WO2014017821A1 - Dispositif de pcr en temps réel pour détection de signaux électrochimiques comprenant un bloc chauffant dans lequel des unités d'élément chauffant sont réparties de manière répétée et procédé de pcr en temps réel l'utilisant - Google Patents

Dispositif de pcr en temps réel pour détection de signaux électrochimiques comprenant un bloc chauffant dans lequel des unités d'élément chauffant sont réparties de manière répétée et procédé de pcr en temps réel l'utilisant Download PDF

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Publication number
WO2014017821A1
WO2014017821A1 PCT/KR2013/006621 KR2013006621W WO2014017821A1 WO 2014017821 A1 WO2014017821 A1 WO 2014017821A1 KR 2013006621 W KR2013006621 W KR 2013006621W WO 2014017821 A1 WO2014017821 A1 WO 2014017821A1
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WIPO (PCT)
Prior art keywords
pcr
heater
real
electrode
chip
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PCT/KR2013/006621
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English (en)
Korean (ko)
Inventor
김성우
이정환
이유진
김덕중
Original Assignee
나노바이오시스(주)
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Publication of WO2014017821A1 publication Critical patent/WO2014017821A1/fr

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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
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control

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 is a heater group having at least one heater, the heater group having at least two and the two or more heater groups are at least two heater units spaced apart so that mutual heat exchange does not occur, at least,
  • a thermal block having a contact surface of a PCR chip containing a sample and a reagent on one surface thereof;
  • a column electrode unit having a column electrode connected to supply electric power to heaters provided in the column block;
  • At least one reaction channel having both an inlet and an outlet at both ends, and a plurality of reaction channels, which are repeatedly spaced apart across the bottom surface in the longitudinal direction of the reaction channel, and are generated by the binding of the amplifying nucleic acid and the active material within the reaction channel.
  • a detection electrode implemented to detect a chemical signal, wherein the detection electrode is disposed between the two or more heater groups in thermal contact with the thermal block;
  • a chip holder mounted with the PCR chip, the chip holder having a connection port configured to be electrically connected to the detection electrode end of the PCR chip;
  • an electrochemical signal measuring module electrically connected to the connection port of the chip holder to measure in real time an electrochemical signal generated in the reaction channel of the PCR chip. do.
  • 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 detection electrode is at least one selected from the group consisting of gold (Au), cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes, graphene, and carbon. Can be.
  • the detection electrode is a two-electrode module having a working electrode (combination of the amplification nucleic acid and the active material) and a reference electrode (combination of the amplification nucleic acid and the active material does not occur), or the indicator electrode It may be implemented as a three-electrode module having a counter electrode for adjusting the electronic balance generated from the reference electrode and the indicator electrode.
  • the electrochemical signal measuring module includes an anodic stripping voltammetry (ASV), a chronoamperometry (CA), a cyclic voltammetry, a square wave voltammetry (SWV), and a pulse voltage It may be selected from the group consisting of differential pulse voltammetry (DPV), and impedance.
  • ASV anodic stripping voltammetry
  • CA chronoamperometry
  • SWV square wave voltammetry
  • DPV differential pulse voltammetry
  • impedance impedance
  • the thermal block may be provided with two to four heater groups.
  • the thermal block has two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group is PCR annealing Maintain extension step temperature and the second heater group may maintain PCR denaturation step temperature.
  • the thermal block has three heater groups, the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing step temperature, and the third heater group maintains the PCR extension step temperature. Or the first heater group maintains a PCR annealing step temperature and the second heater group maintains a PCR extension step temperature and the third heater group maintains a PCR denaturation step temperature, or the first heater group Maintaining the PCR extension step temperature and the second heater group may be to maintain the PCR denaturation step temperature and the third heater group to maintain the PCR annealing step temperature.
  • the one or more reaction channels may be extended so as to pass in a straight longitudinal direction through the upper corresponding part of the heater disposed most optimally and the upper corresponding part of the heater disposed last.
  • the PCR chip may include a first plate provided with the detection electrode; A second plate disposed on the first plate and provided with the one or more reaction channels; And a third plate disposed on the second plate and provided with the inlet and the outlet.
  • the PCR chip may be implemented detachably to the chip holder.
  • the apparatus may further include a power supply unit for supplying power to the column electrode unit.
  • It may further comprise a pump arranged to provide a positive or negative pressure to control the flow rate and flow rate of the fluid flowing in the one or more reaction channels.
  • FIG. 1 to 5 show a column block and a column electrode part of a real-time PCR device according to an embodiment of the present invention.
  • 6 to 8 illustrate a PCR chip of a real-time PCR device according to an embodiment of the present invention.
  • FIG. 9 to 10 illustrate detection electrodes of a PCR chip of a real-time PCR device according to an embodiment of the present invention.
  • FIG. 11 illustrates a chip holder of a real-time PCR device according to an embodiment of the present invention.
  • FIG. 12 shows a real-time PCR device according to an embodiment of the present invention having a PCR chip, a power supply, and a pump.
  • FIG. 13 illustrates a nucleic acid amplification process by a real-time PCR device according to an embodiment of the present invention, and a process of detecting and measuring a nucleic acid amplification signal in real time.
  • FIG. 14 illustrates a series of processes for detecting and measuring nucleic acid amplification and nucleic acid amplification signals in real time using a real time PCR apparatus according to an embodiment of the present invention.
  • PCR device refers to a device used for PCR (Polymerase Chain Reaction) for amplifying a nucleic acid having a specific base sequence.
  • a PCR device may prepare a solution containing a PCR sample and a reagent comprising double stranded DNA as a template nucleic acid at a specific temperature, for example about 95 ° C.
  • An extension (or amplification) step of forming a double strand of DNA on the basis is performed, and the above steps are repeated, for example, 20 to 40 times to exponentially amplify the DNA having the specific base sequence.
  • the PCR device may simultaneously perform the annealing step and the extension (or amplification) step, and in this case, the PCR device performs two steps including the extension step and the annealing and extension (or amplification) step. By doing so, the first circulation can be completed.
  • 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 It is assumed that all of them are provided or are provided in the obvious range.
  • FIG. 1 to 5 show a column block and a column electrode part of a real-time PCR device according to an embodiment of the present invention.
  • the thermal block 100 is a module implemented to supply heat to a sample and a reagent at a specific temperature to perform a PCR, and has at least one surface of a contact surface of a PCR chip containing a sample and a reagent, One side of the PCR chip to be described is contacted to heat the sample and reagents present in one or more reaction channels to perform PCR.
  • the thermal block 100 may be implemented using a substrate as a body.
  • the substrate may be made of any material such that physical and / or chemical properties thereof do not change due to heating and temperature maintenance of a heater disposed in the substrate, and mutual heat exchange does not occur between two or more heaters spaced apart in the substrate. Can be.
  • the substrate may be formed of a material such as plastic, glass, silicon, or the like, or may be implemented to be transparent or translucent.
  • the thermal block 100 may be implemented in a plate shape as a whole, but is not limited thereto.
  • the thermal block 100 includes a heater group including one or more heaters, two or more heater groups, and the two or more heater groups are repeatedly disposed at least two heater units spaced apart from each other so that mutual heat exchange does not occur.
  • the contact surface of the PCR chip is implemented on at least one surface of the thermal block 100, various shapes for efficiently supplying heat to the PCR chip containing the sample and reagents, for example, to increase the surface area of the contact surface It may be implemented in a planar shape or a pillar shape (pillar).
  • the heaters 111, 112, 121, 122, 131, and 132 are heat generating elements, and may be implemented such that a hot wire (not shown) is disposed therein.
  • the heating wire may be operably connected with various heat sources to maintain a constant temperature, and may be operably connected with various temperature sensors for monitoring the temperature of the heating wire.
  • the heating wire may be disposed to be symmetrical in the vertical direction and / or the horizontal direction with respect to the surface center point of the heater in order to maintain the internal temperature of the heater as a whole.
  • the heater may have a thin film heater (not shown) disposed therein.
  • the thin film heaters may be disposed at regular intervals in the vertical direction and / or the left and right directions with respect to the center point of the heater surface in order to maintain the internal temperature of the heater as a whole.
  • the heater is a heating element, and may itself be a metal material, for example, chromium, aluminum, copper, iron, silver, and the like, for even heat distribution and rapid heat transfer over the same area.
  • the heater is a light-transmitting heating element, for example, conductive nanoparticles including an oxide semiconductor material or a material added with impurities selected from the group consisting of In, Sb, Al, Ga, C and Sn to the oxide semiconductor material, And at least one selected from the group consisting of indium tin oxide, conductive polymeric materials, carbon nanotubes, and graphene.
  • conductive nanoparticles including an oxide semiconductor material or a material added with impurities selected from the group consisting of In, Sb, Al, Ga, C and Sn to the oxide semiconductor material, And at least one selected from the group consisting of indium tin oxide, conductive polymeric materials, carbon nanotubes, and graphene.
  • the heater groups 110, 120, and 130 are units including the one or more heaters, and are regions that maintain a temperature for performing a denaturation step, annealing step, and / or extension step for PCR.
  • Two or more heater groups are disposed in the thermal block 100, and the two or more heater groups are spaced apart from each other so that mutual heat exchange does not occur.
  • Two to four heater groups may be included in the thermal block 100. That is, the thermal block includes two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group Maintaining the PCR annealing / extension step temperature and the second heater group may maintain the PCR denaturation step temperature.
  • the thermal block includes three heater groups, wherein the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing step temperature, and the third heater group has the PCR extension step temperature.
  • the first heater group maintains a PCR annealing step temperature and the second heater group maintains a PCR extension step temperature and the third heater group maintains a PCR denaturation step temperature, or the first heater The group may maintain the PCR extension step temperature, the second heater group may maintain the PCR denaturation step temperature, and the third heater group may maintain the PCR annealing step temperature.
  • the heater group may be disposed three times in the thermal block 100 to maintain three temperatures for performing PCR, that is, a temperature for performing a denaturation step, an annealing step, and an extension step, and more preferably, The heater group may be disposed twice in the thermal block 100 to maintain two temperatures for performing PCR, that is, a temperature for performing a denaturation step and an annealing / extension step, respectively, but is not limited thereto.
  • the heater group is disposed in the heat block 100 twice, and when performing two steps for performing PCR, that is, denaturation step and annealing / extension step, three steps for performing PCR, that is, denaturation step, annealing step and extension step It is possible to reduce the reaction time than to perform, there is an advantage of simplifying the structure by reducing the number of heaters.
  • the temperature for performing the denaturation step is 85 °C to 105 °C, preferably 95 °C
  • the temperature for performing the annealing step is 40 °C to 60 °C, preferably 50 °C
  • the temperature for performing the extension step is 50 °C to 80 °C, preferably 72 °C
  • the temperature for performing the denaturation step is 85 °C to 105 °C, preferably 95 ° C.
  • the temperature for performing the annealing / extension step is 50 ° C. to 80 ° C., preferably 72 ° C.
  • the specified temperature and temperature range for performing the PCR can be adjusted within a range feasible in performing the PCR.
  • the heater group may further include a heater that serves as a temperature buffer.
  • the heater units 10 and 20 are units including the two or more heater groups including the one or more heaters, and the first circulation including the denaturation step, annealing step, and / or extension step for performing PCR is completed. Area.
  • the heater unit is repeatedly arranged at least two in the thermal block (100). Preferably, the heater unit may be repeatedly arranged in the heat block 100 10 times, 20 times, 30 times, or 40 times, but is not limited thereto.
  • the heat block 100 includes heater units 10 and 20 repeatedly arranged, two heater groups 110 and 120 included therein, and one heater 111 and 121 respectively included therein.
  • a two-step temperature for performing the PCR that is, one temperature of the denaturation step and one temperature of the annealing / extension step are repeatedly provided sequentially.
  • the first heater 111 maintains one temperature in the range of 85 ° C. to 105 ° C., preferably 95 ° C., so that the first heater group 110 provides a temperature for performing the modification step.
  • the second heater 121 maintains one temperature in the range of 50 ° C. to 80 ° C., preferably 72 ° C. such that the second heater group 120 provides a temperature for performing an annealing / extension step.
  • 100 sequentially and repeatedly provides two-step temperature for performing PCR in the first heater unit 10 and the second heater unit 20.
  • the thermal block 100 includes heater units 10 and 20 repeatedly arranged, two heater groups 110 and 120 included therein, and two heaters 111 and 112 respectively included therein. 121, 122) to provide a two-step temperature for performing PCR, that is, two temperatures of the denaturation step and two temperatures of the annealing / extension step.
  • the first heater 111 has one temperature in the range of 85 ° C to 105 ° C
  • the second heater 112 has one temperature that is the same as or different from the temperature of the first heater 111 in the range of 85 ° C to 105 ° C.
  • the third heater 121 is one temperature in the range of 50 °C to 80 °C
  • the fourth heater 122 is 50 °C to
  • the thermal block 100 is maintained by maintaining a temperature equal to or different from the temperature of the third heater 121 in an 80 ° C range so that the second heater group 120 provides a temperature for performing an annealing / extension step. Is sequentially and repeatedly provided two-step temperature for performing PCR in the first heater unit 10 and the second heater unit 20.
  • the heat block 100 may include heater units 10 and 20 repeatedly arranged, three heater groups 110, 120, and 130 included therein, and one heater 111, respectively included therein.
  • 121, 131 sequentially provide three steps of temperature for performing PCR, that is, one temperature of the denaturation step, one temperature of the annealing step, and one temperature of the extension step.
  • the first heater 111 maintains one temperature in the range of 85 ° C. to 105 ° C., preferably 95 ° C., so that the first heater group 110 provides a temperature for performing the modification step.
  • the second heater 121 maintains one temperature in the range of 40 ° C.
  • the thermal block 100 is provided with a first heater unit. 10 and the second heater unit 20 sequentially and repeatedly provide three step temperatures for performing PCR.
  • heater units 10 and 20 repeatedly arranged, three heater groups 110, 120, and 130 included therein, and two heaters 111, 112, 121, 122, and 131 respectively included therein , 132) to provide three steps of temperature for performing PCR, that is, two temperatures of the denaturation step, two temperatures of the annealing step, and two temperatures of the extension step.
  • the first heater 111 has one temperature in the range of 85 ° C to 105 ° C
  • the second heater 112 has one temperature that is the same as or different from the temperature of the first heater 111 in the range of 85 ° C to 105 ° C.
  • the third heater 121 is in the temperature range of 40 °C to 60 °C 1
  • the fourth heater 122 is 40 °C to
  • the second heater group 120 provides a temperature for performing the annealing step by maintaining one temperature equal to or different from the temperature of the third heater 121 in a range of 60 ° C
  • the fifth heater 131 is 50 ° C.
  • the third heater group 130 extends by maintaining one temperature equal to or different from the temperature of the fifth heater 131 in a temperature range of 50 ° C. to 80 ° C., and the sixth heater 132.
  • the thermal block 100 is configured in three stages for performing PCR in the first heater unit 10 and the second heater unit 20. The system temperature is repeatedly provided sequentially.
  • the rate of change of temperature can be significantly improved.
  • the heater The rate of change of temperature between them is within the range of 20 °C to 40 °C per second can greatly shorten the reaction time.
  • the heaters are spaced apart so that mutual heat exchange does not occur, and as a result, in the nucleic acid amplification reaction that can be greatly affected by minute temperature changes, the denaturation step, annealing step and extension step (or the denaturation step and annealing).
  • the heater unit may be repeatedly arranged 10 times. That is, the heater unit may be repeatedly arranged in 10 times, 20 times, 30 times, 40 times, 50 times, etc. in consideration of the PCR circulation cycle according to the user who intends to perform the PCR or the type of the sample and the reagent. It is not limited.
  • the heater unit may be repeatedly arranged in half of the predetermined PCR cycle.
  • the heater unit may be repeatedly arranged ten times.
  • the sample and reagent solutions may be repeated 10 times of the PCR cycle from the inlet to the outlet in one or more reaction channels to be described in detail below, followed by 10 times of the PCR cycle from the outlet to the inlet. Can be run repeatedly
  • the thermal block 100 includes a heater unit repeatedly disposed ten times, and the heater unit includes a first heater group and a second heater group, and the first heater group and the second heater group. Each includes one heater, that is, the first heater 110 and the second heater 120.
  • the heaters, heater groups, heater units and heat blocks according to FIG. 5 are as described above.
  • the column electrode part 200 is a module for heating the heat block 100 by supplying power to the heat block 100 from a power supply unit (not shown), and the heaters provided in the heat block 100.
  • column electrodes 210 and 220 connected to supply power thereto.
  • the first column electrode 210 of the column block 100 is connected to supply power to the first heater 110
  • the second column electrode 220 is connected to the second heater ( It is connected to supply power to 120, but is not limited thereto.
  • the first heater 110 maintains the PCR denaturation step temperature, for example 85 °C to 105 °C and the second heater 120 maintains the PCR annealing / extension stage temperature, for example 50 °C to 80 °C
  • the second column electrode 220 is supplied with power for maintaining the PCR annealing / extension step temperature from the power supply I can receive it.
  • the first column electrode 210 and the second column electrode 220 are respectively provided to the first heater 110 and the two or more second heaters 120 repeatedly arranged in the column block 100. Can be connected.
  • the first column electrode 210 and the second column electrode 220 may be conductive materials such as gold, silver, and copper, and are not particularly limited.
  • the PCR chip 900 will be described later.
  • FIG. 6 to 8 show a PCR chip 900 of a real-time PCR device according to an embodiment of the present invention.
  • PCR chip 900 is implemented in a plate shape, at least one reaction channel 921, the inlet part 931 and the outlet part 932 is implemented at both ends, and the reaction channel (
  • a detection electrode 950 is disposed to be repeatedly spaced apart across the bottom surface in the longitudinal direction of the 921 and to detect an electrochemical signal generated due to the combination of the amplifying nucleic acid and the active material in the reaction channel 921.
  • the detection electrode 950 may be disposed between the two or more heater groups 110, 120, and 130 when in thermal contact with the thermal block 100.
  • 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. According to FIG. 6, the reaction channel 921 may be extended to pass through the upper corresponding portion of the first heater and the upper corresponding portion of the second heater in the longitudinal direction.
  • 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 outer structure of the PCR chip 900 is implemented to be fixedly mounted in the inner space of the chip holder 300 so as not to be separated from the chip holder 300 to be described later.
  • 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 detection electrode 950 may be formed of various materials to detect an electrochemical signal generated by the combination of an amplifying nucleic acid and an active material in the one or more reaction channels 921, for example, gold (Au ), Cobalt (Co), platinum (Pt), silver (Ag), carbon nanotubes (carbon nanotube), graphene (graphene), and carbon (carbon) may be selected from one or more. 6 to 8, the detection electrode 950 is repeatedly spaced apart across the bottom surface in the longitudinal direction of the reaction channel 921, and the detection electrode 950 is in thermal contact with the thermal block 100. ) Is implemented to be disposed between the two or more heater groups (110, 120, 130). According to FIG.
  • the detection electrode 950 is repeatedly spaced at regular intervals from the inlet 931 to the outlet 932 in the region of the reaction channel 921.
  • the electrochemical signal may be repeatedly detected from the nucleic acid sequentially amplified while passing through the reaction channel 921 in the longitudinal direction.
  • 7 to 8 showing vertical cross-sectional views of the PCR chip 900, it can be seen that the detection electrode 950 is disposed on the bottom surface of the reaction channel 921.
  • the PCR chip 900 may be largely 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 provided with the inlet part 931 and the outlet part 932.
  • An upper surface of the first plate 910 provided with the detection electrode 950 is adhesively disposed on a lower surface of the second plate 920.
  • the first plate 910 is adhered to the second plate 920 having the reaction channel 921 to secure a space with respect to the reaction channel 921, and further, at least the reaction channel 921.
  • the detection electrode 950 is disposed in one region (surface).
  • 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 according to the purpose and range of use of the PCR device according to an embodiment of the present invention.
  • 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.
  • the detection electrode 950 may be implemented in various ways.
  • a working electrode 950a in which the amplification nucleic acid is coupled to the active material and a reference electrode 950b in which the amplification nucleic acid is not coupled to the active material are provided.
  • a two-electrode module or a counter electrode 950c for adjusting the electronic balance generated from the indicator electrode 950a, the reference electrode 950b, and the indicator electrode as shown in FIG. It may be implemented as an electrode module (left side of FIG. 4).
  • the structure of the detection electrode 950 is implemented in the multi-electrode module method as illustrated in FIGS. 9 to 10
  • not only the sensitivity of the electrochemical signal generated inside the reaction channel 921 may be increased but also generated. Detection and measurement of signals can be performed easily.
  • FIG. 11 shows a chip holder 300 of a real-time PCR device according to an 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.
  • 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 may be connected to any driving means (not shown) to move up and down or left and right inside the real-time PCR apparatus.
  • FIG. 12 shows a real-time PCR device according to an embodiment of the present invention having a PCR chip 900, a power supply 400, and a pump 500.
  • the PCR chip 900 is disposed on and in contact with the heat block, and specifically, the detection electrode 950 is disposed between the repeatedly arranged first and second heaters on the heat block 100. It is arranged repeatedly.
  • the PCR chip 900 and the components included therein are as described above.
  • the power supply unit 400 is a module for supplying power to the column electrode unit 200, and may be connected to the first column electrode 210 and the second column electrode 220 of the column electrode unit 200, respectively. have.
  • a first power port (not shown) of the power supply 400 may be connected to the first column electrode (not shown).
  • a second power port (not shown) of the power supply 400 is electrically connected to the second column electrode 220.
  • the power supply unit 400 supplies power to the first column electrode 210 and the second column electrode 220, respectively, so that the first block of the column block 100 is provided.
  • the heater 110 and the second heater 120 can be quickly heated, and when the heaters 110 and 120 reach a predetermined temperature, the power supply is controlled to maintain the predetermined temperature.
  • the predetermined temperature may be a PCR denaturation step temperature (85 ° C. to 105 ° C., preferably 95 ° C.) in the first heater 110 and a PCR annealing / extension step temperature (in the second heater 120). 50 ° C. to 80 ° C., preferably 72 ° C.), or in the first heater 110, a PCR annealing / extension step temperature (50 ° C. to 80 ° C., preferably 72 ° C. or 60 ° C.) and the second heater At 120, the PCR denaturation step temperature (85 °C to 105 °C, preferably 95 °C) may be.
  • the pump 500 is a module for controlling the flow rate and the flow rate of the fluid flowing in one or more reaction channels 921 of the PCR chip 900, may be a positive pressure pump or a negative pressure pump, for example a syringe It may be a syringe pump.
  • the pump 500 may be operably disposed in a portion of the reaction channel 921, but preferably the inlet 931 and / or outlet 932 formed at both ends of the reaction channel 921. Is placed in the connection. When the pump 500 is connected to the inlet 931 and / or the outlet 932, the pump 500 may not only serve as a pump but also provide a sample and the like through the inlet 931 and / or the outlet 932.
  • the pump 500 when it is desired to control the flow rate and flow rate of the fluid flowing in the reaction channel 921, that is, the sample and reagent solutions in one direction, the pump 500 includes the inlet part 931 and the outlet part 932. If only one of the connections, and the remaining one can be a general plug is sealed, the flow of the fluid flowing in the reaction channel 921, that is, if you want to control the flow rate and flow rate of the sample and reagent solution in both directions The pump 500 may be connected to both the inlet part 931 and the outlet part 932.
  • Nucleic acid amplification reaction of the sample and the reagent in the PCR device including the PCR chip 900, the power supply 400 and the pump 500 may be performed through the following steps as an embodiment.
  • oligonucleotide primer having a sequence complementary to the specific nucleotide sequence to be amplified
  • DNA polymerase DNA polymerase
  • deoxyribonucleotide triphosphates dNTP
  • PCR reaction buffer PCR reaction buffer
  • the sample and reagent solutions are introduced into the PCR chip 100.
  • the sample and reagent solutions are disposed in the reaction channel 921 inside the PCR chip 900 through the inlet portion 931.
  • the column electrode unit 200 specifically, the first column electrode 210 and the second column electrode 220 are connected to the power supply 400, respectively, and the inlet of the PCR chip 900 931 and the outlet 932 are sealingly connected to the pump 500.
  • the power supply unit 400 is instructed to supply power to heat the first heater 110 and the second heater 120 through the first column electrode 210 and the second column electrode 220. And a specific temperature, for example, a PCR denaturation step temperature (95 ° C.) for the first heater 110 and a PCR annealing / extension step temperature (72 ° C.) for the second heater 120.
  • a specific temperature for example, a PCR denaturation step temperature (95 ° C.) for the first heater 110 and a PCR annealing / extension step temperature (72 ° C.) for the second heater 120.
  • the sample and reagent solutions are transferred to the reaction channel 921.
  • the flow rate and flow rate of the sample and reagent solutions may be controlled by adjusting the strength of the positive pressure or the negative pressure provided by the pump 500.
  • the sample and reagent solution may be transferred from the inlet 931 end of the reaction channel 921 to the outlet 932 end of the upper corresponding portion 301 and the first heater 110.
  • 2 PCR is performed while moving the upper corresponding portion 302 of the heater 120 in the longitudinal direction.
  • the first sample and reagent solution receives heat from a heat block 100 in which a heater unit including the first heater 110 and the second heater 120 is repeatedly disposed 10 times. 10 PCR cycles are completed while undergoing a PCR denaturation step in the upper counterpart 301 of the heater 110 and a PCR annealing / extension step in the upper counterpart 302 of the second heater 120.
  • the sample and reagent solution is formed from the upper end of the first heater 110 and the second heater 120 from the outlet 931 end of the reaction channel 921 to the end of the inlet 932.
  • PCR can be performed again by moving the upper corresponding part of the back side in the longitudinal direction.
  • FIG. 13 illustrates a nucleic acid amplification process by a real-time PCR device according to an embodiment of the present invention, and a process of detecting and measuring a nucleic acid amplification signal in real time.
  • a PCR device includes a heat block 100, the first heater 110, and the first heater 110 and the second heater 120 repeatedly arranged in a horizontal direction.
  • the PCR chip 900 is repeatedly arranged to correspond to the detection electrode 950 in the space between the second heater 120, but is electrically connected to a connection port (not shown) of the chip holder (not shown).
  • the electrochemical signal measuring module 800 implemented to measure in real time the electrochemical signal generated inside the reaction channel 921 of the PCR chip 900, and other, although not shown, includes a power supply, a pump, etc. .
  • the electrochemical signal measuring module 800 may be electrically connected to the connection port of the chip holder through an electrical connection means 700, for example, a lead wire.
  • electrochemical signals repeatedly generated by sequential nucleic acid amplification inside the reaction channel 921 of the PCR chip 900 are sequentially detected through the detection electrode 950 of the PCR chip 900.
  • the detected signal may be measured and further processed or analyzed by the electrochemical signal measuring module 800 via the connection port of the chip holder and the electrical connection means 700.
  • the electrochemical signal measuring module 800 may vary, but an anode stripping voltammetry (ASV), a chronoamperometry (CA), a cyclic voltammetry, a square wave voltmeter (square) wave voltammetry (SWV), differential pulse voltammetry (DPV), and impedance. Therefore, according to the PCR device according to the embodiment of the present invention according to FIG.
  • the nucleic acid amplification process may be measured and analyzed in real time when performing PCR.
  • the sample and reagent solutions do not need to be added a separate fluorescent material.
  • the step of measuring the nucleic acid amplification reaction in real-time (real-time) by the real-time PCR device according to an embodiment of the present invention can be confirmed.
  • the sample and reagent solutions pass through the upper corresponding portion 301 of the first heater 110 and the upper corresponding portion 302 of the second heater 120 in the reaction channel 921.
  • a PCR denaturation step and a PCR annealing / extension step are performed, in which case the sample and reagent solution is between the first heater 110 and the second heater 120 and between the first heater 110 and the second heater. It passes through the detection electrode 950 region repeatedly disposed between the heater unit including the (120).
  • the flow rate of the sample and reagent solutions is slowed or stopped for a short time, and then due to the binding of the amplifying nucleic acid and the active substance.
  • the generated electrochemical signal may be sequentially detected and measured in real time through the detection electrode 950.
  • the amount of target nucleic acid is monitored in real time by monitoring the result of the reaction by amplification of the nucleic acid in the reaction channel 921 (without the fluorescent substance and the light detection system) in real time during each cycle of PCR. Can be detected and measured in real-time
  • FIG. 14 illustrates a series of processes for detecting and measuring nucleic acid amplification and nucleic acid amplification signals in real time using a real time PCR apparatus according to an embodiment of the present invention.
  • the real-time PCR method using a real-time PCR device comprises the steps of providing the above-described real-time PCR device; 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 PCR sample and the PCR reagent are sequentially moved to the first heater and the second heater to maintain the denaturation stage temperature of the PCR and the annealing and extension (or amplification) stage temperatures of the PCR while moving the reaction channel 921 in the longitudinal direction, respectively. Repeatedly performing thermal contact with PCR; And detecting and measuring, in real time, an electrochemical signal generated due to the binding of the amplifying nu
  • the real time PCR device providing step S1 is a step of preparing the above-described real time PCR device. Therefore, the real-time PCR method according to an embodiment of the present invention below assumes the operation of the real-time PCR device.
  • Sample and reagent injection step (S2) is a material that can generate an electrical signal, 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 temperature of the first heater 110 and the second heater 120 of the thermal block 100 is maintained while heating, and the sample and the reagent are removed from the reaction channel 921 of the PCR chip 900.
  • PCR is performed while moving in the longitudinal direction.
  • target nucleic acid sites are sequentially amplified on the basis of template nucleic acids in samples and reagents moving inside the reaction channel 921, and continuous reaction (combination) with the active material is performed according to continuous amplification of the target nucleic acid sites. This produces an electrochemical signal.
  • 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.

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Abstract

Un mode de réalisation de la présente invention concerne un dispositif de PCR en temps réel pour détection de signaux électrochimiques, le dispositif comprenant un bloc chauffant dans lequel des unités d'élément chauffant sont réparties de manière répétée et concerne un procédé de PCR en temps réel l'utilisant, ce par quoi non seulement il est possible de réaliser une analyse à vitesse ultra-haute simultanée d'une pluralité d'échantillons au moyen du bloc chauffant dans lequel les unités d'élément chauffant sont réparties de manière répétée et d'une puce de PCR en forme de plaque, mais encore il est possible de contribuer de manière significative à la microminiaturisation et à l'aptitude au portage accrue de produits par l'intermédiaire d'une modularisation simple autorisant la détection aisée de signaux électrochimiques continus générés dans le processus d'amplification d'acide nucléique.
PCT/KR2013/006621 2012-07-24 2013-07-24 Dispositif de pcr en temps réel pour détection de signaux électrochimiques comprenant un bloc chauffant dans lequel des unités d'élément chauffant sont réparties de manière répétée et procédé de pcr en temps réel l'utilisant WO2014017821A1 (fr)

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KR1020120080459A KR101950210B1 (ko) 2012-07-24 2012-07-24 히터 유닛이 반복 배치된 열 블록을 포함하는 전기화학적 신호를 검출하기 위한 실시간 ｐｃｒ 장치, 및 이를 이용한 실시간 ｐｃｒ 방법
KR10-2012-0080459 2012-07-24

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