WO2022045811A1 - Photonic pcr system using surface plasmonic phenomenon of conductive polymer, and real-time detection of target nucleic acids using same - Google Patents

Abstract

The present invention relates to a method which manufactures, by means of a roll-to-roll process, a nucleic acid detection device comprising a photonic PCR well based on surface plasmonic of a conductive polymer and detects target nucleic acids in real-time by using the manufactured device, and to a system therefor. When using the present invention, it is possible to detect target nucleic acids (DNA, RNA) present in a sample in real-time, and the nucleic acid detection device of the present invention can be mass-produced at low cost within a short amount of time through the roll-to-roll process.

Description

Photo PCR system using surface plasmonic phenomenon of conductive polymer and real-time detection of target nucleic acid using the same

The present invention relates to a multi-purpose optical PCR system for producing an optical PCR plate using the surface plasmonic phenomenon of a conductive polymer through control of the ultrafine structure of a conductive polymer, which can be easily manufactured using a roll-to-roll device, and for real-time detection of a target nucleic acid.

This application claims priority based on Korean Patent Application No. 10-2020-0109881 filed on August 31, 2020, and all contents disclosed in the specification and drawings of the application are incorporated herein by reference.

Conductive polymers can exhibit properties of non-conductors, semiconductors, and conductors depending on the doping state, and can easily prepare dispersions. Therefore, it has the advantage of being able to print a variety of patterns ranging from microstructures to microstructures desired by printing, while at the same time realizing metallic properties. It has the advantage of inducing light-to-thermal conversion by implementing a surface plasmonic phenomenon.

On the other hand, polymerase chain reaction (PCR) is a molecular biology technique for enzymatically replicating DNA without using living organisms. PCR is routinely used in medical and biological laboratories for various tasks, for example, detection of genetic diseases, identification of genetic fingerprints, diagnosis of infectious diseases, gene cloning, paternity testing, and DNA computing. there is.

PCR is recognized by molecular biologists as the method of choice for nucleic acid detection due to its excellent amplification and precision capability. Since DNA detection after PCR is typically performed at the end-point or plateau phase of a PCR reaction, it is difficult to quantify the starting template.

Real-time PCR or dynamic PCR advances the capabilities of endpoint PCR assays by recording the amplicon concentration as the reaction progresses. Amplicon concentration is frequently recorded via a fluorescent signal change associated with the amplified target. In addition, real-time PCR is advantageous over end-point detection PCR in that contamination is limited because DNA detection can be performed in a closed system.

The present inventors manufactured a conductive polymer-based optical PCR device that induces a nucleic acid amplification reaction using photo-thermal conversion by the surface plasmon phenomenon of the conductive polymer as a roll-to-roll device, while simultaneously manufacturing optical PCR The present invention was completed by developing a multi-purpose optical PCR system capable of sequentially and real-time detection of target nucleic acids using the device.

Accordingly, an object of the present invention is to provide an optical PCR system for manufacturing a conductive polymer-based optical PCR plate using a roll-to-roll device and for real-time detection of a target nucleic acid.

Another object of the present invention is to provide a method for detecting a target nucleic acid in real time using the prepared optical PCR plate while manufacturing a conductive polymer-based optical PCR plate with a roll-to-roll device.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned tasks, and other tasks not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. will be.

In order to achieve the object of the present invention, the present invention provides a conductive polymer-based light having a plurality of reaction spaces in which a nucleic acid amplification reaction occurs by light-to-thermal conversion by the surface plasmon phenomenon of a conductive polymer having a microstructure according to light irradiation. A roll-to-roll device for preparing a PCR (Photonic Polymerase Chain Reaction) plate; a light source for irradiating light for inducing a nucleic acid amplification reaction to a light-to-thermal conversion element using the conductive polymer microstructure manufactured in the roll-to-roll device and a conductive polymer-based optical PCR plate using the same; and a photodetector for detecting the nucleic acid amplification reaction in the reaction space in real time, wherein the conductive polymer-based optical PCR plate prepared in the roll-to-roll device is transferred from the roll-to-roll device to the light source, and from the light source in the transport process Light is irradiated into the reaction space, and the roll-to-roll device forms a reaction space including a plasmonic thin film for plasmonic photothermal light-to-heat conversion of a conductive polymer into a microwell. an imprinting unit for imprinting on a substrate in an nxm array in the form; And it provides an optical PCR system for producing a conductive polymer-based optical PCR plate using a roll-to-roll device and real-time detection of a target nucleic acid using a roll-to-roll device, characterized in that it comprises a laminating unit for laminating an upper portion of the reaction space with a film.

In addition, the present invention uses a roll-to-roll device for (a) a conductive polymer-based optical PCR plate having a plurality of reaction spaces in which a nucleic acid amplification reaction occurs by photo-thermal conversion due to the surface plasmon phenomenon of the conductive polymer according to light irradiation. to manufacture; (b) transferring the prepared conductive polymer-based optical PCR plate to a light source and irradiating light into the reaction space; and (c) detecting the nucleic acid amplification reaction in the reaction space in real time with a photodetector, wherein the manufacturing of the conductive polymer-based photoPCR plate using a roll-to-roll device comprises: (a-1) plasmonic Imprinting a reaction space including a plasmonic conductive polymer thin film for plasmonic photothermal light-to-heat conversion on a substrate in an nxm array in the form of microwells; (a-2) injecting a biological sample and a reagent for amplification of a target nucleic acid in the biological sample into the reaction space; And (a-3) while preparing a conductive polymer-based optical PCR plate with a roll-to-roll device, characterized in that it comprises the step of laminating the upper portion of the reaction space with a film, using the prepared optical PCR plate A method for detecting nucleic acids in real time is provided.

In one embodiment of the present invention, the substrate is PVDF (Polyvinylidene fluoride), PET (Polyethylene terephthalate), PEN (Polyethylene naphthalate), PMMA (Poly (methyl methacrylate)), PE (Polyethylene), PP (Polypropylene), PC (Polycarbonate), PI (Polyimide), PES (Polyethersulfone), polyester (Polyester), PS (Polystyrene), PDMS (Polydimethylsiloxane), PEO (Polyethylene oxide), and may be selected from the group consisting of a combination thereof.

In another embodiment of the present invention, the conductive polymer may be selected from the group consisting of PEDOT-PSS (Polyethylenedioxythiophene-Polystyrene sulfonic acid), PPY (Polypyrrole), PAN (Polyaniline), PA (Polyacetylene), and combinations thereof. .

In another embodiment of the present invention, the substrate may be made of a translucent or transparent material.

In another embodiment of the present invention, the light source includes LEDs, diode lasers, diode laser arrays, quantum well (vertical)-cavity lasers, and It may be selected from the group consisting of combinations thereof.

In another embodiment of the present invention, the photo detector may include a photodiode or a spectrophotometer.

In another embodiment of the present invention, the photodiode or spectrophotometer may be configured to detect light emitted from a fluorescent label oligonucleotide probe hybridized to a nucleic acid amplicon.

In another embodiment of the present invention, the photodiode or spectrophotometer may be configured to detect light emitted from a fluorescent label primer extension product hybridized to a nucleic acid amplicon.

In another embodiment of the present invention, a primer having a nucleotide sequence complementary to a target nucleic acid in a sample, a fluorescent die, a dNTP molecule, and a polymerase for the nucleic acid amplification reaction may be placed in the reaction space.

In another embodiment of the present invention, the plasmonic thin film is a conductive polymer (CP), QD (quantum dot), QP (quantum particle), CQ (carbon quantum dot), GQ (graphene quantum dot), GNP (gold) nanoparticle), SNP (silica nanoparticle), MNP (magnetic nanoparticle), ANP (silver nanoparticle), SiO ₂ , metal nanoparticles, graphene nanoparticles, and combinations thereof may be a nanoparticle thin film.

In another embodiment of the present invention, the conductive polymer-based optical PCR plate produced in the roll-to-roll device is transferred in the direction of the light source, and as light is irradiated from the light source to the reaction space in the process of being transferred, nucleic acid amplification in the reaction space The reaction is induced, and after induction and detection of the nucleic acid amplification reaction in the reaction space of the nth column of the optical PCR plate is completed, the induction and detection of the nucleic acid amplification reaction in the reaction space of the n+1st column can be sequentially performed.

The present invention relates to a method and a system for real-time detection of a target nucleic acid using a roll-to-roll process of manufacturing a nucleic acid detection device having a surface plasmonic-based optical PCR well implemented in a conductive polymer, and using the manufactured device, By using the present invention, target nucleic acids (DNA, RNA) present in a sample can be detected in real time, and the nucleic acid detection device of the present invention can be mass-produced at low cost within a short time through a roll-to-roll process.

1 is a diagram schematically illustrating components of an optical PCR system for manufacturing a conductive polymer-based optical PCR plate using a roll-to-roll device and for real-time detection of a target nucleic acid, according to an embodiment of the present invention.

Figure 2 shows a process in which the detection of nucleic acid amplification products is sequentially performed by the optical PCR system according to an embodiment of the present invention, the upper part shows a conductive polymer-based optical PCR plate, and the green mark is nucleic acid amplification by the optical detector It indicates that the detection of the product has been completed, and the red mark indicates that the detection process has not yet been performed.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. Embodiments of the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. This example is provided to more completely explain the present invention to those of ordinary skill in the art. Accordingly, the shapes of elements in the drawings are exaggerated in some cases to emphasize a clearer description. In addition, the terms or words used in the present specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventor appropriately defines the concept of the term in order to best describe his invention. Based on the principle that it can be done, it should be interpreted as meaning and concept consistent with the technical idea of the present invention.

When describing with reference to the drawings, the same or corresponding components are given the same reference numerals, and overlapping descriptions thereof will be omitted.

1 is a diagram schematically illustrating components of an optical PCR system for manufacturing a conductive polymer-based optical PCR plate using a roll-to-roll device and for real-time detection of a target nucleic acid, according to an embodiment of the present invention.

1, the conductive polymer-based optical PCR system 100 according to an embodiment of the present invention is a plurality of reaction spaces (S) in which a nucleic acid amplification reaction occurs by light-to-thermal conversion by a surface plasmon phenomenon according to light irradiation. ) A light source irradiating light for inducing a nucleic acid amplification reaction to a roll-to-roll device for manufacturing a photonic polymerase chain reaction (PCR) plate 10, and a conductive polymer-based optical PCR plate 10 manufactured in a roll-to-roll device (20) and a photodetector 30 for detecting the nucleic acid amplification reaction in the reaction space (S) in real time.

The roll-to-roll device forms a reaction space (S) including a conductive polymer-based plasmonic thin film for plasmonic photothermal light-to-heat conversion in an nxm array in the form of microwells. It includes an imprinting unit 11 for imprinting on the surface and a laminating unit 12 for laminating an upper portion of the reaction space S with a film.

The roll-to-roll device is a device for performing various types of processes on a roll-type film or web, and an unwinder for unwinding a film wound in a roll shape, and printing on a film It includes process units for performing various processes, such as a process, and a rewinder for rewinding the film in a roll form, and may include various transfer units for transferring the film therebetween.

The material of the substrate of the photo-PCR plate 10 is not particularly limited, and materials (eg, plastic, glass, etc.) commonly used for manufacturing devices for detecting biomolecules such as biosensors, biochips, and microfluidic chips are limited. can be used without The substrate may be made of a translucent or transparent material (eg, polymethyl methacrylate (PMMA)) so that light irradiated from the outside can reach the plasmonic thin film in the reaction space S.

For example, the optical PCR plate 10 of the present invention is PVDF (Polyvinylidene fluoride), PET (Polyethylene terephthalate), PEN (Polyethylene naphthalate), PMMA (Poly (methyl methacrylate)), PE (Polyethylene), PP (Polypropylene) , PC (Polycarbonate), PI (Polyimide), PES (Polyethersulfone), polyester (Polyester), PS (Polystyrene), PDMS (Polydimethylsiloxane), PEO (Polyethylene oxide), or a combination thereof can be manufactured using as a material for the substrate. there is.

In addition, the conductive polymer microstructure that generates the plasmonic light-to-thermal conversion is prepared using PEDOT-PSS (Polyethylenedioxythiophene-Polystyrene sulfonic acid), PPY (Polypyrrole), PAN (Polyaniline), PA (Polyacetylene), or a combination thereof. can be

The reaction space S is a plasmonic well in the form of a microwell having a plasmonic thin film for plasmonic light-to-thermal light-to-thermal conversion. This is the space where the polymerase chain reaction (nucleic acid amplification reaction) takes place. The nucleic acid sample placed in the reaction space (S) generates heat through a plasmonic phenomenon by light irradiated to the plasmonic well, and 91°C (heating) and 60°C (cooling) are performed within a short time (<3 minutes). By performing 30 cycles, nucleic acid amplification can be achieved through photo-PCR.

When light is irradiated, heat is generated by light-to-heat conversion of polaron and dipolaron of the conductive polymer, heating and cooling the nucleic acid and PCR sample solution located in the reaction space (S) As this rapid cycle occurs, the nucleic acid amplification reaction occurs rapidly. In this case, temperature control for nucleic acid amplification may be performed by controlling light irradiation by the light source 20 .

For the nucleic acid amplification reaction, a sample solution for PCR such as a primer, a probe, a fluorescent die, a dNTP molecule, and a polymerase having a base sequence complementary to a target nucleic acid in the sample may be placed in the reaction space (S).

In the imprinting unit 11 , a reaction space S having a plasmonic thin film is formed on the substrate of the optical PCR plate 10 . The plasmonic thin film is a conductive polymer (CP), QD (quantum dot), QP (quantum particle), CQ (carbon quantum dot), GQ (graphene quantum dot), GNP (gold nanoparticle), SNP (silica nanoparticle), MNP (magnetic nanoparticles), ANP (silver nanoparticles), SiO ₂ , metal nanoparticles, graphene nanoparticles, or a combination thereof may be used. For example, metal microwells can be printed on a substrate using conductive polymers, gold or silver nanoparticles-based conductive inks using R2R gravure or offset. In this case, microwells of conductive polymer, gold or silver can be printed from several hundred nanometers to several microns in diameter and to several microns in depth.

A conductive polymer-based optical PCR plate 10 is manufactured by laminating the substrate on which the reaction space S is formed by the imprinting unit 11 with a film on the upper part of the reaction space, and the manufactured optical PCR plate 10 is a light source (20) is transferred in the direction. For subsequent nucleic acid amplification and detection of the target nucleic acid, a biological sample and a reagent for amplification of the target nucleic acid in the biological sample may be injected into the reaction space S before the lamination process.

The light source 20 may be implemented as an LED, diode lasers, diode laser array, quantum well (vertical)-cavity laser, and the like. In addition, the emission wavelength of the light source 20 may be ultraviolet (UV) light, visible light or infrared (IR) light.

The conductive polymer-based optical PCR plate 10 manufactured in the roll-to-roll device is transferred in the direction of the light source 20 in the roll-to-roll device, and as the optical PCR plate 10 passes the light source 20, the reaction space (S) ), and the nucleic acid amplification reaction occurs according to the light-to-heat conversion by the surface plasmon phenomenon by the irradiated light. This nucleic acid amplification reaction is called photo PCR or photonic polymerase chain reaction (PCR).

The photodetector 30 detects a nucleic acid amplification reaction in the reaction space S in real time. The photodetector 30 may detect the intensity of the emitted light based on the intensity of the light emitted from the reaction space S, and convert it into an electronic signal. The photodetector 30 may be implemented as a digital camera, a photodiode, a spectrophotometer, or a similar imaging device that detects a fluorescence signal in real time.

In one specific example, the photodiode or spectrophotometer may be configured to detect light emitted from a fluorescently labeled oligonucleotide probe hybridized to a nucleic acid amplicon. In another specific example, the photodiode or spectrophotometer may be configured to detect light emitted from a fluorescent label primer extension product hybridized to a nucleic acid amplicon.

2 shows a process in which nucleic acid amplification products are sequentially detected by the conductive polymer-based optical PCR system according to an embodiment of the present invention, the upper part shows the optical PCR plate, and the green mark is the nucleic acid amplification by the optical detector It indicates that the detection of the product has been completed, and the red mark indicates that the detection process has not yet been performed.

Referring to Figure 2, as the optical PCR system 100 according to an embodiment of the present invention is driven, the reaction space (S) of the nth column of the conductive polymer-based optical PCR plate 10 manufactured in the roll-to-roll device After the induction and detection of the nucleic acid amplification reaction in , the induction and detection of the nucleic acid amplification reaction in the reaction space of the n+1th column are sequentially performed.

The optical PCR system according to an embodiment of the present invention may further include a temperature sensor for monitoring the temperature of the PCR sample solution for nucleic acid amplification. The temperature sensor may be coupled to or face the platform for measuring the temperature of the sample. Such temperature sensors may include multiple sensor types, such as thermocouples or cameras (eg, IR cameras) facing the platform.

The sensor and light source may be coupled to a computing unit for obtaining sensor data and controlling the light source. In general, a computing device obtains data from a sensor, including a processor and a memory stored in application software executable by the processor to drive the light source (eg, to control LED timing, intensity/injection current, etc.) and/or process data from the diagnostic device, such as digital camera real-time detection of fluorescent signals of nucleic acids and/or sample solutions. The computing device may include a separate computer or device, or may be integrated into a microcontroller module having the remaining components.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

[Explanation of code]

10: light PCR plate

11: Imprinting unit

12: laminating unit

20: light source

30: photo detector

S: reaction space

100: conductive polymer-based optical PCR system

A nucleic acid detection device having a surface plasmonic-based optical PCR well of a conductive polymer of the present invention is manufactured using a roll-to-roll process, and the manufactured device and an optical PCR system using the same are target nucleic acids (DNA, RNA) present in the sample. It is expected to be usefully used to detect in real time.

Claims (12)

1. Roll-to-roll manufacturing a conductive polymer-based photonic polymerase chain reaction (PCR) plate having a plurality of reaction spaces in which nucleic acid amplification reaction occurs by light-to-thermal conversion by the surface plasmon phenomenon of the conductive polymer according to light irradiation to-roll) device;

   a light source irradiating light for inducing a nucleic acid amplification reaction to the conductive polymer-based optical PCR plate manufactured in the roll-to-roll device; and

   A photodetector for detecting the nucleic acid amplification reaction in the reaction space in real time,

   The conductive polymer-based optical PCR plate manufactured in the roll-to-roll device is transferred from the roll-to-roll device toward the light source, and light is irradiated from the light source to the reaction space during the transport process,

   In the roll-to-roll device, a reaction space including a plasmonic thin film for plasmonic photothermal light-to-heat conversion of a conductive polymer is placed on a substrate in an nxm arrangement in the form of a microwell. Imprinting unit for printing; and

   An optical PCR system for manufacturing a conductive polymer-based optical PCR plate using a roll-to-roll device and real-time detection of a target nucleic acid, characterized in that it comprises a laminating unit for laminating an upper portion of the reaction space with a film.
2. The method of claim 1,

   The substrate is PVDF (Polyvinylidene fluoride), PET (Polyethylene terephthalate), PEN (Polyethylene naphthalate), PMMA (Poly (methyl methacrylate)), PE (Polyethylene), PP (Polypropylene), PC (Polycarbonate), PI (Polyimide), PES (Polyethersulfone), polyester (Polyester), PS (Polystyrene), PDMS (Polydimethylsiloxane), PEO (Polyethylene oxide), characterized in that selected from the group consisting of, and combinations thereof, optical PCR system.
3. According to claim 1,

   The optical PCR system, characterized in that the substrate is translucent or transparent.
4. According to claim 1,

   The conductive polymer is PEDOT-PSS (Polyethylenedioxythiophene-Polystyrene sulfonic acid), PPY (Polypyrrole), PAN (Polyaniline), PA (Polyacetylene), characterized in that selected from the group consisting of, and combinations thereof, an optical PCR system.
5. According to claim 1,

   The light source is selected from the group consisting of LEDs, diode lasers, diode laser arrays, quantum well (vertical)-cavity lasers, and combinations thereof. , an optical PCR system.
6. According to claim 1,

   An optical PCR system, characterized in that the photodetector comprises a photodiode or a spectrophotometer.
7. 7. The method of claim 6,

   The photodiode or spectrophotometer is characterized in that it is configured to detect light emitted from a fluorescent label oligonucleotide probe hybridized to a nucleic acid amplicon.
8. 7. The method of claim 6,

   wherein the photodiode or spectrophotometer is configured to detect light emitted from a fluorescent label primer extension product hybridized to a nucleic acid amplicon.
9. According to claim 1,

   In the reaction space, a primer, a fluorescent die, a dNTP molecule, and a polymerase having a base sequence complementary to a target nucleic acid in the sample are placed for the nucleic acid amplification reaction, the optical PCR system.
10. According to claim 1,

    The plasmonic thin film is a conductive polymer (CP), QD (quantum dot), QP (quantum particle), CQ (carbon quantum dot), GQ (graphene quantum dot), GNP (gold nanoparticle), SNP (silica nanoparticle), MNP (magnetic nanoparticle), ANP (silver nanoparticle), SiO ₂ , Metal nanoparticles, graphene nanoparticles and nanoparticle thin films made of a combination thereof, characterized in that the optical PCR system.
11. According to claim 1,

    As light is irradiated from the light source to the reaction space while the conductive polymer-based optical PCR plate produced in the roll-to-roll device is transported in the direction of the light source, a nucleic acid amplification reaction is induced in the reaction space, the nth column of the optical PCR plate An optical PCR system, characterized in that induction and detection of a nucleic acid amplification reaction in the reaction space of the n+1th column are sequentially performed after induction and detection of the nucleic acid amplification reaction in the reaction space is completed.
12. (a) manufacturing a conductive polymer-based optical PCR plate having a plurality of reaction spaces in which a nucleic acid amplification reaction occurs by light-to-thermal conversion by a surface plasmon phenomenon of a conductive polymer according to light irradiation using a roll-to-roll device;

    (b) transferring the prepared conductive polymer-based optical PCR plate to a light source and irradiating light into the reaction space; and

    (c) detecting the nucleic acid amplification reaction in the reaction space in real time with a photodetector,

    The step of manufacturing the conductive polymer-based optical PCR plate using a roll-to-roll device,

    (a-1) imprinting a reaction space including a plasmonic conductive polymer thin film for plasmonic photothermal light-to-thermal conversion on a substrate in an n x m array in the form of a fine well;

    (a-2) injecting a biological sample and a reagent for amplification of a target nucleic acid in the biological sample into the reaction space; and

    (a-3) while preparing a conductive polymer-based optical PCR plate with a roll-to-roll device, characterized in that it comprises the step of laminating the upper portion of the reaction space with a film, using the prepared optical PCR plate to perform real-time how to detect it.

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