WO2014014268A1 - Appareil d'amplification en chaîne par polymérase en temps réel pour détecter un signal électrochimique à l'aide de nanoparticules métalliques - Google Patents

Appareil d'amplification en chaîne par polymérase en temps réel pour détecter un signal électrochimique à l'aide de nanoparticules métalliques Download PDF

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WO2014014268A1
WO2014014268A1 PCT/KR2013/006387 KR2013006387W WO2014014268A1 WO 2014014268 A1 WO2014014268 A1 WO 2014014268A1 KR 2013006387 W KR2013006387 W KR 2013006387W WO 2014014268 A1 WO2014014268 A1 WO 2014014268A1
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Prior art keywords
pcr
real
electrode
chip
time pcr
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English (en)
Korean (ko)
Inventor
김성우
박현규
이정환
이유진
김덕중
원병연
백송이
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Korea Advanced Institute of Science and Technology KAIST
Nanobiosys Inc
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Korea Advanced Institute of Science and Technology KAIST
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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • 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
    • 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
    • C12Q2561/00—Nucleic acid detection characterised by assay method
    • C12Q2561/113—Real time assay

Definitions

  • One embodiment of the present invention relates to a real-time PCR device and a real-time PCR method using the same.
  • 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 entire PCR execution time due to repeated heating or cooling of one reaction chamber. There is a problem with this lengthening.
  • the PCR device since the PCR device requires the use of a separate detection device to identify the PCR final product, the working time is delayed and is always exposed to contamination of the PCR final product.
  • 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 structure for accurate temperature control is not required, but a long flow path for passing the high and low temperature reaction chambers is required, and the structure is complicated.
  • a separate control device for controlling the flow rate of a sample solution comprising nucleic acid flowing in a channel passing therethrough.
  • the recent PCR device is developing a method for grasping the PCR process in real time as well as efforts to increase the yield of the PCR final product. This technique of real-time PCR process is called "real-time PCR", a real-time PCR device is a fluorescent material in the PCR chamber to measure the optical signal generated by coupling with the amplification product Technology is applied.
  • 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. It must be adopted, it is difficult to miniaturize the device, there are a lot of difficulties in developing a portable device.
  • an embodiment of the present invention rationally improves the PCR time and yield for a plurality of samples, and furthermore, a real-time PCR device capable of miniaturizing and carrying a product, and a real-time PCR method using the same. I would like to propose.
  • a first embodiment of the present invention is directed to one or more column blocks;
  • a housing having a thermal block contact region in contact with the at least one thermal block, a reaction chamber formed within the housing, a capture disposed in one region within the reaction chamber and capable of complementarily binding to one region of the amplifying target nucleic acid
  • a probe having a surface-treated fixed layer and an electrode disposed in another region within the reaction chamber and configured to detect an electrochemical signal, the probe being connected to the metal nanoparticle and the metal nanoparticle,
  • a PCR vessel containing a complex having a signal probe capable of complementarily binding to another region;
  • an electrochemical signal measuring module electrically connected to an electrode of the PCR vessel and configured to measure in real time an electrochemical signal generated in the reaction chamber of the PCR vessel.
  • the metal nanoparticles are selected from the group consisting of zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu), gallium (Ga), indium (In), gold (Au), and silver (Ag). Can be selected.
  • the electrochemical signal may be due to a change in current generated as the amplification target nucleic acid complementarily binds to the capture probe and the signal probe of the complex.
  • the 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 amplification target nucleic acid, the capture probe, and the signal probe may be single stranded DNA.
  • 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
  • a second 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 inlets and outlets at both ends, a fixed layer disposed on one region of the reaction channel, and having a capture probe surface-treated with a capture probe capable of complementarily binding to one region of the amplifying target nucleic acid, and An electrode disposed in another region inside the reaction channel and configured to detect an electrochemical signal, the electrode being coupled to the metal nanoparticle and the metal nanoparticle, and complementarily binding to the other region of the amplification target nucleic acid.
  • a plate-shaped PCR chip comprising a complex having a signal probe present;
  • 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 metal nanoparticles are selected from the group consisting of zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu), gallium (Ga), indium (In), gold (Au), and silver (Ag). Can be selected.
  • the electrochemical signal may be due to a change in current generated as the amplification target nucleic acid complementarily binds to the capture probe and the signal probe of the complex.
  • the 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 (oxidation or reduction reaction) and a reference electrode (oxidation or reduction) does not occur, or the indicator electrode, the reference electrode, and the It may be implemented as a three-electrode module having a counter electrode for adjusting the electronic balance generated from 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, the third plate having a fixed layer on which the inlet and the outlet and the capture probe are surface treated.
  • FIGS. 1 to 2 are schematic diagrams of a real-time PCR device according to a first embodiment of the present invention.
  • FIG 3 shows the binding between the capture probe and the signal probe and the amplification target nucleic acid in the reaction chamber of the real-time PCR device according to the first embodiment of the present invention.
  • Figure 4 shows the electrochemical signal generation process of the real-time PCR device according to a first embodiment of the present invention.
  • FIG. 5 shows a real-time PCR device according to a second embodiment of the present invention.
  • FIG. 6 shows a driving principle of a real-time PCR device according to a second embodiment of the present invention.
  • FIG. 7 to 10 show a PCR chip according to a second embodiment of the present invention.
  • FIG. 11 shows a chip holder according to a second embodiment of the present invention.
  • FIG. 12 is a layout view of an electrochemical signal measurement module of a real-time PCR device according to a second embodiment of the present invention.
  • FIG. 13 illustrates a process of performing real-time PCR using a real-time PCR device according to a second embodiment of the present invention.
  • 17 to 18 are electrophoresis pictures showing the results of performing the real-time PCR method according to an embodiment of the present invention.
  • FIG. 1 to 2 show schematic diagrams of a real-time PCR device according to a first embodiment of the present invention.
  • the real-time PCR device 1 comprises at least one column block 10; A housing 22 having a thermal block contact region 21 in contact with the at least one thermal block 10, a reaction chamber 23 formed in the housing 22, and a region inside the reaction chamber 23.
  • 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 thermal block 10 is a module that maintains a temperature for performing a denaturation step, an annealing step and an extension (or amplification) step to amplify the nucleic acid. Accordingly, the thermal block 10 may include or be operably connected with various modules for providing the PCR vessel 20 with the temperatures required for the respective steps.
  • the heat block 10 may be a heating wire (not shown) disposed therein, the heating wire is a variety of heat sources to maintain a temperature for performing the denaturation step, annealing step and extension (or amplification) step It may be connected to and driven in conjunction with various temperature sensors for monitoring the temperature of the hot wire.
  • the heating wires may be symmetrically disposed in the vertical direction and / or the left and right directions with respect to the center point of the surface in order to keep the internal temperature of the heat block 10 as a whole.
  • the thermal block 10 may have a thin film heater (not shown) disposed therein.
  • the thin film heaters may be spaced apart at regular intervals in the vertical direction and / or the left and right directions with respect to the center point of the surface in order to keep the internal temperature of the thermal block 10 as a whole.
  • the heat block 10 may be implemented in a plate shape for uniform heat distribution and rapid heat transfer of the same area in the PCR vessel 20, and may be implemented in a metal material, for example, aluminum, but is not limited thereto. It is not.
  • the thermal block 10 maintains an appropriate temperature for performing the denaturation step or the annealing and extension (or amplification) step.
  • the thermal block 10 may maintain 50 ° C. to 100 ° C., preferably 90 ° C. to 100 ° C., more preferably 95 ° C. to perform the denaturation step, and the annealing and extension (or 55 ° C. to 75 ° C., preferably 72 ° C., may be maintained to effect the amplification) step.
  • the column block 10 may be implemented in two or more, the real-time PCR device according to a second embodiment of the present invention to be described later is implemented in two of the column block 10.
  • the PCR vessel 20 includes a housing 22 having a thermal block contact region 21 in contact with the thermal block 10, a reaction chamber 23 formed inside the housing 22, and the reaction chamber ( 23) a fixed layer 25 disposed in a region inside the surface of the capture probe 24, which is capable of complementarily binding to a region of the amplifying target nucleic acid 2, and the reaction chamber 23.
  • An electrode 26 disposed in the other region and configured to detect an electrochemical signal, and connected to the metal nanoparticle 27 and the metal nanoparticle 27 in the reaction chamber 23, wherein the amplification target nucleic acid And a complex 29 having a signal probe 28 capable of complementarily binding to another region of.
  • the housing 22 is a main body of the PCR vessel 20, and may be made of various materials, for example, plastic, metal, glass, silicon, and the like.
  • the housing 22, although not shown in Figure 1, having a separate inlet or outlet for introducing a PCR sample and reagents, or discharge the PCR final product or waste, or at the same time serves as an inlet and outlet It may be provided with a single opening to be carried out.
  • the thermal block contact area 21 refers to an area that is in thermal contact with the one or more thermal blocks 10 to receive heat from the thermal block 10 to transfer heat into the reaction chamber 23. .
  • the thermal block contact area 21 may be implemented in various shapes and structures according to the shape and structure of the housing 22 or the thermal block 10.
  • the reaction chamber 23 is a space in which PCR is performed by a PCR sample and a reagent, and is formed in the housing 22.
  • the reaction chamber 23 may be implemented in various shapes and structures, such as a hollow cylinder shape, a bar shape, and a square column shape.
  • FIG. 2 is a detailed view of the reaction chamber 23 and the complex 29 accommodated in the real-time PCR device 1 according to the first embodiment of the present invention.
  • the reaction chamber 23 is disposed in one region therein, and the fixed layer 25 having the capture probe 24 surface-combined with the capture probe 24 capable of complementarily binding to one region of the amplification target nucleic acid 2, and the interior thereof.
  • an electrode 26 disposed in another region of the electrode and configured to detect an electrochemical signal, wherein the fixed layer 25 and the electrode 26 may be disposed at various positions within the reaction chamber 23. However, it is preferable to be disposed to face each other as shown in FIG.
  • the reaction chamber 23 is connected to the metal nanoparticles 27 and the metal nanoparticles 27 therein, the signal probe 28 that can be complementarily bonded to another region of the amplification target nucleic acid (28) Housed with a composite 29 having a.
  • the complex 29 may be previously accommodated in a real-time PCR device according to the first embodiment of the present invention before introduction of a PCR sample including a template nucleic acid and the like, and may be included in a PCR reagent including a primer, a polymerase, and the like. It may be introduced together into the reaction chamber 23 in a state.
  • the pinned layer 25 may be formed of various materials, for example, silicon, plastic, glass, and metal, such that the capture probe 24 may be deposited and exposed on one surface thereof.
  • the surface of the fixed layer 25 may be first surface-treated with a material such as amine NH 3 + , aldehyde COH, carboxyl group COOH, or the like before the capture probe 24 is deposited.
  • the capture probe 24 is implemented to complementarily bind to a portion (region) of the amplification target nucleic acid, and combines with the metal nanoparticles 27 to form a complex 29.
  • the metal nanoparticles 27 may vary, but zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu), gallium (Ga), indium (In), gold (Au), and silver One or more from the group consisting of (Ag).
  • the signal probe 28 is implemented to bind to a region of the amplification target nucleic acid complementarily, in this case, in the amplification target nucleic acid, the complementary binding region of the signal probe 28 is the capture probe ( Different from the complementary binding region of 24).
  • the capture probe 24 and signal probe 28 may bind complementarily to the amplification target nucleic acid (see FIG. 3).
  • the left figure shows a case in which the amplification target nucleic acid is not present before the PCR
  • the right figure shows a case in which the amplification target nucleic acid is present after the PCR is performed.
  • the target nucleic acid is amplified inside the amplification target nucleic acid (2) is complementary to the capture probe 24 surface-treated in the fixed layer 25, as shown in the right figure, and to the metal nanoparticle (27) Complementarily binds to the connected signal probe 28 to concentrate the metal nanoparticles 27 in a region proximate the pinned layer 25.
  • the metal nanoparticles 27 do not reach the electrode 26 and cause a current change (decrease) between the metal nanoparticles 27 and the electrode 26 to amplify the target nucleic acid 2. Resulting in a detectable electrochemical signal. Meanwhile, the amplification target nucleic acid 2, the capture probe 24, and the signal probe 28 may be single stranded DNA.
  • the electrode 26 is disposed in at least one region of the reaction chamber 23, and is implemented to detect an electrochemical signal generated inside the reaction chamber 23.
  • the electrode 26 may be made of various materials to perform the above functions, but for example, gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes (carbon nanotubes) ), Graphene, and carbon may be selected from one or more selected from the group consisting of.
  • the electrode 26 may be implemented in various shapes and structures to efficiently detect electrochemical signals generated in the reaction chamber 23. For example, as shown in FIG. It may be implemented in a plate shape of a metal material disposed along the inner surface.
  • the electrochemical signal measuring module 30 is electrically connected to the electrode 26 of the PCR vessel 20 to measure in real time the electrochemical signal generated in the reaction chamber 23 of the PCR vessel 20. It is implemented to be.
  • 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.
  • the electrochemical signal may be due to a change in current that occurs as the amplification target nucleic acid complementarily binds with the capture probe 24 and the signal probe 28.
  • Figure 4 shows the electrochemical signal generation process of the real-time PCR device according to a first embodiment of the present invention.
  • the step S1 is a complex including a capture probe 24, a signal probe 28, and metal nanoparticles 27, which are surface-treated on a fixed layer 25, a gold matrix, before PCR is started.
  • the step S2 is the current change (signal, signal) generated by the reduction or oxidation between the electrode (26, GC electrode) and the metal nanoparticles (27, yellow particles)
  • the amplification target nucleic acid (2, H1N1 DNA) is captured by the capture probe 24 and the signaling probe 28 of the complex 29 (signaling probe-AuNP). In combination with and causes a decrease in current change (signal reduction).
  • the metal nanoparticles 27 (AuNP) of the complex 29 are proximal surfaces of the electrodes 26 (GC electrodes). Is accumulated while accumulating to form an accumulation layer (Accumulation of AuNP), followed by applying a voltage to the electrode (26, GC electrode) to oxidize the reducing metal nanoparticles (27, AuNP) as a current change, that is, a signal Is generated, and the current change can be easily measured by the voltage value indicated by the oxidation current peak (S2).
  • the current change value that is, the electrochemical signal, represents the maximum value in the reaction chamber 23.
  • the target nucleic acid (2, H1N1 DNA) is amplified from the template nucleic acid, and the amplified target nucleic acid (2, H1N1 DNA) is captured by the capture probe (24) and the complex (29, signaling probe).
  • FIG. 5 shows a real-time PCR device according to a second embodiment of the present invention.
  • the real-time PCR device 1 may include a first column block 100 and a second column block 200 spaced apart from each other on a substrate 400; At least one reaction channel having inlets and outlets at both ends, a fixed layer disposed on one region of the reaction channel, and having a capture probe surface-treated with a capture probe capable of complementarily binding to one region of the amplifying target nucleic acid, and An electrode disposed in another region inside the reaction channel and configured to detect an electrochemical signal, the electrode being coupled to the metal nanoparticle and the metal nanoparticle, and complementarily binding to the other region of the amplification target nucleic acid.
  • a plate-shaped PCR chip 900 comprising a complex with a signal probe present;
  • 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).
  • the real-time PCR apparatus 1 may include a first column block 100 disposed on the substrate 400 and spaced apart from the first column block 100 on the substrate 400.
  • the second column block 200 is included.
  • 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 column block 100 of the real time PCR apparatus 1 may maintain 50 ° C.
  • 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.
  • the annealing and extension (or amplification) step is performed in the second thermal block, 55 ° C. to 75 ° C. may be maintained, and preferably 72 ° C. may be maintained.
  • 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 can 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 The yield may not be reduced or the reaction may occur, and when the denaturation step temperature is higher than 100 ° C., the activity of the enzyme used in PCR is reduced or disappeared, so that the denaturation step temperature may be 90 ° C. to 100 ° C., preferably 95 May be ° C.
  • the second row block 200 may maintain the annealing / extension temperature of the annealing and extension (or amplification) of the 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.
  • 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 disposed to be slidably movable in the left and right directions through the rail 510 extending in the left and right directions, and the rail 510, and the connecting member 520 that is slidably movable in the vertical 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 accommodating PCR samples and reagents, the PCR chip 900 is implemented to be detachable to the chip holder 300 Details of the PCR chip 900 and the chip holder 300 will be described later.
  • FIG. 6 shows the driving principle of the real-time PCR device 1 according to the second 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.
  • 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 e.g., a DNA polymerase, and a triphosphate deoxyribo
  • dNTP deoxyribonucle
  • 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).
  • FIG. 7 to 10 are detailed views of the PCR chip 900 according to the second embodiment of the present invention.
  • the PCR chip 900 has a plate shape and is formed of one or more reaction channels 921 having inlets 931 and outlets 932 formed at both ends thereof, and in one region inside the reaction channel 921.
  • a capture probe disposed on the fixed layer 940 that is surface treated and capable of complementarily binding to one region of the amplification target nucleic acid, and the other region within the reaction channel 921, to detect an electrochemical signal.
  • a complex having a metal nanoparticle and a signal probe connected to the metal nanoparticle and complementarily binding to another region of the amplification target nucleic acid.
  • the reaction channel 921 should be understood as a kind of reaction chamber 23 according to the first embodiment of the present invention.
  • the reaction channel 921 is disposed in one region of the reaction channel 921 and is one region of the amplification target nucleic acid.
  • Composite having a will be as described in the first embodiment of the present invention described above.
  • 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 electrode 950 is disposed in at least one region of the reaction channel 921 and is implemented to detect an electrochemical signal generated 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 center region of the reaction channel 921 as shown in FIG.
  • working electrode 950a oxygen or reduction reaction
  • a reference electrode oxygen or reduction
  • It may be implemented as a three-electrode module (left side of FIG. 8) having an electrode 950c.
  • the structure of the electrode 950 is implemented in the multi-electrode module method as illustrated in FIG. 8, not only the sensitivity of the electrochemical signal generated in the reaction channel 921 may be increased, but the detection of the generated signal may be performed. And measurement can be easily performed.
  • the PCR chip 900 may be divided into three layers based on the vertical cross-sectional view.
  • 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 having an inlet 931 and an outlet 932 and a fixing layer 940 on which the capture probe is surface treated. Can be.
  • 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 having the one or more reaction channels 921 is disposed in contact with the lower surface of the third plate 930.
  • the second plate 920 includes the reaction channel 921.
  • the reaction channel 921 is connected to a portion corresponding to the inlet 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 having the inlet portion 931 and the outlet portion 932 and the fixed layer 940 on which the capture probe is surface treated is provided on the upper surface of the second plate 920. Is placed.
  • 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.
  • the pinned layer 940 is formed in one region of the reaction channel 921.
  • the fixed layer 940 may be disposed to face the electrodes 950 of the first plate 910.
  • 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. 11 shows a chip holder according to a second 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 due to the binding of the amplification target nucleic acid to the capture probe and the signal probe in the reaction channel 921 of the PCR chip 900 is transferred to the electrochemical signal measurement module 800.
  • 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. 12 is a layout view of an electrochemical signal measurement module of a real-time PCR device according to a second 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 that the reaction channel 921 of the PCR chip 900 is provided.
  • An electrochemical signal measuring module 800 is implemented to measure the electrochemical signal generated therein in real time.
  • 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. 13 illustrates a step-by-step method for performing real-time PCR using a real-time PCR device according to a second embodiment of the present invention.
  • the real-time PCR method includes providing the real-time PCR device 1 described above; 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 real time PCR device 1 according to the second embodiment of the present invention. Therefore, the following real-time PCR method is based on the operation of the real-time PCR device 1 according to the second embodiment of the present invention. In this case, a metal-probe including metal nanoparticles and a signal probe may already be accommodated in the reaction chamber 921 of the real-time PCR device 1.
  • Sample and reagent injection step (S2) is a step of injecting the PCR sample and reagent to the PCR chip 900.
  • the metal-probe is not accommodated in the reaction chamber 921 of the real-time PCR device 1 in the step S1, the metal-probe including the metal nanoparticles and the signal probe in this step.
  • the metal-probe including the metal nanoparticles and the signal probe in this step can be accommodated in the reaction chamber 921 of the real-time PCR device (1).
  • 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.
  • the target nucleic acid region is amplified based on the template nucleic acid in the reaction channel 921, and the binding between the amplifying target nucleic acid, the capture probe, and the signal probe increases according to the continuous amplification of the target nucleic acid region. An electrochemical signal is generated.
  • 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 ends thereof in a plastic plate shape.
  • the electrode 950 was fabricated using carbon nanotubes and silver (Ag), and the electrochemical signal measuring module 800 adopted an anode stripping voltammetry (ASV). Gold (Au) was used as the metal nanomaterial.
  • the detection conditions of the electrochemical signal of the anode peeling voltammeter were assumed as follows.
  • a PCR sample 0.1 ng / ⁇ l of double-stranded template DNA of H1N1 virus is prepared, and a pair of primers that can complementarily bind to the template DNA as a PCR reagent, specifically, a forward primer, 0.125 ⁇ l, 1 pmole), reverse primer (0.125 ⁇ l, 1 pmole), 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 After preparing mM KCl, 1.5 mM MgCl 2 , 30 mM salt), the PCR sample and reagent solution were introduced into the PCR chip 900, and the PCR chip 900 was mounted on the chip holder 300.
  • a forward primer 0.125 ⁇ l, 1 pmole
  • reverse primer 0.125 ⁇ l, 1 pmole
  • 0.2 mL of dNTP 0.2 m
  • 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.
  • ASV anodizing stripping voltammetry
  • FIG. 14 to 16 are graphs showing the results of the real-time PCR method
  • FIGS. 17 to 18 are electrophoretic photographs showing the results of the real-time PCR method.
  • FIG. 14 is an actual graph illustrating changes in ASV peak signals due to gold (Au) nanoparticles according to a PCR cycle
  • FIGS. 15 to 16 are processed graphs quantified in FIG.

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KR102166494B1 (ko) * 2019-05-27 2020-10-15 김은근 Pcr 장치
KR102375991B1 (ko) * 2021-09-29 2022-03-18 주식회사 위즈바이오솔루션 분자 진단용 핵산 증폭장치
KR102790515B1 (ko) * 2021-12-31 2025-04-09 고려대학교 산학협력단 코로나 바이러스 검출용 키트 및 코로나 바이러스 검출 방법
KR20240171383A (ko) * 2023-05-30 2024-12-09 홍익대학교 산학협력단 전기화학적 방식을 이용한 중합효소 연쇄 반응의 피로인산 이온 농도 변화 측정 방법

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