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WO2014007557A1 - Dispositif de pcr en temps réel pour détecter des signaux électrochimiques, et procédé de pcr en temps réel l'utilisant - Google Patents

Dispositif de pcr en temps réel pour détecter des signaux électrochimiques, et procédé de pcr en temps réel l'utilisant Download PDF

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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
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Prior art keywords
pcr
real
chip
electrode
time pcr
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English (en)
Korean (ko)
Inventor
김성우
이정환
이유진
김덕중
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Nanobiosys Inc
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Nanobiosys Inc
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    • 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.

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PCT/KR2013/005939 2012-07-04 2013-07-04 Dispositif de pcr en temps réel pour détecter des signaux électrochimiques, et procédé de pcr en temps réel l'utilisant Ceased WO2014007557A1 (fr)

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WO2020122922A1 (fr) 2018-12-13 2020-06-18 Hewlett-Packard Development Company, L.P. Détection d'acide nucléique multiplex
CN111925931A (zh) * 2020-08-25 2020-11-13 墨卓生物科技(上海)有限公司 Pcr仪的加热结构及芯片定位加热方法
CN114308161A (zh) * 2021-12-31 2022-04-12 上海中航光电子有限公司 微流控芯片及其制作方法

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KR102336308B1 (ko) * 2014-12-26 2021-12-09 주식회사 미코바이오메드 반복 슬라이딩 구동 수단을 구비하는 pcr 장치 및 이를 이용하는 pcr 방법
GB2596672A (en) * 2019-03-15 2022-01-05 Green Monster Offshore Pty Ltd Connection test apparatus

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WO2020122922A1 (fr) 2018-12-13 2020-06-18 Hewlett-Packard Development Company, L.P. Détection d'acide nucléique multiplex
EP3824099A4 (fr) * 2018-12-13 2021-08-11 Hewlett-Packard Development Company, L.P. Détection d'acide nucléique multiplex
CN111925931A (zh) * 2020-08-25 2020-11-13 墨卓生物科技(上海)有限公司 Pcr仪的加热结构及芯片定位加热方法
CN114308161A (zh) * 2021-12-31 2022-04-12 上海中航光电子有限公司 微流控芯片及其制作方法

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