Hereinafter, an embodiment of the present invention will be described in detail with reference to the accompanying drawings. The following description is only for easily understanding the embodiments of the present invention, but is not intended to limit the protection scope of the present invention.
PCR device according to an embodiment of the present invention is a device for performing PCR to amplify a nucleic acid having a specific base sequence, in addition to the components described in the specification, other components generally required to perform PCR And may be understood within the scope of the already disclosed or obvious of the prior art.
1 illustrates a structure of a column block 100 of a PCR device according to an embodiment of the present invention.
The thermal block 100 is a module implemented to supply heat to a target sample at a specific temperature to perform a PCR. The thermal block 100 includes a contact surface of a PCR chip that accommodates a target sample on at least one side, and will be described in detail below. One side of the PCR chip is contacted to heat the target sample present in one or more reaction channels to perform PCR. The thermal block 100 is based on a substrate. The substrate may be made of any material such that physical and / or chemical properties thereof do not change due to heating and temperature maintenance of a heater disposed in the substrate, and mutual heat exchange does not occur between two or more heaters spaced apart in the substrate. Can be. For example, the substrate may be made of plastic, glass, silicon, or the like, and may be implemented transparently or semitransparently. It is preferable to implement with (transparent) material. The thermal block 100 may have a planar shape as a whole, but is not limited thereto. Heat block 100 of the PCR device according to an 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 disposed to be spaced apart so that mutual heat exchange does not occur The unit is repeatedly arranged at least two. In addition, the contact surface of the PCR chip is implemented on at least one surface of the thermal block 100, and may be implemented in various shapes for efficiently supplying heat to the PCR chip containing the target sample, the surface area of the contact surface The planar shape or the pillar shape is preferable so that it may be wide.
The heaters 111, 112, 121, 122, 131, and 132 are heat generating elements, and a heating wire (not shown) may be 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 a constant internal temperature of the heater as a whole. Also, the heater may have a thin film heater (not shown) disposed therein. The thin film heaters may be disposed at regular intervals in the vertical direction and / or the left and right directions with respect to the center point of the heater surface in order to maintain the internal temperature of the heater as a whole. In addition, the heater is a heating element, and may itself be a metal material, for example, chromium, aluminum, copper, iron, silver, and the like, for even heat distribution and rapid heat transfer over the same area. In addition, the heater is a light-transmitting heating element, for example, conductive nanoparticles including an oxide semiconductor material or a material added with impurities selected from the group consisting of In, Sb, Al, Ga, C and Sn to the oxide semiconductor material, And at least one selected from the group consisting of indium tin oxide, conductive polymeric materials, carbon nanotubes, and graphene. If the PCR device according to an embodiment of the present invention is used for real-time PCR, the heater is preferably a light transmitting heating element.
The heater groups 110, 120, and 130 are units including the one or more heaters, and are regions that maintain a temperature for performing a denaturation step, annealing step, and / or extension step for PCR. Two or more heater groups are disposed in the thermal block 100, and the two or more heater groups are spaced apart from each other so that mutual heat exchange does not occur. Two to four heater groups may be included in the thermal block 100. That is, the thermal block includes two heater groups, the first heater group maintains the PCR denaturation step temperature and the second heater group maintains the PCR annealing / extension step temperature, or the first heater group Maintaining the PCR annealing / extension step temperature and the second heater group may maintain the PCR denaturation step temperature. In addition, the thermal block includes three heater groups, wherein the first heater group maintains the PCR denaturation step temperature, the second heater group maintains the PCR annealing step temperature, and the third heater group has the PCR extension step temperature. Or the first heater group maintains a PCR annealing step temperature and the second heater group maintains a PCR extension step temperature and the third heater group maintains a PCR denaturation step temperature, or the first heater The group may maintain the PCR extension step temperature, the second heater group may maintain the PCR denaturation step temperature, and the third heater group may maintain the PCR annealing step temperature. Preferably, the heater group may be disposed three times in the thermal block 100 to maintain three temperatures for performing PCR, that is, a temperature for performing a denaturation step, an annealing step, and an extension step, and more preferably, The heater group may be disposed twice in the thermal block 100 to maintain two temperatures for performing PCR, that is, a temperature for performing a denaturation step and an annealing / extension step, respectively, but is not limited thereto. The heater group is disposed in the heat block 100 twice, and when performing two steps for performing PCR, that is, denaturation step and annealing / extension step, three steps for performing PCR, that is, denaturation step, annealing step and extension step It is possible to shorten the reaction time rather than to perform, there is an advantage of simplifying the structure by reducing the number of heaters. In this case, in the three steps for performing the PCR, the temperature for performing the denaturation step is 85 ℃ to 105 ℃, preferably 95 ℃, the temperature for performing the annealing step is 40 ℃ to 60 ℃, preferably 50 ℃, the temperature for performing the extension step is 50 ℃ to 80 ℃, preferably 72 ℃, in two steps for performing the PCR, the temperature for performing the denaturation step is 85 ℃ to 105 ℃, preferably 95 ° C., and the temperature for performing the annealing / extension step is 50 ° C. to 80 ° C., preferably 72 ° C. However, the specified temperature and temperature range for performing the PCR can be adjusted within a range feasible in performing the PCR. On the other hand, the heater group may further include a heater that serves as a temperature buffer.
The heater unit 10, 20 is a unit including the two or more heater groups including the one or more heaters and includes a region in which one cycle including a denaturation step, an annealing step, and / or an extension step for performing PCR is completed. to be. The heater unit is repeatedly arranged at least two in the thermal block (100). Preferably, the heater unit may be repeatedly arranged in the heat block 100 10 times, 20 times, 30 times, or 40 times, but is not limited thereto.
According to FIG. 1A, the heat block 100 includes heater units 10 and 20 repeatedly arranged, two heater groups 110 and 120 included therein, and one heater 111 and 121 respectively included therein. By providing a two-step temperature for performing the PCR, that is, one temperature of the denaturation step and one temperature of the annealing / extension step are repeatedly provided sequentially. For example, the first heater 111 maintains one temperature in the range of 85 ° C. to 105 ° C., preferably 95 ° C., so that the first heater group 110 provides a temperature for performing the modification step. The second heater 121 maintains one temperature in the range of 50 ° C. to 80 ° C., preferably 72 ° C. such that the second heater group 120 provides a temperature for performing an annealing / extension step. 100 sequentially and repeatedly provides two-step temperature for performing PCR in the first heater unit 10 and the second heater unit 20.
According to FIG. 1B, the heat block 100 may include heater units 10 and 20 repeatedly arranged, two heater groups 110 and 120 included therein, and two heaters 111 and 112 respectively included therein. 121, 122) to provide a two-step temperature for performing PCR, that is, two temperatures of the denaturation step and two temperatures of the annealing / extension step. For example, the first heater 111 has one temperature in the range of 85 ° C to 105 ° C, and the second heater 112 has one temperature that is the same as or different from the temperature of the first heater 111 in the range of 85 ° C to 105 ° C. By maintaining the first heater group 110 provides a temperature for performing the modification step, the third heater 121 is one temperature in the range of 50 ℃ to 80 ℃, the fourth heater 122 is 50 ℃ to The thermal block 100 is maintained by maintaining a temperature equal to or different from the temperature of the third heater 121 in an 80 ° C range so that the second heater group 120 provides a temperature for performing an annealing / extension step. Is sequentially and repeatedly provided two-step temperature for performing PCR in the first heater unit 10 and the second heater unit 20.
According to FIG. 1C, the heat block 100 includes heater units 10 and 20 repeatedly arranged, three heater groups 110, 120, and 130 included therein, and one heater 111, respectively included therein. 121, 131, sequentially provide three steps of temperature for performing PCR, that is, one temperature of the denaturation step, one temperature of the annealing step, and one temperature of the extension step. For example, the first heater 111 maintains one temperature in the range of 85 ° C. to 105 ° C., preferably 95 ° C., so that the first heater group 110 provides a temperature for performing the modification step. The second heater 121 maintains one temperature in the range of 40 ° C. to 60 ° C., preferably 50 ° C., so that the second heater group 120 provides a temperature for performing the annealing step, and the third heater 131. Is maintained at a temperature in the range of 50 ° C to 80 ° C, preferably 72 ° C, so that the third heater group 130 provides a temperature for performing the extension step, whereby the thermal block 100 is provided with a first heater unit. 10 and the second heater unit 20 sequentially and repeatedly provide three step temperatures for performing PCR.
According to FIG. 1D, heater units 10 and 20 repeatedly arranged, three heater groups 110, 120, and 130 included therein, and two heaters 111, 112, 121, 122, and 131 respectively included therein , 132) to provide three steps of temperature for performing PCR, that is, two temperatures of the denaturation step, two temperatures of the annealing step, and two temperatures of the extension step. For example, the first heater 111 has one temperature in the range of 85 ° C to 105 ° C, and the second heater 112 has one temperature that is the same as or different from the temperature of the first heater 111 in the range of 85 ° C to 105 ° C. By maintaining the first heater group 110 provides a temperature for performing the modification step, the third heater 121 is in the temperature range of 40 ℃ to 60 ℃ 1, the fourth heater 122 is 40 ℃ to The second heater group 120 provides a temperature for performing the annealing step by maintaining one temperature equal to or different from the temperature of the third heater 121 in a range of 60 ° C, and the fifth heater 131 is 50 ° C. The third heater group 130 extends by maintaining one temperature equal to or different from the temperature of the fifth heater 131 in a temperature range of 50 ° C. to 80 ° C., and the sixth heater 132. By providing a temperature for performing the step, the thermal block 100 is configured in three stages for performing PCR in the first heater unit 10 and the second heater unit 20. The system temperature is repeatedly provided sequentially.
As shown in Figs. 1A to 1D, the temperature change rate can be greatly improved by repeatedly disposing two or more heaters maintaining a constant temperature. For example, according to the conventional single heater method using only one heater, while the temperature change rate is within the range of 3 ℃ to 7 ℃ per second, according to the repeating heater arrangement method according to an embodiment of the present invention, the heater The rate of change of temperature between them is within the range of 20 ℃ to 40 ℃ per second can greatly shorten the reaction time. The heaters are spaced apart so that mutual heat exchange does not occur, and as a result, in the nucleic acid amplification reaction that can be greatly affected by minute temperature changes, the denaturation step, annealing step and extension step (or the denaturation step and annealing). Accurate temperature control), and it is possible to maintain the desired temperature or temperature range only at the site where heat is supplied from the heaters. In addition, two or more heater units are repeatedly arranged in the thermal block 100, and the number of repeated arrangements of the heater units 10 and 20 may vary according to the type of a user or a target sample to perform PCR. . For example, when the PCR apparatus according to an embodiment of the present invention is to be applied to a PCR having 10 cycles, the heater unit may be repeatedly arranged 10 times. That is, the heater unit may be repeatedly arranged in 10 times, 20 times, 30 times, 40 times, 50 times, etc. in consideration of the PCR circulation cycle according to the type of the user or target sample to be PCR, which is particularly limited. It doesn't happen. On the other hand, the heater unit may be repeatedly arranged in half of the predetermined PCR cycle. For example, when the PCR apparatus according to an embodiment of the present invention is to be applied to a PCR having a circulation cycle of 20 cycles, the heater unit may be repeatedly arranged ten times. In this case, the target sample solution is repeated 10 times in a PCR cycle from the inlet 304 to the outlet 305 in the reaction channel 303 disposed in the nucleic acid amplification reaction unit 300 to be described in detail below. On the contrary, the PCR cycle may be repeated 10 times from the outlet 305 toward the inlet 304.
2 shows a structure of a PCR device 1 according to an embodiment of the present invention including a PCR chip 300. Specifically, the upper part of FIG. 2 shows a lateral cross-sectional view of the PCR chip 300 and the column block 100 of the PCR device according to an embodiment of the present invention, and the lower part of FIG. 2 is an embodiment of the present invention. The top plan view of the PCR chip 300 and the column block 100 of the PCR apparatus is shown.
According to FIG. 2, the heat block 100 includes a heater unit that is 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 heater, the heater group, the heater unit and the heat block according to FIG. 2 are as described above.
The electrode unit 200 is a module that receives power from a power supply unit (not shown) and supplies power to heat the heat block 100, and supplies power to heaters provided in the heat block 100. Electrodes 210 and 220 connected to each other. According to FIG. 2, the first electrode 210 of the thermal block 100 is connected to supply power to the first heater 110, and the second electrode 220 is connected to the second heater 120. The electrode unit 200 is connected to supply power to the power source, but is not limited thereto. The electrode unit 200 may be disposed to be driven to the outside of the thermal block 100. If the first heater 110 maintains the PCR denaturation step temperature, for example 85 ℃ to 105 ℃ and the second heater 120 maintains the PCR annealing / extension stage temperature, for example 50 ℃ to 80 ℃ In this case, the first electrode 210 may receive power from the power supply for maintaining the PCR denaturation stage temperature, and the second electrode 220 may receive power from the power supply for maintaining the PCR annealing / extension stage temperature. have. The first electrode 210 and the second electrode 220 may be connected to the first heater 110 and two or more second heaters 120 repeatedly arranged in the thermal block 100 (see FIG. 2). ). The first electrode 210 and the second electrode 220 may be a conductive material such as gold, silver, copper, and the like, and are not particularly limited.
The PCR chip 300 is a module in which PCR is performed by amplifying a nucleic acid present in a target sample by receiving heat from the heat block 100. The target sample is preferably a solution in liquid form, and the target sample is a nucleic acid, eg, double stranded DNA, an oligonucleotide primer having a sequence complementary to a particular nucleotide sequence to be amplified, DNA polymerase, deoxyribolic Nucleotides (deoxyribonucleotide triphosphates, dNTP), PCR reaction buffer (PCR reaction buffer) may be included. The PCR chip 300 may be supplied with heat from the first heater 110 and the second heater 120 in contact with the heat block 100 to enable heat exchange with the heat block 100. have. In addition, the PCR chip 300 is one or more reactions extending to extend in the longitudinal direction through the upper corresponding portion 301 of the first heater 110 and the upper corresponding portion 302 of the second heater 120 Channel 303 may be provided. According to the upper part of FIG. 2, the reaction channel 303 may include an upper corresponding portion 301 of the first heater 110 and an upper corresponding portion of the second heater 120 disposed in the thermal block 100. 302 extends in fluid communication and extends in the longitudinal direction. The upper corresponding portion 301 of the first heater 110 of the reaction channel 303 is a region where a PCR denaturation reaction of nucleic acid present in the target sample occurs, and the upper corresponding portion 302 of the second heater 120 occurs. ) May be a region where PCR annealing / extension reactions of nucleic acids present in a target sample occur. That is, PCR is performed while a target sample flowing through the reaction channel 303 passes through the upper corresponding portion 301 of the first heater 110 and the upper corresponding portion 302 of the second heater 120 in succession. Can be. In addition, the PCR chip 300 may include inlets 304 and outlets 305 at both ends of the one or more reaction channels 303, respectively. The inlet 304 and the outlet 305 are portions in which a target sample is introduced into and / or discharged from the PCR chip 300, and the order thereof is not limited. In addition, according to the bottom of FIG. 2, five reaction channels 303 are disposed in the nucleic acid amplification reaction unit 300, but the reaction channels 303 may be arranged in various numbers without limitation. On the other hand, as shown in the lower end of Figure 2, the one or more reaction channels 303 is the upper side of the upper side corresponding portion 301 of the first heater 110 disposed most of the heater unit and the upper side of the second heater 120 disposed last. It is preferably arranged to extend through the corresponding portion 302 in the straight longitudinal direction. Accordingly, the target sample introduced through the inlet 304 passes through the reaction channel 303 in the longitudinal direction, and the upper corresponding portion 301 of the first heater 110 and the upper side of the second heater 120. In the corresponding portion 302, the PCR denaturation step and the PCR annealing / extension step may be repeatedly performed and discharged to the outside through the outlet. On the other hand, the PCR chip 300 may be a planar shape as a whole, but is not limited thereto. On the other hand, the PCR chip 300 may be implemented with a light transmissive material, and if the PCR device according to an embodiment of the present invention is used for real-time (PC) use, the PCR chip 300 is light transmissive It is preferable that the material is implemented.
3 shows a detailed structure of a PCR chip 300 according to an embodiment of the present invention.
According to FIG. 3, the PCR chip 300 includes a first plate 310 disposed on the thermal block 100, a first plate 310 disposed on the first block 310, and the one or more reaction channels 303. A third plate 320 and a third plate disposed on the second plate 320 and having inlets 304 and outlets 305 at both ends of the one or more reaction channels 303, respectively. 330 may include. As such, by implementing the PCR chip 300 in a thin film-like stacked structure, the manufacturing method is simple, the cost can be greatly reduced, and heat exchange with the heat block 100 is very easy. The PCR chip 300 may be implemented in various materials, but is preferably implemented in a thin film shape of a plastic material as a whole. In addition, the PCR chip 300 may be implemented with a light transmissive material. If the PCR device according to an embodiment of the present invention is used for real-time PCR, the PCR chip 300 is light transmissive. It is preferable that the material is implemented.
The first plate 310 is disposed on the thermal block 100 to receive heat from the thermal block 100. The first plate 310 may be implemented with various materials, but preferably, polydimethylsiloxane (PDMS), cycloolefin copolymer (COC), polymethylmethacrylate (PMMA), or the like. ), Polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof The material may be. In addition, the top surface of the first plate 310 may be treated with a hydrophilic material (not shown), which allows the PCR to be performed smoothly. By treating the hydrophilic material, a single layer including a hydrophilic material may be formed on the first plate 310. 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 320 is disposed on the first plate 310. The second plate 320 includes one or more reaction channels 303. The one or more reaction channels 303 are connected to portions corresponding to the inlet 304 and the outlet 305 formed in the third plate 330. Therefore, the PCR reaction proceeds after the target sample solution to be amplified is introduced into the one or more reaction channels 303. In addition, the one or more reaction channels 303 may be present in two or more according to the purpose and range of use of the PCR chip 300 according to an embodiment of the present invention. According to FIG. 1, five reaction channels 303 may be provided. It is illustrated. In addition, the second plate 320 may be formed of various materials, but preferably, polymethylmethacrylate (PMMA), polycarbonate (PC), cycloolefin copolymer (cycloolefin copolymer, COC) , Polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM) Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinylchloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (polybutylene terephthalate , PBT), fluorinated ethylenepropylene (FEP), perfluoroalkoxyalkane (PFA), and combinations thereof It is chosen or a thermoplastic resin may be a thermosetting resin material. In addition, the thickness of the second plate 320 may vary, but may be selected from 0.01 μm to 5 mm. In addition, the width and length of the reaction channel 303 may vary, but preferably the width of the reaction channel 303 is selected from 0.001 mm to 10 mm, the length of the reaction channel 303 is 1 mm To 100 mm. In addition, the inner wall of the second plate 320 may be coated with a material such as silane-based and Bovine Serum Albumin (BSA) to prevent DNA and protein adsorption. Treatment of the materials can be carried out according to methods known in the art.
The third plate 330 is disposed on the second plate 320. The third plate 330 includes an inlet portion 304 formed in one region on one or more reaction channels 303 formed in the second plate 320 and an outlet portion 305 formed in the other region. The inlet 304 is a portion to which the target sample solution containing the nucleic acid to be amplified is introduced. The outlet 305 is a portion where the target sample solution is discharged after the PCR is completed. Thus, the third plate 330 covers at least one reaction channel 303 formed in the second plate 320, wherein the inlet 304 and the outlet 305 are at least one reaction channel ( It serves as an inlet and outlet of 303). In addition, the third plate 330 may be implemented with various materials, but preferably, polydimethylsiloxane (PDMS), cycloolefin copolymer (COC), polymethylmethacrylate (polymethylmetharcylate) , PMMA), polycarbonate (PC), polypropylene carbonate (PPC), polyether sulfone (PES), and polyethylene terephthalate (PET), and combinations thereof It may be a material selected from. In addition, the inlet 304 may have various sizes, but preferably may be selected from a diameter of 0.001 mm to 10 mm. In addition, the outlet portion 305 may have a variety of sizes, but preferably may be selected from 0.001 mm to 10 mm in diameter. In addition, the inlet 304 and the through-opening outlet 305 are provided with separate cover means (not shown) to target the PCR when the target sample solution proceeds in the one or more reaction channels 303. The sample solution can be prevented from leaking. The cover means may be implemented in various shapes, sizes or materials. In addition, the thickness of the third plate 330 may vary, but preferably may be selected from 0.001 mm to 10 mm. In addition, the inlet 304 and the outlet 305 may be present in two or more.
The PCR chip 300 to form the inlet 304 and the outlet 305 through mechanical processing to provide the third plate (330); The plate having a size corresponding to the bottom surface of the third plate 330 from the portion corresponding to the inlet portion 304 of the third plate 330 to the outlet portion 305 of the third plate 330 Forming one or more reaction channels 303 through mechanical processing to a corresponding portion to provide a second plate 320; Providing a first plate 310 by forming a surface made of a hydrophilic material (not shown) through a surface treatment on an upper surface of a plate having a size corresponding to a lower surface of the second plate 320. ; And bonding a lower surface of the third plate 330 to an upper surface of the second plate 320 through a bonding process, and attaching a lower surface of the second plate 320 to an upper portion of the first plate 310. It can be easily produced by a method comprising the step of bonding to the surface through a bonding process.
Inlet 304 and outlet 305 of the third plate 330, and one or more reaction channels 303 of the second plate 320 are injection molded, hot-embossing, casting It can be formed by a processing method selected from the group consisting of casting, and laser ablation. In addition, the hydrophilic material on the surface of the first plate 310 may be treated by a method selected from the group consisting of oxygen and argon plasma treatment, corona discharge treatment, and surfactant application and performed according to methods known in the art. Can be. In addition, the lower surface of the third plate 330 and the upper surface of the second plate 320, and the lower surface of the second plate 320 and the upper surface of the first plate 310 are 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 or a thermoplastic or thermosetting resin (not shown) may be processed between the third plate 330 and the second plate 320 and between the second plate 320 and the first plate 310. .
4 shows a PCR device 1 according to an embodiment of the present invention comprising a PCR chip, a power supply and a pump.
According to FIG. 4, a PCR device according to an embodiment of the present invention includes a PCR chip, a power supply unit 400, and a pump 500. Specifically, the PCR chip is placed in contact on the heat block. The PCR chip and the components included therein are as described above.
The power supply unit 400 is a module for supplying power to the electrode unit 200, and may be connected to the first electrode 210 and the second electrode 220 of the electrode unit 200, respectively. For example, when the PCR chip is placed in contact on the thermal block to perform PCR, a first power port (not shown) of the power supply 400 is electrically connected to the first electrode 210. The second power port (not shown) of the power supply unit 400 is electrically connected to the second electrode 220. Subsequently, when there is a user instruction to perform PCR, the power supply unit 400 supplies power to the first electrode 210 and the second electrode 220, respectively, so that the first heater 110 and The second heater 120 may be quickly heated, and when the heaters 110 and 120 reach a predetermined temperature, the amount of power is controlled to maintain the predetermined temperature. For example, the predetermined temperature may be a PCR denaturation step temperature (85 ° C. to 105 ° C., preferably 95 ° C.) in the first heater 110 and a PCR annealing / extension step temperature (in the second heater 120). 50 ° C. to 80 ° C., preferably 72 ° C.), or in the first heater 110, a PCR annealing / extension step temperature (50 ° C. to 80 ° C., preferably 72 ° C.) and the second heater 120 In the PCR denaturation step temperature (85 ℃ to 105 ℃, preferably 95 ℃).
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 303 of the PCR chip, may be a positive pressure pump or a negative pressure pump, for example a syringe (syringe) It may be a pump. The pump 500 may be operably disposed in a portion of the reaction channel 303, but preferably the inlet 304 and / or outlet 305 formed at both ends of the reaction channel 303. Is placed connected to). When the pump 500 is connected to the inlet 304 and / or outlet 305, it serves as a pump as well as a target sample through the inlet 304 and / or outlet 305. It can also act as a stopper to prevent leakage of the solution. In addition, when it is desired to control the flow rate and the flow rate of the fluid flowing in the reaction channel 303, that is, the target sample solution in one direction, the pump 500 may include one of the inlet 304 and the outlet 305. It can be connected to only one, and a general stopper can be sealedly connected to the remaining one, the pump (if you want to control the flow rate and flow rate of the fluid flowing in the reaction channel 303, ie the target sample solution in both directions) 500 may be connected to both the inlet 304 and the outlet 305.
The nucleic acid amplification reaction of the target sample in the PCR device including the PCR chip, the power supply 400, and the pump 500 may be performed through the following steps.
1.Double-stranded target DNA, oligonucleotide primer having a sequence complementary to the specific nucleotide sequence to be amplified, DNA polymerase, deoxyribonucleotide triphosphates (dNTP), PCR reaction buffer Prepare a target sample solution containing.
2. Introduce the target sample solution into the PCR chip. In this case, the target sample solution is disposed in the reaction channel 303 inside the PCR chip 300 through the inlet 304.
3. The electrode part 200, specifically, the first electrode 210 and the second electrode 220 are connected to the power supply 400, respectively, the inlet 304 of the PCR chip 300 And the outlet 305 is sealingly connected to the pump 500.
4. The power supply unit 400 is instructed to supply power to heat the first heater 110 and the second heater 120 through the first electrode 210 and the second electrode 220, and A specific temperature, for example, the PCR denaturation step temperature (95 ° C.) for the first heater 110 and the PCR annealing / extension step temperature (72 ° C.) for the second heater 120 are maintained.
5. When the positive pressure is provided by the pump 500 connected with the inlet 304 or the negative pressure is provided by the pump 500 connected with the outlet 305, the target sample solution is transferred to the reaction channel 303. Allow it to flow horizontally from the inside. In this case, the flow rate and flow rate of the target sample solution may be controlled by adjusting the intensity of the positive pressure or the negative pressure provided by the pump 500.
By performing the above steps, the target sample solution is transferred from the end of the inlet 304 of the reaction channel 303 to the end of the outlet 305 of the upper corresponding portion 301 and the second of the first heater 110. PCR is performed while moving the upper corresponding portion 302 of the heater 120 in the longitudinal direction. According to FIG. 2, the target sample 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 at the upper corresponding part 301 of 110 and a PCR annealing / extending step at the upper corresponding part 302 of the second heater 120. Subsequently, optionally, the target sample solution comprises an upper corresponding portion 301 of the first heater 110 and the second heater from the outlet 305 end of the reaction channel 303 to the end of the inlet 304. PCR may be performed again while the upper corresponding portion 302 of the 120 is moved backward in the longitudinal direction.
5 is a PCR device according to an embodiment of the present invention including a PCR chip made of a light transmissive material and a heat block including a light source disposed between a first heater and a second heater, a power supply unit, a pump, and a light detector. Illustrated.
According to FIG. 5, a PCR device according to an embodiment of the present invention includes a PCR chip 300 made of a light transmissive material and a light source 150 disposed between the first heater 110 and the second heater 120. The thermal block 100, the power supply unit 400, the pump 500, and the light detector 600. The power supply and the pump are as described above.
The PCR chip 300 according to FIG. 5 is made of a light transmissive material, and the light source 150 is disposed between the first heater 110 and the second heater 120 of the thermal block 100. . In addition, the PCR apparatus further includes a light detector 600 for detecting the light emitted from the light source 150. Therefore, the PCR device according to the embodiment of the present invention according to FIG. 5 may measure and analyze the nucleic acid amplification process in real-time when performing PCR. In this case, a separate fluorescent substance may be further added to the target sample solution, which induces a measurable and analytical light signal by emitting light with a specific wavelength according to the generation of the PCR product. The light source 150 may be disposed in a spaced space between the first heater 110 and the second heater 120 and may emit the same light. The light source 150 may be operably connected to a lens (not shown) that collects light emitted from the light source 150 and an optical filter (not shown) that filters light of a specific wavelength band.
According to FIG. 5, the nucleic acid amplification reaction may be checked by a PCR device according to an embodiment of the present invention in real-time. For example, the target sample solution may be PCR while successively passing 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 303. A denaturation step and PCR annealing / extension step are performed, in which case the target sample solution is between the first heater 110 and the second heater 120, and the first heater 110 and the second heater 120. Between the heater unit including a) passes through the upper corresponding portion of the light source 150. When the target sample solution passes through the upper corresponding portion of the light source 150, the fluid control slows or briefly stops the flow rate of the target sample solution and then emits light from the light source 150, and emits the light. The light is passed through the optically transparent PCR chip 300, specifically, the reaction channel 330, and the optical detector 600 measures and analyzes an optical signal generated by nucleic acid amplification in the reaction channel 330. can do. Accordingly, the amount of target nucleic acid is monitored in real-time by monitoring the result of the reaction by amplification of nucleic acid (with fluorescent substance bound) in the reaction channel 303 during each cycle of PCR. -time) can be measured and analyzed.
FIG. 6 illustrates a PCR device according to an embodiment of the present invention including a heat block made of a light transmissive material and a PCR chip, a power supply unit, a pump, a light supply unit, and a light detector.
According to FIG. 6, a PCR device according to an embodiment of the present invention includes a heat block 100 and a PCR chip 300, a power supply unit 400, a pump 500, a light supply unit 700, and a light transmitting material. And a light detector 800. The power supply 400 and the pump 500 are as described above.
The PCR device according to FIG. 6 is characterized in that the thermal block 100 and the PCR chip 300 are implemented with a light transmissive material. In addition, the PCR apparatus further includes a light providing unit 700 arranged to provide light to the PCR chip 300 and a light detecting unit 800 arranged to receive light emitted from the PCR chip 300. . Therefore, the PCR device according to the embodiment of the present invention according to FIG. 5 may measure and analyze the nucleic acid amplification process in real-time when performing PCR. In this case, a separate fluorescent substance may be further added to the target sample solution, which induces a measurable and analytical light signal by emitting light with a specific wavelength according to the generation of the PCR product.
In real-time PCR, it is necessary to increase the sensing efficiency of the optical signal as much as possible in order to accurately monitor the PCR product in real-time. Since the thermal block 100 of the light transmissive material has a light transmittance as a whole, the excitation light derived from the light source may be transmitted as it is, thereby increasing the sensing efficiency of the optical signal. However, some of the excitation light may be reflected on the heat block 100 of the light transmissive material or reflected after passing through the heat block 100 of the light transmissive material to act as noise of the optical signal. Therefore, the light absorbing material may be formed on the lower surface of the heat block 100 of the light transmissive material to form a light absorbing layer (not shown), thereby further increasing the sensing efficiency. The light absorbing material may be, for example, mica, but is not limited to a material having a property of absorbing light. Therefore, the light absorbing layer absorbs a part of the light derived from the light source, and the generation of reflected light acting as noise of the optical signal can be suppressed as much as possible. Alternatively, the light reflection prevention layer (not shown) may be formed on the upper surface of the heat block 100 made of the light transmissive material to further increase the sensing efficiency. The anti-reflective material may be, for example, a fluoride such as MgF 2, an oxide such as SiO 2 or Al 2 O 3, but is not limited as long as the material has a property of preventing light reflection. Further, more preferably, the light absorbing material is processed on the lower surface of the heat block 100 made of the light transmissive material, and at the same time, the light reflection preventing material is treated on the upper face of the heat block 100 made of the light transmissive material for sensing. The efficiency can be further increased. That is, in order to effectively monitor real-time PCR, the ratio of the optical signal to the noise should have the maximum possible value, and the ratio of the optical signal to the noise may be improved as the reflectance of the excitation light from the PCR chip is lower. For example, the reflectance of the excitation light of the conventional heaters of a general metallic material is about 20% to 80%, but the light reflectance is 0.2 when using the light transmitting material thermal block 100 including the light absorbing layer or the light reflecting layer. The light reflectance can be reduced to within 4% and less than 0.2%.
The light providing unit 700 is a module for providing light to the PCR chip 300 of the light transmissive material, the light detector 800 receives the light emitted from the PCR chip 300 of the light transmissive material. By the module for measuring the PCR progress performed in the light transmitting material PCR chip 300. Light is emitted from the light providing unit 700, and the emitted light passes through or reflects the PCR chip 300 of the light transmissive material, specifically, the reaction channel 330, and in this case, the reaction channel 330. The optical detection unit 800 measures and analyzes an optical signal generated by nucleic acid amplification in the C). Therefore, according to the PCR device according to FIG. 6, the amplification of the nucleic acid (phosphorescent material bound) in the reaction channel 330 during each cycle of the PCR in the light transmitting material PCR chip 1 By monitoring the result of the reaction in real-time (real-time), it is possible to measure and analyze in real-time whether the target nucleic acid amplification and the degree of amplification. In addition, the light providing unit 700 and the light detecting unit 800 may be disposed above or below the heat block 100 of the light transmissive material, or may be disposed respectively. However, the arrangement of the light providing unit 700 and the light detecting unit 800 may be varied in consideration of the arrangement relationship with other modules for the optimal implementation of the PCR apparatus. Study 700 and light detector 800 may be disposed on the heat block 100 of the light transmissive material.
The light providing unit 700 may include a light emitting diode (LED) light source or a laser light source, a first light filter for selecting light having a predetermined wavelength from light emitted from the light source, and light emitted from the first light filter. And a first optical lens configured to collect light, and further comprising a first aspherical lens disposed to spread light between the light source and the first light filter. The light source includes all light sources capable of emitting light, and according to an embodiment of the present invention, includes a light emitting diode (LED) light source or a laser light source. The first light filter selects and emits light having a specific wavelength among incident light having various wavelength bands, and may be variously selected according to the predetermined light source. For example, the first light filter may pass only light in a wavelength band of 500 nm or less of the light emitted from the light source. The first optical lens collects the incident light and increases the intensity of the emitted light, and the intensity of the light irradiated onto the PCR chip 300 of the light transmissive material through the heat block 100 of the light transmissive material. Can be increased. In addition, the light providing unit further includes a first aspherical lens disposed to spread light between the light source and the first light filter. By adjusting the arrangement direction of the first aspherical lens, the light range emitted from the light source is extended to reach the measurable area.
The light detector 800 selects a second optical lens for collecting light emitted from the PCR chip 1 of the light transmissive material and a second light for selecting a light having a predetermined wavelength from the light emitted from the second optical lens. A light filter, and an optical analyzer that detects an optical signal from light emitted from the second light filter, and disposed between the second light filter and the light analyzer to integrate light emitted from the second light filter. And a second aspherical lens, disposed between the second aspherical lens and the optical analyzer to remove noise of light emitted from the second aspherical lens and to amplify the light emitted from the second aspherical lens. It may further include a photodiode integrated circuit (PDIC) (not shown). The second optical lens collects the incident light and increases the intensity of the emitted light, and the second optical lens collects the light emitted from the PCR chip 300 of the light transmissive material through the heat block 100 of the light transmissive material. The intensity is increased to facilitate optical signal detection. The second light filter selects and emits light having a specific wavelength among incident light having various wavelength bands, and is predetermined from the PCR chip 300 of the light transmissive material through the heat block 100 of the light transmissive material. It may be variously selected depending on the wavelength of light. For example, the second light filter may pass only light in a wavelength range of 500 nm or less among predetermined light emitted from the PCR chip 1 of the light transmissive material through the heat block 100 of the light transmissive material. . The optical analyzer is a module for detecting an optical signal from the light emitted from the second optical filter, and converts the fluorescent light expressed from the target sample solution into an electrical signal to enable qualitative and quantitative measurement. In addition, the light detector 800 may further include a second aspherical lens disposed between the second light filter and the light analyzer to integrate light emitted from the second light filter. By adjusting the arrangement direction of the second aspherical lens, the light range emitted from the second light filter is extended to reach the measurable area. In addition, the light detector 800 is disposed between the second aspherical lens and the optical analyzer to remove noise of light emitted from the second aspherical lens and to amplify the light emitted from the second aspherical lens. It may further include a photodiode integrated circuit (PDIC). By using the photodiode integrated device, the PCR device can be further miniaturized, and noise can be minimized to measure a reliable optical signal.
The PCR apparatus may adjust one or more dichroic filters 750a to adjust a light propagation direction so that the light emitted from the light providing unit 700 reaches the light detecting unit 800 and separate light having a predetermined wavelength. 750b). The dichroic filters 750a and 750b are modules that reflect light at an angle selectively transmitted or selectively adjusted according to the wavelength. According to FIG. 6, the dichroic filter 750a is disposed to be inclined at an angle of about 45 degrees with respect to the optical axis of the light emitted from the light providing unit 700, and selectively transmits the light according to its wavelength and transmits the short wavelength component. Is reflected at right angles to reach the PCR chip 300 of the light transmissive material disposed on the heat block 100 of the light transmissive material. In addition, the dichroic filter 750b is disposed to be inclined at an angle of about 45 degrees with respect to the optical axis of the light reflected from the PCR chip 300 and the heat block 100 of the light transmissive material, and selectively shifts the light according to its wavelength. The short wavelength component is transmitted and the long wavelength component is reflected at right angles to reach the light detector 800. The light reaching the light detector 800 is converted into an electrical signal in the optical analyzer to indicate whether the nucleic acid is amplified and the degree of amplification.
According to Figure 6, it can be confirmed that the nucleic acid amplification reaction is measured in real time (real-time) by the PCR device according to an embodiment of the present invention. For example, the target sample solution may be PCR while successively passing 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 303. A denaturation step and a PCR annealing / extension step are performed. In this case, when the target sample solution passes through any position in the reaction channel 303, the reaction from the light provider 700 after the fluid control slows down or maintains the flow rate of the target sample solution for a while. The light detector 800 may receive light at an arbitrary position in the channel 303, and the light detector 800 may receive light emitted by an amplification reaction of a target nucleic acid in the reaction channel 303 to measure and analyze an optical signal. have. According to FIG. 6, although the light providing unit 700 and the light detecting unit 800 are disposed in pairs, in order to measure and analyze a plurality of optical signals generated at arbitrary positions in the reaction channel 303. It can be arranged in pairs at appropriate locations. Accordingly, the amount of target nucleic acid is monitored in real-time by monitoring the result of the reaction by amplification of nucleic acid (with fluorescent substance bound) in the reaction channel 303 during each cycle of PCR. -time) can be measured and analyzed.
