Real-time quantitative PCR apparatus using photothermal effect

Abstract

The present invention relates to a real-time quantitative PCR device using a photothermal effect, wherein heating is performed using the photothermal effect when performing PCR, thereby reducing the overall apparatus size, and even an unskilled user can conveniently use same. The present invention provides a real-time quantitative PCR apparatus using a photothermal effect, the PCR apparatus comprising a sample injection unit, a sample movement unit, and a reaction chamber. The PCR apparatus comprises a temperature control means capable of heating the inside of the reaction chamber, and the temperature control means comprises a photothermal material pattern.

Description

Real-time quantitative PCR device using photothermal effect

[0001] The present invention relates to a real-time quantitative PCR device utilizing a photothermal effect, and more specifically, to a real-time quantitative PCR device utilizing a photothermal effect that performs heating using the photothermal effect during PCR, thereby reducing the overall size of the device and enabling easy use even by unskilled users.

[0002] PCR (Polymerase Chain Reaction) is a molecular biology technique that amplifies specific DNA fragments millions of times. Developed by Kary Mullis in 1983, it has revolutionized various research and diagnostic fields. PCR amplifies DNA through repeated cycles.

[0003] Among these PCR technologies, real-time quantitative polymerase chain reaction (RT-qPCR) is used to quantify the amount of specific DNA or RNA sequences in real time. This technique boasts extremely high sensitivity and specificity and is widely used in molecular biology, medicine, and life science research.

[0004] RT-qPCR technology, in particular, is a crucial tool in the field of diagnostics. Because it can detect very low concentrations of nucleic acids, it can detect pathogens even in the early stages of infection. Furthermore, it can target highly specific DNA or RNA sequences using specific primers and probes, and the absolute or relative amount of target genes can be quantified in real time via fluorescence. Therefore, it is useful for assessing the extent of infection or analyzing the expression levels of specific genes.

[0005] Due to these advantages, RT-qPCR technology is being utilized in diverse fields beyond diagnostics, including gene expression analysis, drug response studies, food safety and quality control, and environmental monitoring. However, traditional RT-qPCR devices have several limitations due to their complex structure, high price, and the high level of skill required for use.

[0006] RT-qPCR equipment consists of various components, including an optical system requiring high precision, a thermal cycling system capable of rapid heating and cooling, and data analysis software.

[0007] In particular, the thermal cycling system used in the RT-qPCR must incorporate rapid and precise temperature control. Typically, a Peltier element is used for rapid and accurate temperature changes, enabling rapid heating and cooling by controlling the flow of current. Furthermore, the thermal cycling system must maintain a very uniform temperature distribution to ensure that each sample tube is exposed to the same temperature conditions, a requirement essential for ensuring accurate PCR amplification.

[0008] Meanwhile, various studies are being conducted to reduce the size of existing bulky RT-qPCR equipment and increase its portability. Miniaturized devices, in particular, are easier to transport and can be used in a variety of settings. Furthermore, systems are being developed that automate the entire process, from sample preparation and nucleic acid extraction to PCR reaction and data analysis, with the goal of providing consistent results regardless of user skill level.

[0009] In particular, new thermal cycling technologies are being developed that enable rapid PCR cycles while reducing volume. These technologies are not limited to using existing bulky Peltier elements, but are also utilizing photothermal effects and microfluidic technologies.

[0010] In order to solve the above-mentioned problem, the present invention aims to provide a real-time quantitative PCR device utilizing the photothermal effect, which can reduce the overall size of the device by performing heating using the photothermal effect during PCR, and can also be easily used even by unskilled users.

[0011] In order to solve the above-described problem, the present invention provides a real-time quantitative PCR device utilizing a photothermal effect, characterized in that the device includes a sample injection unit, a sample movement unit, and a reaction chamber, and includes a temperature control means capable of heating the inside of the reaction chamber, wherein the temperature control means includes a photothermal material pattern.

[0012] In one embodiment, the photothermal material pattern may be formed on the outer or inner side of the bottom surface of the reaction chamber.

[0013] In one embodiment, the photothermal material pattern may be formed to occupy 20 to 80% of the total area of ​​the bottom surface of the reaction chamber.

[0014] In one embodiment, the photothermal material pattern can be formed in a circular, oval, donut-shaped or polygonal shape.

[0015] In one embodiment, the photothermal material pattern may contain 1 to 10% of the photothermal material.

[0016] In one embodiment, the upper or lower portion of the reaction chamber may include a photothermal effect light source capable of exciting the photothermal material pattern and a fluorescent light source capable of exciting a fluorescent material within the reaction chamber.

[0017] In one embodiment, the apparatus further includes a fluorescence sensor for detecting fluorescence excited by the fluorescence light source, wherein the photothermal effect light source may be installed in the same direction as the fluorescence light source with respect to the reaction chamber, and the fluorescence sensor may be installed in the opposite direction of the fluorescence light source.

[0018] In one embodiment, a temperature-sensitive material may be applied to the upper portion of the reaction chamber.

[0019] In one embodiment, the temperature-sensitive material may be moved to the upper surface of the sample inside the reaction chamber when heated using the photothermal material pattern to spatially isolate the sample.

[0020] In one embodiment, the sample injected into the sample injection unit can be supplied toward the reaction chamber by centrifugal force.

[0021] The real-time quantitative PCR device utilizing the photothermal effect according to the present invention can be easily used by users without expertise through popularization of RT-qPCR technology.

[0022] In addition, the real-time quantitative PCR device utilizing the photothermal effect according to the present invention can minimize the use of expensive parts by simplifying the optical structure and significantly reduce the manufacturing cost of the entire equipment.

[0023] FIG. 1 illustrates the optical structure of an RT-qPCR device according to one embodiment of the present invention. The device has a structure in which a circular chip rotates around a motor rotation axis, so that fluorescent substances can be induced and the intensity measured in multiple wells using only a single optical measurement system.

[0024] Figure 2 sequentially illustrates the process from injecting nucleic acid into a diagnostic chip to sealing according to one embodiment of the present invention (showing a top view and side view for each process).

[0025] FIG. 3 illustrates a micropatterned chip for simultaneous photothermal reaction and fluorescence measurement according to one embodiment of the present invention, showing (a) a top view of one channel and reaction chamber within the chip and (b) a side view of the reaction chamber, respectively.

[0026] Figure 4 shows the structure of the excitation light and dichroic mirror of a conventional fluorescence measurement system.

[0027] Figure 5 illustrates a fluorescence measurement system that does not use a dichroic mirror according to one embodiment of the present invention.

[0028] Figure 6 shows the fluorescence transmission results of a photothermal material pattern according to one embodiment of the present invention.

[0029] Figure 7 shows an experiment on light transmittance using a photothermal material pattern according to one embodiment of the present invention.

[0030] Figure 8 compares the light transmittance at a wavelength of 495 nm by a photothermal material pattern according to one embodiment of the present invention.

[0031] Figure 9 shows the results of a heating experiment using a photothermal material pattern according to one embodiment of the present invention.

[0032] Hereinafter, preferred embodiments of the present invention will be described in detail. In describing the present invention, if a detailed description of related known technology is judged to obscure the gist of the present invention, the detailed description will be omitted. Throughout the specification, singular expressions should be understood to include plural expressions unless the context clearly indicates otherwise, and terms such as “comprise” or “have” should be understood to indicate the presence of described features, numbers, steps, operations, components, parts, or combinations thereof, but do not preemptively exclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof. In addition, when performing a method or a manufacturing method, each step constituting the method may occur in a different order from the stated order, unless the context clearly indicates a specific order. That is, each step may occur in the same order as the stated order, may be performed substantially simultaneously, or may be performed in the opposite order.

[0033] The technology disclosed in this specification is not limited to the implementation examples described herein and may be embodied in other forms. However, the implementation examples introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the technical spirit of the present technology can be sufficiently conveyed to those skilled in the art. In the drawings, the dimensions of each device component, such as width and thickness, are somewhat enlarged to clearly represent the components. The drawings are described from the perspective of an observer, and when an element is mentioned as being positioned above another element, this includes the meaning that the element is positioned directly above the other element or that additional elements may be interposed between them. Furthermore, those skilled in the art will be able to implement the spirit of the present invention in various other forms without departing from the technical spirit of the present invention. In addition, the same reference numerals in multiple drawings indicate substantially the same elements.

[0034] As used herein, the term "and / or" includes a combination of multiple listed items or any one of multiple listed items. As used herein, "A or B" can include "A," "B," or "both A and B."

[0035] The present invention relates to a real-time quantitative PCR device utilizing a photothermal effect, characterized in that the device comprises a sample injection unit, a sample movement unit, and a reaction chamber, and includes a temperature control means capable of heating the inside of the reaction chamber, wherein the temperature control means includes a photothermal material pattern.

[0036] The sample injection unit may include an opening that is open upwards, which is a portion for injecting a sample requiring PCR. The sample used at this time may be a part of the human body, such as blood, tears, sweat, urine, feces, body fluids, semen, skin, or tissue, or a part or all of a living organism existing in nature, such as an animal, plant, virus, or bacteria. In addition, the sample may be simply injected as it is collected, but if it is processed through an appropriate preprocessing process, the size and operating time of the real-time quantitative PCR device of the present invention can be reduced. In particular, it is preferable to use a sample that contains only nucleic acid or has a high nucleic acid content after the sample has been collected through an appropriate processing process.

[0037] The sample injected as described above can be moved through the sample moving unit. At this time, the sample can be moved without limitation as long as it is a method that can move the sample as described above, and centrifugal force can be preferably used. That is, when manufacturing the real-time quantitative PCR device of the present invention, the sample injection unit can be placed at a point close to the rotation axis, and the reaction chamber to be described later can be placed at a point far from the rotation axis, and then the sample injection unit and the reaction chamber can be connected through the sample moving unit. In this way, when the rotation axis is rotated, the sample injected into the sample injection unit can be moved to the reaction chamber through the sample moving unit by centrifugal force.

[0038] At this time, the sample moving unit may simply be a passage connecting the sample injection unit and the reaction chamber, but a filter or a structure having a similar effect may be installed to perform purification or concentration of the sample, and reaction reagents, diluents, etc. may be placed inside to be mixed before reaching the reaction chamber.

[0039] As described above, the sample passing through the sample transport section reaches the reaction chamber. The reaction chamber is where the PCR reaction takes place, and reagents for the PCR reaction are injected inside. While these reagents can be additionally injected after the sample is supplied, it is preferable to inject them in advance, and in particular, it is preferable to lyophilize them to minimize changes in the reagents due to the external environment.

[0040] Additionally, the reagent may be additionally supplied from outside after being previously injected as described above. In this case, the injection may be performed using a separate injection port, or may be performed through the sample injection port.

[0041] A temperature-sensitive material may be applied to the upper part of the above reaction chamber.

[0042] The above-mentioned temperature-sensitive material refers to a material that changes from a solid to a liquid state or maintains a liquid state but has a changed viscosity depending on the temperature change inside the reaction chamber. As described below, as the reaction chamber is heated, the material moves to the sample surface inside the reaction chamber, thereby spatially isolating the sample. This isolation prevents the sample from being contaminated, and also prevents the evaporation of the solvent contained in the sample, thereby obtaining consistent results.

[0043] Looking into this in detail, the sample used in the present invention can be mixed with body fluid or water as described above. After such a sample is supplied to the reaction chamber, it can be heated multiple times for the PCR reaction. In particular, since the PCR reaction occurs at high temperatures, evaporation of the solvent, such as water, mixed with the sample may occur at such high temperatures. In the case of existing PCR devices, the PCR reaction is performed by physically closing the injection port to minimize changes in the sample amount due to evaporation or by injecting an excessive amount of solvent considering the amount of evaporation. However, simply closing the port does not prevent condensation in the sample moving portion during the cooling stage of the PCR step, and if such condensation occurs, the sample amount may change by the amount of condensation. Furthermore, even if an excessive amount of solvent is used, different condensation may occur at each point, which means that the sample amount may vary each time it is measured.

[0044] However, in the case of the present invention, the sample surface is covered with the temperature-sensitive material, and through this, not only can evaporation of the sample itself be minimized, but also external contamination can be prevented from moving toward the sample and contaminating the sample.

[0045] At this time, the temperature-sensitive material may be applied alone, but may also be in the form of a film attached to the upper portion of the reaction chamber to which the temperature-sensitive material is applied. In this case, when the temperature inside the reaction chamber rises, the viscosity of the temperature-sensitive material changes, resulting in a decrease in the adhesive strength of the film, causing the film to fall onto the upper portion of the sample and cover the surface of the sample.

[0046] The temperature-sensitive material used at this time is preferably coconut oil, triglycerides, fatty acids, mink oil, wax, or a mixture thereof.

[0047] The above coconut oil is a fat extracted from the kernel of the fruit of a palm tree that grows wild along the coasts of tropical regions such as the Philippines, Indonesia, and Malaysia. It is also called copra oil. It has a melting point of 23 to 28°C and is composed of saturated fatty acids of C10, C12, and C14. In particular, the above coconut oil has low biotoxicity, so changes in the sample used in the present invention can be minimized.

[0048] Furthermore, since the melting point of the above oils may be below room temperature, it is desirable to mix them with other types of oils to adjust their melting points. In particular, mink oil has a melting point of 80-95°C, and wax can reach 100°C. Therefore, by mixing them appropriately and using them, the temperature-sensitive material can be moved to the surface of the sample at the desired time.

[0049] Alternatively, the temperature-sensitive material may be a triglyceride, fatty acid, or mixture thereof that is solid at room temperature but melts and becomes liquid at high temperatures. Examples of the triglyceride include tricaprin, trilaurin, and trimyristin, alone or in combination. Furthermore, the fatty acid may include capric acid, lauric acid, and myristic acid, alone or in combination.

[0050] As described above, in order to perform a PCR reaction within the reaction chamber, it is necessary to heat the interior of the reaction chamber. Conventional methods involve heating the entire PCR device or the entire reaction chamber. However, this method has the disadvantages of lowering thermal efficiency and increasing the volume of the heating device, thereby lowering the efficiency of the entire PCR device and increasing its size.

[0051] To improve this, there have been cases where heating wires were inserted around the reaction chamber, but simply inserting the heating wires can cause the reaction chamber itself to melt due to the heating wires, and there is also the problem of increasing the price of the chamber used for disposable purposes.

[0052] A widely used method today involves installing a Peltier element at the bottom of the chamber to precisely control its temperature. However, installing such a Peltier element blocks the optical path, making simultaneous heating and observation impossible. Furthermore, the large size of the Peltier element limits its miniaturization.

[0053] In order to overcome these shortcomings, the present invention forms a photothermal material pattern on the outer or inner side of the bottom surface of the reaction chamber, thereby enabling heating to be performed simply by supplying light, thereby providing a real-time quantitative PCR device utilizing the photothermal effect that can perform accurate heating while having a very small volume.

[0054] The photothermal material used at this time may be any material that can generate heat when excited by supplied light, and may preferably include a carbon-based material, metal particle, or metal foil. Specifically, the carbon-based material may include carbon black, carbon fiber, carbon nanotube, or graphene, and the metal particle may be a nanoparticle, microparticle, microwire, or flake of gold, silver, copper, iron, or aluminum.

[0055] The photothermal material pattern may be formed on the inner or outer bottom surface of the reaction chamber. In the case of the reaction chamber, since a sample is loaded inside, heating through the bottom surface can have the highest efficiency. Therefore, the photothermal material pattern is preferably located on the bottom surface of the reaction chamber. In this case, for the highest efficiency, it may be located on the inner bottom surface of the reaction chamber, and even if the efficiency is low, if there is a possibility of sample contamination, it is preferable to use a protective film when located on the outer bottom surface of the reaction chamber or the inner bottom surface.

[0056] As described below, in the case of the photothermal effect light source that excites the photothermal material pattern and the fluorescent light source that excites the fluorescent material, the same direction may be positioned with respect to the reaction chamber, and in particular, it is preferable that both light sources be positioned at the bottom of the reaction chamber. However, in this case, the excitation light generated from the fluorescent light source may be blocked by the photothermal material pattern and may not be properly transmitted into the sample.

[0057] Therefore, it is desirable to form the photothermal material pattern to have a certain shape, thereby maintaining the heating efficiency by the photothermal material pattern while facilitating excitation of the fluorescent material by a fluorescent light source.

[0058] The photothermal material pattern may be formed to occupy 20 to 80% of the total area of ​​the bottom surface of the reaction chamber. If the photothermal material pattern is less than 20% of the total area of ​​the bottom surface of the reaction chamber, heating by the photothermal material pattern is not easy, and if it exceeds 80%, the fluorescent light source may be blocked by the photothermal material pattern, making fluorescence observation impossible.

[0059] In addition, in order to further increase the efficiency of the above-mentioned fluorescent light source, the photothermal material pattern may be formed in a circular, oval, donut, or polygonal shape. In the case of such a pattern, a pattern having a single shape may be formed, but it is also possible for a plurality of patterns having the same shape to be formed repeatedly. That is, when the photothermal material pattern is formed in a circular shape, a pattern having a single circle may occupy a part of the bottom surface of the reaction chamber, and also a pattern having a plurality of circles may be arranged at regular intervals on the bottom surface of the reaction chamber. In addition, in the case of having a pattern as described above, a part of the fluorescent light source may be blocked by the photothermal material pattern, but since the light source supplied by the photothermal material pattern is diffracted, the decrease in efficiency during fluorescence observation can be minimized.

[0060] The photothermal material pattern may have a constant transmittance. In the case of the photothermal material pattern, as described above, it must be able to transmit or diffract a fluorescent light source while performing heating by a photothermal effect light source. In this case, it is possible to form the photothermal material pattern in a constant shape as described above to transmit and diffract between each pattern, but it is also possible to transmit and diffract by controlling the transmittance of the photothermal material pattern. For this purpose, the photothermal material pattern may contain 1 to 10% of the photothermal material. If the photothermal material is contained less than 1%, the heating effect by the photothermal material pattern may be reduced, and if it exceeds 10%, the light source for exciting the fluorescent material may not be transmitted, thereby reducing the fluorescence efficiency.

[0061] In the case of the photothermal material pattern, as will be described later, a fluorescent light source can be transmitted above a certain level to excite the fluorescent material inside the well. Therefore, it is preferable that the photothermal material pattern have a certain light transmittance. In particular, when the photothermal material pattern is formed over the entire bottom surface of the well, it is preferable that the photothermal material pattern have a certain light transmittance.

[0062] In the present invention, light transmittance refers to the ratio of light incident on the bottom surface of the well and light observed from the top surface of the well among the light generated from the light source. If the light transmittance is 20%, this means that 20% of the light incident on the bottom surface is transmitted and can be observed from the top surface.

[0063] In the present invention, the light transmittance by the photothermal material pattern may be 10 to 30%. If the light transmittance is less than 10%, the amount of transmitted light may decrease, which may lower the efficiency of the fluorescent light source. If the light transmittance exceeds 30%, the heating efficiency by the photothermal material pattern may decrease.

[0064] In addition, in the case of the present invention, the above-mentioned light transmittance is preferably targeted at the entire wavelength, but may also be the light transmittance of the specific wavelength when the fluorescent material inside the well is excited by light of the specific wavelength. For example, Sybr green, a fluorescent material widely used in PCR, is excited by light of 495 nm. In this case, the photothermal material pattern of the present invention preferably has a light transmittance of 10 to 30% at all wavelengths, but may also have a light transmittance of 10 to 30% for a wavelength of 495 nm.

[0065] The reaction chamber may include a photothermal effect light source capable of exciting the photothermal material pattern and a fluorescent light source capable of exciting the fluorescent material within the reaction chamber, located above or below the reaction chamber. In the case of a PCR device, after nucleic acid replication by heating as described above is completed, excitation light is supplied to perform fluorescence observation. At this time, the fluorescent light source may be located at the upper or lower portion of the reaction chamber, but in the case of the present invention, it is preferably located on the opposite side of the fluorescent sensor, as will be described later.

[0066] In addition, as described above, in the case of the photothermal material pattern of the present invention, it is preferable to be positioned at the bottom of the reaction chamber in order to increase heating efficiency, and in order to increase the efficiency of the photothermal material pattern, the photothermal effect light source may also be positioned at the bottom of the reaction chamber. That is, the photothermal effect light source and the fluorescent light source may be positioned in the same direction with respect to the reaction chamber, and preferably may be positioned at the bottom of the reaction chamber. However, the photothermal material pattern and the photothermal effect light source may be positioned at the top of the reaction chamber depending on the configuration of the reaction chamber and the type of specimen.

[0067] In addition, in the case of the fluorescent sensor for detecting the fluorescence, it is preferable to be located on the opposite side from the fluorescent light source, and therefore, the fluorescent sensor may be located on the opposite side from the photothermal effect light source with respect to the reaction chamber, preferably on the upper side of the reaction chamber.

[0068] Due to the arrangement as described above, the heating efficiency by the photothermal material pattern of the present invention can be maximized, and although some fluorescent light sources may be blocked, this can be minimized through optimization of the shape of the photothermal material pattern as described above. In addition, since the excitation light generated from the fluorescent light source can be scattered and diffracted between the fluorescent material patterns, this decrease in fluorescence efficiency can be minimized.

[0069] Below, the present invention is explained through the configuration of PCR equipment.

[0070] 1. Overall system configuration

[0071] The present invention comprises an RT-qPCR device, a compatible diagnostic chip, and software for operating the device and analyzing data. The system's main components and operating principles are as follows.

[0072] RT-qPCR equipment

[0073] 1) Thermal Circulation System: RT-qPCR equipment can be equipped with a thermal circulation system, including a photothermal material pattern and a photoluminescent light source, for precise and rapid temperature control. This system can achieve rapid and precise temperature changes in a localized area, allowing PCR reactions to proceed quickly and efficiently in the desired chamber.

[0074] 2) Light source and fluorescence measurement system: The equipment may include a fluorescent light source (LED or laser) that induces fluorescence and a detector (fluorescence sensor) that detects the fluorescence signal. In the present invention, a rotating structure allows for the measurement of multiple wells with a single fluorescent light source and detector. This minimizes the need for expensive components, such as dichroic mirrors, thereby reducing costs.

[0075] A closer look reveals that conventional PCR equipment often uses Peltier elements for heating. However, since these Peltier elements are large and non-transparent, the supply of excitation light and observation of fluorescence must be performed from the same direction. At this time, a dichroic mirror is typically used to supply the excitation light (see Fig. 4). However, in the case of the present invention, as shown in Fig. 5, since the photothermal material pattern is transparent to excitation light, the supply of excitation light is possible from a different direction from the fluorescence sensor, especially from the bottom, so fluorescence observation can be performed smoothly without the use of an expensive dichroic mirror. Consequently, in the case of the present invention, not only does the manufacturing and use of the equipment require less cost because the dichroic mirror is not used, but the structure can also be simplified.

[0076] 3) Rotating Mechanism: The diagnostic chip containing the sample rotates, allowing each well to be positioned between the fluorescence light source and detector. This simplifies complex optical paths and allows multiple wells to be measured with a single optical system.

[0077] 4) Data Analysis Software: Software built into the device or configured separately can analyze fluorescence signals in real time, generate PCR amplification curves, and calculate Ct (cycle threshold) values. This allows users to view and analyze results in real time.

[0078] 1-1 Compatibility Microfluidic Chip

[0079] 1) Sample storage and evaporation prevention function: The diagnostic chip according to the present invention can store a sample for PCR reaction and prevent evaporation of the sample during the reaction process. The reagent (PCR premix) is lyophilized and pre-packaged inside the chip, which allows the user to perform PCR by simply injecting nucleic acid.

[0080] 2) Reaction Chamber and Channel Design: The diagnostic chip is equipped with a channel (sample transport section) connecting the reaction chamber and the sample injection section. The injected sample can be moved to the reaction chamber using centrifugal force to initiate the PCR reaction. Each chamber contains a temperature-sensitive material to prevent sample evaporation during the PCR process, or a film that detaches due to heat can be installed to prevent sample evaporation.

[0081] 3) Micropatterned photothermal material: The reaction chamber is micropatterned with photothermal materials, such as carbon black or gold foil, enabling rapid thermal circulation using the photothermal effect. This allows the PCR reaction to proceed quickly and efficiently.

[0082]

[0083] 1-2. Equipment operation and configuration

[0084] The device can be rotated using a motor around a central rotational axis, and a chip can be attached to part or all of this rotational axis. The device is configured with a structure that can engage the joint portion with the rotational axis to enable the chip to rotate, and this structure enables the chip to rotate stably. Through the rotation, centrifugal force is applied to the fluid (sample) within the chip, allowing the fluid to move in a desired direction. In addition to moving the fluid through the rotation, the chamber can be positioned at a desired location by rotating it by a certain angle.

[0085] The RT-qPCR equipment has a thermal cycling function for nucleic acid amplification, a rotation function for moving liquid, and a fluorescence measurement function for confirming the amount of nucleic acid amplification and whether it has been amplified. The microfluidic chip compatible with the equipment can replace the tube used in conventional PCR and perform the functions of sample support and evaporation prevention.

[0086] Unlike existing systems, such as 48-well and 96-well systems, which amplify dozens of isolated samples, the present invention is designed to amplify 1 to 20 samples, making it suitable for fields such as point-of-care diagnostics. Therefore, by eliminating the complex optical paths of existing systems and implementing a rotating optical structure, costs can be reduced.

[0087] The optical structure proposed in the present invention has the advantage of enabling observation of multiple wells while the light source and fluorescence sensor are fixed. To this end, the equipment may be configured with two or more fluorescence light sources and two or more fluorescence sensors.

[0088] The photothermal material pattern for applying heat to a material within the chamber and the heating inside the reaction chamber using the same are as described above.

[0089] The PCR device of the present invention may also be configured as a nucleic acid measurement system comprising a nucleic acid amplification system capable of amplifying nucleic acids inside a reaction chamber using the photothermal material pattern and a photothermal effect light source, and a light source of a wavelength capable of exciting the internal material to measure the amount of fluorescence generated as the nucleic acid inside the chamber is amplified, and a sensor measuring the emitted fluorescence.

[0090] Each chamber is moved for fluorescence measurement after each cycle of thermal cycling, which can be accomplished using the rotation function of the instrument. For example, if 35 cycles of amplification are performed, the instrument can be positioned 35 times at each light source.

[0091]

[0092] Hereinafter, the present invention will be described in detail through the operation of a PCR chip.

[0093] Figure 2 is a schematic diagram illustrating the sequential process of nucleic acid injection into a chip and amplification.

[0094] (a) Nucleic acid extracted from a preprocessing structure is injected through a sample injection unit. At this time, the injected nucleic acid may be mixed with a material for a PCR reaction. Reagents for a PCR reaction, such as primers, polymerase, and fluorescent substances, may be stored in the PCR reaction chamber. At this time, the reagents may be in a liquid or dry state, and preferably in a freeze-dried state. A temperature-sensitive material (oil or wax) may be present in a liquid or solid state at the upper part of the reaction chamber to prevent evaporation of the liquid during the PCR process. If solidified, the temperature-sensitive material may be liquefied at a condition above a specific temperature.

[0095] (b) The injected nucleic acid moves toward the reaction chamber within the sample moving section due to the centrifugal force generated as the PCR chip rotates, and is mixed with the pre-packaged reagents within the reaction chamber.

[0096] (c) During the PCR thermal cycling process, heat is applied to the reaction chamber, and the temperature-sensitive material liquefies and covers the PCR solution. This prevents evaporation during the thermal cycling process.

[0097] (d) Nucleic acid amplification is performed while preventing evaporation of the sample. At this time, heating for nucleic acid amplification is performed using a photothermal material pattern, which is the same as described above.

[0098] (e) The fluorescent light source and fluorescent sensor are positioned in a straight line in the reaction chamber where the sample is located (Fig. 3). The photothermal material pattern and its effect for simultaneously enabling this are the same as described above, so description is omitted.

[0099]

[0100] Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily implement them. Furthermore, when describing the present invention, detailed descriptions of related, known functions or configurations will be omitted if they are deemed to unnecessarily obscure the gist of the present invention. Furthermore, certain features presented in the drawings may be enlarged, reduced, or simplified for ease of explanation, and the drawings and their components are not necessarily drawn to scale. However, those skilled in the art will readily understand these details.

[0101]

[0102] Examples 1-6

[0103] An experiment was conducted to verify the effects of the shape and concentration of the photothermal material pattern. To simulate a reaction chamber, a 5-mm-diameter well was formed in the center of a PDMS (polydimethylsiloxane) plate. The PDMS plate was 5 mm thick and the well was 4 mm deep.

[0104] A pattern was formed using conductive carbon paste (conductive tech) as a photothermal material on a 100㎛ thick silicone film (IS solution). At this time, in order to control the amount applied, acetone and the conductive carbon paste were mixed in a ratio of 8:1 (containing 15% conductive carbon paste).

[0105] The photothermal material patterns formed on the above silicon film are as follows.

[0106] Full coverage (5 mm diameter) - Example 1

[0107] Midopo - Example 2

[0108] Central uncoated area (50% of total area, central diameter of approximately 3.57 mm uncoated) - Example 3

[0109] Apply only to the central area (apply only 50% of the total area, approximately 3.57 mm in diameter to the central area) - Example 4

[0110] Mix acetone and conductive carbon paste in a ratio of 40:1 and apply to the entire surface (containing 5% conductive carbon paste) - Example 5

[0111] After mixing acetone and conductive carbon paste at a ratio of 60:1, apply to the entire surface (containing 3% conductive carbon paste) - Example 6

[0112] The film produced as described above was cut to a diameter of 5 mm and then attached to the inner lower part of the well to form a reaction chamber.

[0113]

[0114] Experimental Example 1

[0115] The following reaction solution was injected into the well manufactured in the above example.

[0116] Target Microorganism Primer Sequence Hepatitis B Virus Forward GTC CTC CAA TTT GTC CTG GReverse TGA GGC ATA GCA GCA GGA T

[0117] 2X RbTaq PreMIX-HOT (Enzynomics): 5 μl

[0118] Primer (5 pmol / μl): 2 μl

[0119] Target DNA (105-106 copies / μl):1μl

[0120] 10x SYBR: 1μl

[0121] Distilled water: 1 μl

[0122] Total: 10 μl

[0123] For the experiments below, the well-known virus hepatitis B virus and its corresponding primers were used.

[0124] The amount of fluorescence that increased with each PCR cycle (5, 15, 25, 30) was measured using a fluorescence sensor, and the intensity of the voltage measured according to the amount of light was described.

[0125] In this measurement method, a small voltage (V) (large absolute value) means that the amount of light emitted by the excitation light is small. In other words, the amount of Sybr green observed is small, which means that the intensity of the excitation light passing through the photothermal material pattern is small.

[0126] Cycle Example 1 Example 2 Example 3 Example 4 Example 5 65-71.0-19.9-68.0-48.0-38.2-24.915-62.5-10.6-51.3-35.5-35.0-18.925-55.8-7.1-38.3-13.4-27.1-18.230-45.8-5.3-30.4-4.7-23.4-15.8

[0127] As shown in Table 2 and Figure 6, Example 2, in which nothing was applied, was found to have the highest fluorescence. However, even in Examples 2 and 4, in which only a portion was applied, it was confirmed that the fluorescence increased above a certain level, and in Examples 5 and 6, in which the amount of photothermal material was controlled, it was confirmed that the magnitude of the fluorescence was controlled.

[0128]

[0129] Experimental Example 2

[0130] A light transmittance experiment was conducted on the above examples 1, 2, and 5.

[0131] As shown in Fig. 7, the results of the light transmittance experiment at a wavelength of 380 to 780 nm showed that Example 2, which used only the film, had a light transmittance of 40 to 70% depending on the wavelength, and Example 5 had a light transmittance of 14 to 25%. However, Example 1 was found to have a light transmittance of less than 5%, which was consistent with the excitation light experiment of Experimental Example 1.

[0132] In addition, when comparing the transmittance at a wavelength of 498 nm, which is the excitation light that excites Sybr green (see Fig. 8), Example 2 had a light transmittance of 50-60%, and Example 5 had a light transmittance of 15-25%, confirming that a fluorescent light source can be transmitted when a photothermal material pattern of a certain concentration is used. However, Example 1 had a light transmittance of less than 5%, indicating that a photothermal material pattern exceeding a certain concentration significantly reduces the efficiency of the fluorescent light source.

[0133]

[0134] Experimental Example 3

[0135] Temperature control experiments were conducted for the above Examples 1-6. An OSRAM LZ4-00R708 photothermal light source was installed at the bottom of each well, and light was supplied. The temperature (℃) inside the well was measured at 10-second intervals. If the temperature inside the well exceeded 90℃, the light source was turned off, and the experiment was terminated when the temperature inside the well decreased below 40℃.

[0136] Time (seconds) Example 1 Example 2 Example 3 Example 4 Example 5 60 25.5 25.227.226.124.5 25.8 10 67.7 45.9 46.66 4.35 3.55 1.0 20 10 2.56 1.26 7.28 4.0 77.77 0.0 30 67.9 68.68 1.1 10 1.0 89.9 75.84 048.874.889.15 8.0 96.78 1.65 04 2.879.491.847.683.185.46038.680.373.042.058.492.87081.562.132.942.876.48083.554.332.151.19081.248.740.010080.544.511082.441.112083.538.5

[0137] As shown in Table 3 and Fig. 9, the heating pattern was found to be different depending on the shape and concentration of the photothermal pattern. In the case of Example 1, in which the entire area was coated, it was found that heating could be achieved to 95°C or higher in a short period of time, but as shown in Table 2, the fluorescence efficiency was greatly reduced, making it difficult to use. On the other hand, in the case of Example 6, in which the area was coated at a low concentration, the fluorescence efficiency was excellent, as shown in Table 2, but it was found that heating required a long time, making it difficult to use.

[0138] In addition, in the case of Example 2, which did not use a photothermal material pattern, slow heating was possible by the heat supplied from the LED itself, but the temperature increase rate was very slow and it was difficult to increase to the desired temperature, so it was confirmed that additional heating was required in PCR.

[0139] In addition, in the case of Example 3, since heating occurred from the part close to the wall of the well, it took a long time for heating due to the specific heat of PDMS and the temperature also decreased slowly. In contrast, in the case of Example 4, which used a circular photothermal pattern of the same area, heating occurred from the center of the well, so a fast heating rate was observed and the temperature decrease rate was also confirmed to be faster than in Example 3.

[0140]

[0141] Examples 7-13

[0142] Based on the above Experimental Example 1, an additional experiment was conducted on the shape of the photothermal material pattern.

[0143] 7. Acetone and the conductive carbon paste were mixed in a ratio of 8:1 (containing 15% of the conductive carbon paste) and used, with each pattern having a circular shape with a diameter of 0.2 mm, and the spacing between each pattern was adjusted so that the photothermal material pattern occupied 10% of the total floor area (Example 7).

[0144] 8. Same as Example 7, but adjust the spacing between each pattern so that the photothermal material pattern occupies 20% of the total floor area (Example 8)

[0145] 9. Same as Example 7, but adjust the spacing between each pattern so that the photothermal material pattern occupies 50% of the total floor area (Example 9)

[0146] 10. Same as Example 7, but adjust the spacing between each pattern so that the photothermal material pattern occupies 80% of the total floor area (Example 10)

[0147] 11. Same as Example 7, but adjust the spacing between each pattern so that the photothermal material pattern occupies 90% of the total floor area (Example 11)

[0148] 12. Same as Example 7, but the photothermal material pattern is formed to have a square shape (Example 12)

[0149] 13. Same as Example 7, but the photothermal material pattern is formed to have a triangle (Example 13)

[0150]

[0151] Experimental Example 4

[0152] For the above Examples 7 to 13, the transmittance (%) of a light source having a wavelength of 495 nm, the voltage (V) of the fluorescent sensor after 5 cycles of PCR, and the time (seconds) until the internal temperature of the well exceeded 90°C after exposure to the photothermal effect light source were measured. Each experiment was performed in the same manner as Experimental Examples 1 to 3.

[0153] Transmittance (%) Voltage (V) Time (sec) Example 7 35.8-28.57 2 Example 8 26.4-31.8 31 Example 9 24.5-35.4 23 Example 10 22.5-41.9 21 Example 118.2-84.2 18 Example 12 25.1-33.8 22 Example 13 24.3-34.9 25

[0154] As shown in Table 4, in Example 7, where the pattern occupies 10% of the total floor area, the heating efficiency due to the photothermal material pattern was found to be significantly reduced. Conversely, in Example 11, where the pattern occupies 90% of the floor area, the heating efficiency was increased, but the light transmittance was significantly reduced, resulting in a decrease in the fluorescence efficiency.

[0155] In addition, it was confirmed that examples 12 and 13, which had different pattern shapes, had similar effects to example 9.

[0156]

[0157] Examples 14-18

[0158] Based on the above Experimental Example 1, an additional experiment was conducted on the concentration of the photothermal material pattern.

[0159] 14. Acetone and the above conductive carbon paste were used, but the amount of acetone was adjusted to contain 15% of the conductive carbon paste.

[0160] 15. Acetone and the above conductive carbon paste were used, but the amount of acetone was adjusted to contain 10% of the conductive carbon paste.

[0161] 16. Acetone and the above conductive carbon paste were used, but the amount of acetone was adjusted to contain 5% of the conductive carbon paste.

[0162] 17. Acetone and the above-mentioned conductive carbon paste were used, but the amount of acetone was adjusted to contain 1% of the conductive carbon paste.

[0163] 18. Acetone and the above conductive carbon paste were used, but the amount of acetone was adjusted to contain 0.5% of the conductive carbon paste.

[0164]

[0165] Experimental Example 5

[0166] The same experiment as Experimental Example 4 was conducted using Examples 14 to 18 above.

[0167] Transmittance (%) Voltage (V) Time (sec) Example 142.3-71.021 Example 1518.4-41.629 Example 1622.3-38.236 Example 1738.6-28.645 Example 1845.8-21.4-

[0168] As shown in Table 5, when the content of conductive carbon paste exceeded 15%, the light transmittance decreased and the fluorescence efficiency decreased rapidly. Conversely, when the content of conductive carbon paste was less than 0.5%, the light transmittance and fluorescence efficiency increased, but it was confirmed that the interior of the well could not be heated above 90℃.

[0169]

[0170] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. In a PCR device including a sample injection unit, a sample movement unit, and a reaction chamber, It includes a temperature control means capable of heating the inside of the reaction chamber, A real-time quantitative PCR device utilizing a photothermal effect, characterized in that the temperature control means includes a photothermal material pattern.

2. In paragraph 1, A real-time quantitative PCR device utilizing the photothermal effect, characterized in that the photothermal material pattern is formed on the outer or inner side of the bottom surface of the reaction chamber.

3. In paragraph 1, A real-time quantitative PCR device utilizing the photothermal effect, characterized in that the photothermal material pattern is formed to occupy 20 to 80% of the total area of ​​the bottom surface of the reaction chamber.

4. In paragraph 1, A real-time quantitative PCR device utilizing the photothermal effect, characterized in that the photothermal material pattern is formed in a circular, oval, donut or polygonal shape.

5. In paragraph 1, A real-time quantitative PCR device utilizing the photothermal effect, characterized in that the photothermal material pattern above contains 1 to 10% of the photothermal material.

6. In paragraph 1, A real-time quantitative PCR device utilizing the photothermal effect, characterized in that the upper or lower portion of the reaction chamber includes a photothermal effect light source capable of exciting the photothermal material pattern and a fluorescent light source capable of exciting a fluorescent material within the reaction chamber.

7. In paragraph 6, It further includes a fluorescent sensor for detecting fluorescence excited by the fluorescent light source, A real-time quantitative PCR device using a photothermal effect, characterized in that, based on the above reaction chamber, the photothermal effect light source is installed in the same direction as the fluorescent light source, and the fluorescent sensor is installed in the opposite direction of the fluorescent light source.

8. In paragraph 1, A real-time quantitative PCR device utilizing the photothermal effect, characterized in that a temperature-sensitive material is applied to the upper part of the reaction chamber.

9. In paragraph 8, A real-time quantitative PCR device utilizing the photothermal effect, characterized in that the temperature-sensitive material moves to the upper surface of the sample inside the reaction chamber when heated using the photothermal material pattern, thereby spatially isolating the sample.

10. In paragraph 1, A real-time quantitative PCR device utilizing a photothermal effect, characterized in that a sample injected into the sample injection unit is supplied toward the reaction chamber by centrifugal force.

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