Pre-denaturation, 95 ℃, 30 sec, 1회; Denaturation 95 ℃, 4 sec, 40회; Annealing & Extension 72 ℃, 30 sec, 40회). 상기 PCR 장치(1)가 구동됨에 따라 1 순환 단계마다 핵산 증폭 여부를 양극 벗김 전압전류계(anodic stripping voltammetry, ASV)로 측정하였다. 그 결과, 핵산 증폭 단계에 따라 전류의 피크 값 변화(감소)가 감지되었고, PCR을 완료한 후 산물을 전기영동 한 결과, 3개의 반응 채널(921) 모두에서 증폭 핵산을 확인할 수 있었다. 도 10은 본 발명의 일시예에 따른 실시간 PCR 방법을 수행한 결과를 나타내는 그래프이고, 도 11은 본 발명의 일 실시예에 따른 실시간 PCR 방법을 수행한 결과를 나타내는 전기영동 사진이다.0.1 ng / μl of double stranded template DNA (template ds-DNA) is prepared as a PCR sample, and a pair of primers complementarily binding to a specific nucleotide sequence as a PCR reagent, specifically, a forward primer (0.125 μl, 1 pmole), Reverse primer (0.125 μl, 1pmole), 0.2 mL of dNTP, 0.2 mL of polymerase (i-starmax II polymerase, iNtRON Biotechnology), PCR buffer (pH 9, 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl) 2 , 30 mM salt), and the like, the PCR sample and reagent solution are introduced into the PCR chip 900 according to an embodiment of the present invention, and the PCR chip 900 is mounted on the chip holder 300. It was. Meanwhile, the first heat block 100 is heated and maintained at 95 ° C., that is, the allowable temperature range is 90 ° C. to 100 ° C., and the second heat block 200 is 72 ° C., that is, the allowable temperature range is 55 ° C. Heated to and maintained at 75 ° C. Then, the PCR device 1 was operated to perform 40 cycles of PCR (Pre-denaturation, 95 ° C., 30 sec, once; Denaturation 95 ° C., 4 sec, 40 times; Annealing & Extension 72 ° C.). , 30 sec, 40 times). As the PCR device 1 was driven, nucleic acid amplification was measured by anodizing stripping voltammetry (ASV) at every cycle. As a result, a change in the peak value of the current was detected according to the nucleic acid amplification step. As a result of electrophoresis of the product after completing the PCR, the amplified nucleic acid was identified in all three reaction channels 921. 10 is a graph showing a result of performing a real-time PCR method according to an embodiment of the present invention, Figure 11 is an electrophoresis picture showing the result of performing a real-time PCR method according to an embodiment of the present invention.

Claims (18)

  1. 기판 상부에 이격 배치된 제1 열 블록 및 제2 열 블록;First and second row blocks spaced apart from each other on the substrate;
    양 말단에 유입부 및 유출부가 구현된 1 이상의 반응 채널, 및 상기 반응 채널의 적어도 일 영역에 배치되되 상기 반응 채널 내부에서 증폭 핵산과 활성물질의 결합으로 인해 발생하는 전기화학적 신호를 검출하도록 구현된 전극을 구비하는, 판 형상의 PCR 칩;At least one reaction channel having both an inlet and an outlet at both ends, and disposed in at least one region of the reaction channel, and configured to detect an electrochemical signal generated by the binding of the amplifying nucleic acid and the active material within the reaction channel. A plate-shaped PCR chip having an electrode;
    상기 PCR 칩이 장착되되 상기 PCR 칩의 전극 말단과 전기적으로 연결되도록 구현된 연결 포트를 구비하는 칩 홀더;A chip holder mounted with the PCR chip, the chip holder having a connection port configured to be electrically connected to an electrode end of the PCR chip;
    상기 PCR 칩이 장착된 칩 홀더를 상하 또는 좌우로 이동하여 상기 PCR 칩이 상기 제1 열 블록 또는 상기 제2 열 블록에 열 접촉할 수 있도록 구현된 구동 수단; 및Drive means implemented to move the chip holder on which the PCR chip is mounted vertically or horizontally so that the PCR chip is in thermal contact with the first row block or the second row block; And
    상기 칩 홀더의 연결 포트와 전기적으로 연결되어 상기 PCR 칩의 반응 채널 내부에서 발생하는 전기화학적 신호를 실시간으로 측정하도록 구현된 전기화학적 신호 측정 모듈;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;
    을 포함하는 실시간 PCR 장치.Real time PCR device comprising a.
  2. 제1항에 있어서,The method of claim 1,
    상기 활성물질은 이온결합성 물질의 이온화 산물 중 양이온 물질인 것을 특징으로 하는 실시간 PCR 장치.The active material is a real-time PCR device, characterized in that the cationic material in the ionization product of the ion-bonding material.
  3. 제2항에 있어서,The method of claim 2,
    상기 이온결합성 물질은 메틸렌 블루(methylene blue)인 것을 특징으로 하는 실시간 PCR 장치.The ion-coupled material is methylene blue (methylene blue), characterized in that the real-time PCR device.
  4. 제1항에 있어서,The method of claim 1,
    상기 전기화학적 신호는 상기 증폭 핵산의 음 전하와 상기 활성물질의 양 전하의 결합에 인한 총 전류값 변화에 기인하는 것을 특징으로 하는 실시간 PCR 장치.The electrochemical signal is a real-time PCR device, characterized in that due to the change in the total current value due to the combination of the negative charge of the amplified nucleic acid and the positive charge of the active material.
  5. 제1항에 있어서,The method of claim 1,
    상기 전극은 금(Au), 코발트(Co), 백금(Pt), 은(Ag), 탄소나노튜브(carbon nanotube), 그래핀(graphene), 및 탄소(Carbon)로 구성된 군으로부터 1 이상 선택되는 것을 특징으로 하는 실시간 PCR 장치.The electrode is 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.
  6. 제1항에 있어서,The method of claim 1,
    상기 전극은 상기 증폭 핵산과 활성물질의 결합이 일어나는 지시 전극(working electrode) 및 상기 증폭 핵산과 활성물질의 결합이 일어나지 않는 기준 전극(reference electrode)을 구비하는 2-전극 모듈, 또는 상기 지시 전극, 상기 기준 전극, 및 상기 지시 전극으로부터 발생하는 전자 밸런스를 조절하는 카운터 전극(counter electrode)을 구비하는 3-전극 모듈로 구현되는 것을 특징으로 하는 실시간 PCR 장치.The electrode is a two-electrode module having a working electrode (combination of the amplification nucleic acid and the active material) and a reference electrode (combination of the amplification nucleic acid and the active material does not occur), or the indicator electrode, And a reference electrode and a three-electrode module having a counter electrode for adjusting an electronic balance generated from the indicator electrode.
  7. 제1항에 있어서,The method of claim 1,
    상기 전기화학적 신호 측정 모듈은 양극 벗김 전압전류계(anodic stripping voltammetry, ASV), 대시간 전류계 (chronoamperometry, CA), 순환 전압전류계(cyclic voltammetry), 네모파 전압전류계(square wave voltammetry, SWV), 펄스 전압전류계(differential pulse voltammetry, DPV), 및 임피던스계(impedance)로 구성된 군으로부터 선택되는 것을 특징으로 하는 실시간 PCR 장치.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).
  8. 제1항에 있어서,The method of claim 1,
    상기 제1 열 블록 및 제2 열 블록은 상기 제1 열 블록 및 제2 열 블록의 온도를 전체적으로 일정하게 유지하기 위해 각 열 블록의 중심점을 기준으로 상하 및/또는 좌우 방향으로 대칭되도록 내부에 열선이 배치된 것을 특징으로 하는 실시간 PCR 장치.The first thermal block and the second thermal block are heated to be symmetrical in the vertical direction and / or the left and right directions with respect to the center point of each thermal block so as to maintain the temperature of the first thermal block and the second thermal block as a whole. Real-time PCR device characterized in that the arrangement.
  9. 제1항에 있어서, The method of claim 1,
    상기 제1 열 블록 및 상기 제2 열 블록 중 어느 하나는 PCR의 변성 단계 온도를 유지하고, 다른 하나는 PCR의 어닐링 및 연장 (혹은 증폭) 단계 온도를 유지하도록 구현된 것을 특징으로 하는 실시간 PCR 장치.Any one of the first row block and the second row block is configured to maintain the denaturation step temperature of the PCR, and the other is to maintain the annealing and extension (or amplification) step temperature of the PCR .
  10. 제9항에 있어서,The method of claim 9,
    상기 변성 단계 온도는 90℃ 내지 100℃이고, 상기 연장 (혹은 증폭) 단계 온도는 55℃ 내지 75℃인 것을 특징으로 하는 실시간 PCR 장치.The denaturation step temperature is 90 ℃ to 100 ℃, the extended (or amplified) step temperature is a real-time PCR device, characterized in that 55 ℃ to 75 ℃.
  11. 제1항에 있어서,The method of claim 1,
    상기 제1 열 블록과 제2 열 블록은 상호 열 교환이 일어나지 않도록 미리 결정된 거리로 이격 배치된 것을 특징으로 하는 실시간 PCR 장치.The first thermal block and the second thermal block is a real-time PCR device, characterized in that spaced apart at a predetermined distance so that mutual heat exchange does not occur.
  12. 제1항에 있어서,The method of claim 1,
    상기 구동 수단은 좌우 방향으로 연장된 레일, 및 상기 레일을 통해 좌우 방향으로 슬라이딩 이동가능하게 배치되고 상하 방향으로 슬라이딩 이동 가능한 연결 부재를 포함하고, 상기 연결 부재의 일 말단에는 상기 칩 홀더가 배치된 것을 특징으로 하는 실시간 PCR 장치.The driving means includes a rail extending in a left and right direction, and a connecting member slidably movable in a left and right direction through the rail and slidably movable in an up and down direction, wherein one end of the connecting member includes the chip holder. Real time PCR device, characterized in that.
  13. 제1항에 있어서,The method of claim 1,
    상기 PCR 칩은 상기 칩 홀더에 탈착 가능하게 구현된 것을 특징으로 하는 실시간 PCR 장치.The PCR chip is a real-time PCR device, characterized in that detachable implementation in the chip holder.
  14. 제1항에 있어서,The method of claim 1,
    상기 PCR 칩은 상기 전극이 구비된 제1 판; 상기 제1 판 상에 배치되되 상기 1 이상의 반응 채널이 구비된 제2 판; 및 상기 제2 판 상에 배치되되 상기 유입부 및 유출부가 구비된 제3 판을 포함하는 것을 특징으로 하는 실시간 PCR 장치.The PCR chip may include a first plate provided with the 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.
  15. 제1항 내지 제14항 중 어느 한 항에 따른 실시간 PCR 장치를 제공하는 단계;Providing a real-time PCR device according to any one of claims 1 to 14;
    주형 핵산을 포함하는 PCR 시료 및 상기 활성물질을 포함하는 PCR 시약을 상기 PCR 칩의 반응 채널에 주입하는 단계;Injecting a PCR sample comprising a template nucleic acid and a PCR reagent comprising the active material into a reaction channel of the PCR chip;
    상기 PCR 칩의 전극 말단이 상기 연결 포트에 전기적으로 연결되도록 상기 PCR 시료 및 PCR 시약이 주입된 PCR 칩을 상기 칩 홀더에 장착하는 단계;Mounting a PCR chip in which the PCR sample and the PCR reagent are injected into the chip holder such that an electrode end of the PCR chip is electrically connected to the connection port;
    상기 구동 수단을 가동하여 상기 칩 홀더에 장착된 PCR 칩이 PCR의 변성 단계 온도 및 PCR의 어닐링 및 연장(혹은 증폭) 단계 온도를 각각 유지하는 상기 제1 열 블록 및 상기 제2 열 블록에 반복적으로 열 접촉하여 PCR을 수행하는 단계; 및By operating the driving means, the PCR chip mounted on the chip holder is repeatedly applied to the first row block and the second row block to maintain the denaturation step temperature of PCR and the annealing and extension (or amplification) step temperature of PCR, respectively. Performing PCR in thermal contact; And
    상기 PCR 수행 중 상기 PCR 칩 내부에서 증폭 핵산과 상기 활성물질의 결합으로 인해 발생하는 전기화학적 신호를 실시간으로 검출 및 측정하는 단계;Detecting and measuring, in real time, an electrochemical signal generated by the binding of the amplifying nucleic acid and the active material in the PCR chip during the PCR;
    를 포함하는, 실시간 PCR 방법.Including, real-time PCR method.
  16. 제15항에 있어서,The method of claim 15,
    상기 활성물질은 이온결합성 물질의 이온화 산물 중 양이온 물질인 것을 특징으로 하는 실시간 PCR 방법.The active material is a real-time PCR method, characterized in that the cationic material in the ionization product of the ion-bonding material.
  17. 제16항에 있어서,The method of claim 16,
    상기 이온결합성 물질은 메틸렌 블루(methylene blue)인 것을 특징으로 하는 실시간 PCR 방법.The ion-bonding material is methylene blue (methylene blue), characterized in that the real-time PCR method.
  18. 제1항에 있어서,The method of claim 1,
    상기 전기화학적 신호는 상기 증폭 핵산의 음 전하와 상기 활성물질의 양 전하의 결합에 인한 총 전류값 변화에 기인하는 것을 특징으로 하는 실시간 PCR 방법.The electrochemical signal is a real-time PCR method, characterized in that due to the change in the total current value due to the combination of the negative charge of the amplified nucleic acid and the positive charge of the active material.
PCT/KR2013/005939 2012-07-04 2013-07-04 Real-time pcr device for detecting electrochemical signals, and real-time pcr method using same WO2014007557A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020120072610A KR101404455B1 (en) 2012-07-04 2012-07-04 Real-time PCR device for detecting electrochemcial signal, and Real-time PCR using the same
KR10-2012-0072610 2012-07-04

Publications (1)

Publication Number Publication Date
WO2014007557A1 true WO2014007557A1 (en) 2014-01-09

Family

ID=49882254

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2013/005939 WO2014007557A1 (en) 2012-07-04 2013-07-04 Real-time pcr device for detecting electrochemical signals, and real-time pcr method using same

Country Status (2)

Country Link
KR (1) KR101404455B1 (en)
WO (1) WO2014007557A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020122922A1 (en) 2018-12-13 2020-06-18 Hewlett-Packard Development Company, L.P. Multiplex nucleic acid detection
CN111925931A (en) * 2020-08-25 2020-11-13 墨卓生物科技(上海)有限公司 Heating structure of PCR instrument and chip positioning heating method
CN114308161A (en) * 2021-12-31 2022-04-12 上海中航光电子有限公司 Microfluidic chip and manufacturing method thereof

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102336308B1 (en) * 2014-12-26 2021-12-09 주식회사 미코바이오메드 Device for polymerase chain reaction comprising driving element for reciprocating sliding, and method for polymerase chain reaction using the same
WO2020186298A1 (en) * 2019-03-15 2020-09-24 Green Monster Offshore Pty Ltd Connection test apparatus

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050117811A (en) * 2004-06-11 2005-12-15 에이엔디티 주식회사 Polymerase chain reaction chip
KR20060017283A (en) * 2004-08-20 2006-02-23 삼성테크윈 주식회사 Apparatus for polymerase chain reaction
US20110070578A1 (en) * 2009-06-04 2011-03-24 Lockheed Martin Corporation DNA analyzer
KR20110118572A (en) * 2010-04-23 2011-10-31 나노바이오시스 주식회사 Device for amplify nucleic acid comprising two heating block
US20120088696A1 (en) * 2010-10-08 2012-04-12 Yi-Chiuen Hu Micro electrochemical multiplex real-time pcr platform

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050117811A (en) * 2004-06-11 2005-12-15 에이엔디티 주식회사 Polymerase chain reaction chip
KR20060017283A (en) * 2004-08-20 2006-02-23 삼성테크윈 주식회사 Apparatus for polymerase chain reaction
US20110070578A1 (en) * 2009-06-04 2011-03-24 Lockheed Martin Corporation DNA analyzer
KR20110118572A (en) * 2010-04-23 2011-10-31 나노바이오시스 주식회사 Device for amplify nucleic acid comprising two heating block
US20120088696A1 (en) * 2010-10-08 2012-04-12 Yi-Chiuen Hu Micro electrochemical multiplex real-time pcr platform

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020122922A1 (en) 2018-12-13 2020-06-18 Hewlett-Packard Development Company, L.P. Multiplex nucleic acid detection
EP3824099A4 (en) * 2018-12-13 2021-08-11 Hewlett-Packard Development Company, L.P. Multiplex nucleic acid detection
CN111925931A (en) * 2020-08-25 2020-11-13 墨卓生物科技(上海)有限公司 Heating structure of PCR instrument and chip positioning heating method
CN114308161A (en) * 2021-12-31 2022-04-12 上海中航光电子有限公司 Microfluidic chip and manufacturing method thereof

Also Published As

Publication number Publication date
KR20140004986A (en) 2014-01-14
KR101404455B1 (en) 2014-06-10

Similar Documents

Publication Publication Date Title
WO2015119470A1 (en) Pcr device provided with unidirectional sliding means and pcr method using same
CN102083533B (en) Microfluidic chip devices and their use
WO2011132977A2 (en) Pcr device including two heating blocks
WO2014007557A1 (en) Real-time pcr device for detecting electrochemical signals, and real-time pcr method using same
KR102041205B1 (en) Heating block for polymerase chain reaction comprising repetitively disposed patterned heater and device for polymerase chain reaction comprising the same
US8702948B2 (en) Method and apparatus using electric field for improved biological assays
US7851184B2 (en) Droplet-based nucleic acid amplification method and apparatus
US6929730B2 (en) Two dimensional microfluidic gene scanner
WO2016105073A1 (en) Pcr apparatus comprising repeated sliding means, and pcr method using same
KR20140095107A (en) Nanogap transducers with selective surface immobilization sites
WO2014035167A1 (en) Pcr chip comprising thermal block in which heater units are repeatedly arranged for detecting electrochemical signals, pcr device comprising same, and real-time pcr method using pcr device
BRPI0415342A (en) apparatus for detecting labeling nucleic acid in a sample for detecting biological material, methods for performing field-assisted hybridization, attracting or repelling electrically charged entities to accelerate a detection process for biological molecules, and for forming a reaction cell arrangement to perform biological analysis
WO2014014268A1 (en) Real-time polymerase chain reaction apparatus for detecting electrochemical signal using metallic nano particles
WO2014035164A1 (en) Pcr chip comprising thermal block in which heater units are repeatedly arranged for detecting electrochemical signals, pcr device comprising same, and real-time pcr method using pcr device
WO2017135505A1 (en) Fet-based biosensor using nanowire as sensing channel and using membrane as flow channel, and detection method using same
WO2014017821A1 (en) Real-time pcr device for detecting electrochemical signals comprising heating block in which heater units are repeatedly disposed, and real-time pcr method using same
WO2014035163A1 (en) 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
JP7253045B2 (en) Biopolymer analyzer and biopolymer analysis method
WO2012070809A2 (en) Apparatus, system and method for detecting target nucleic acids
WO2021215783A1 (en) Biosensor having channel into which fluid is easily introduced
KR102193197B1 (en) Automatic nucleic acid detection device using urine
KR20120045909A (en) Apparatus and method for detecting multiplex target nucleic acids in real time
Zhu Genetic analysis and cell manipulation on microfluidic surfaces
Foglieni et al. Molecular diagnostics by microelectronic microchips
ITRM20070574A1 (en) THERMAL CYCLE MINIATURE FOR AMPLIFICATION AND ANALYSIS OF BIOLOGICAL MOLECULES BIOCHIP OPERATING THROUGH ELECTROSMOSIS AC

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13813186

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13813186

Country of ref document: EP

Kind code of ref document: A1