WO2022114816A1

WO2022114816A1 - Thermal cycler

Info

Publication number: WO2022114816A1
Application number: PCT/KR2021/017536
Authority: WO
Inventors: Jae Young Kim
Original assignee: Seegene, Inc.
Priority date: 2020-11-26
Filing date: 2021-11-25
Publication date: 2022-06-02
Also published as: KR20230088831A; KR20230088830A; WO2022114814A1

Abstract

Provided is a thermal cycler. A thermal module heats or cools one area of a sample holder containing a sample. A thermal pusher includes a pressure part for press-contacting the sample holder to the thermal module and a heating part heating an opposite area of the sample holder opposite to the one area. A slot space is defined between the thermal module and the thermal pusher, and is capable of positioning one or more sample holders therein.

Description

THERMAL CYCLER

The present disclosure relates to a thermal cycler for a nucleic acid reaction.

Polynucleotide chain reaction (PCR) is most commonly used as a nucleic acid amplification reaction. PCR includes a repeated cycle process comprised of denaturation of a double-stranded DNA, annealing of an oligonucleotide primer to a DNA template, and primer extension by DNA polymerase (Mullis et al, U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159; and Saiki et al., 1985, Science 230, PP. 1350-1354). The denaturation of DNA proceeds at a temperature of about 95°C, and the annealing and the extension of the primer proceed at a temperature of 55°C to 75°C, which is lower than 95°C.

A typical real-time PCR device includes a thermal cylinder in which a reaction container is positioned and a nucleic acid amplification reaction of a sample held in the reaction container is performed and an optics mechanism analyzing (or monitoring) the nucleic acid amplification reaction in real time. The PCR device is required to have high energy efficiency and a light emission and detection scheme able to accurately detect a target nucleic acid in the operation of the thermal cycler and the optics mechanism.

Referring to the structure of a PCR device of the related art, the thermal cycler in which a reaction container is positioned is provided in the lower portion of the device, and the optics mechanism is provided in the upper portion of the device and configured to be moved in the top-bottom direction by a motor or the like and is positioned adjacent to while being spaced apart from the top area of the reaction container. In addition, the thermal cycler performs an amplification reaction by heating or cooling the reaction container from below the reaction container. The optics mechanism includes a light source emitting excitation light to the top area of the reaction container and an optical detector detecting light emitted from a sample solution, and analyzes the amplification reaction in real time.

However, the-above described PCR device is a device for simultaneously detecting a nucleic acid from a large amount of samples. The thermal cycler of the PCR device of the related art has a structure suitable to a reaction container in the shape of a well plate. It is disadvantageously difficult to apply the PCR device to a point-of-care (POC) system for processing an extraction and a nucleic acid detection obtained from a small amount of samples.

Therefore, there is a need to develop a thermal cycler suitable to a POC system and configured to obtain high energy efficiency, block external light, and accurately detect a target nucleic acid.

In consideration of the above-described background, the present disclosure is intended to provide a thermal cycler suitable to a POC system.

In addition, the present disclosure is intended to provide a thermal cycler including a thermal pusher and configured to obtain energy efficiency when heating or cooling a sample holder containing a sample. The thermal pusher includes a pressure part for press-contacting a sample holder to a thermal module for heating or cooling one area of the sample holder and a heating part heating an opposite area of the sample holder opposite to the one area.

In addition, the present disclosure is intended to provide a thermal cycler in which the thermal push includes an optic path extending through the pressure part and the heating part, with one end of the optic path being open in the direction of the thermal module. It is therefore possible to accurately provide excitation light to the sample held in the sample holder and detect emission light emitted from the sample in real time during operation of the thermal module.

In addition, the present disclosure is intended to provide a thermal cycler in which one or more optic fibers connected to an optics mechanism are inserted into the optic path. It is therefore possible to remove a movement part for moving the optics mechanism, improve structural stability of the POC system, and reduce fabrication costs.

Furthermore, the present disclosure is intended to provide a thermal cycler including a cover part configured to cover at least a portion of areas of the sample holder not covered with the thermal module or the pressure part. It is therefore possible to block external light from being incident on the sample or the optic path or internal light from being transmitted to the outside.

According to an embodiment of the present disclosure, a thermal cycler may include: a thermal module to heat or cool one area of a sample holder containing a sample; a thermal pusher comprising a pressure part for press-contacting the sample holder to the thermal module and a heating part heating an opposite area of the sample holder opposite to the one area; and a slot space defined between the thermal module and the thermal pusher and capable of positioning one or more sample holders therein.

The present disclosure may provide the thermal cycler suitable to a POC system for processing a small amount of a sample.

In addition, according to the present disclosure, both areas of the sample holder are press-contacted to the heating part of the thermal module and the heating part of the thermal pusher, respectively, by the press part of the thermal pusher. Thus, it is possible to prevent a loss of heat during heating or cooling of the sample holder, thereby securing energy efficiency.

In addition, according to the present disclosure, it is possible to accurately provide excitation light to a sample in the sample holder through an optic path provided in the thermal pusher and open in the direction of the thermal module and to detect emission light emitted from the sample.

Furthermore, according to the present disclosure, excitation light is radiated on the sample or emission light emitted from the sample is detected through the optic fiber inserted into the optic path. A movement part for moving an optics mechanism connected to the optic fiber is unnecessary. It is therefore possible to improve the structural stability of the POC system and reduce fabrication costs.

In addition, according to the present disclosure, the areas of the sample holder positioned in a slot space are covered with the thermal module, the press part, and the cover part. Thus, the sample and the optic path can be concealed, and thus, target nucleic acid can be accurately detected.

FIGS. 1 and 2 are perspective views illustrating a thermal cycler according to an embodiment of the present disclosure.

FIG. 3 is an exploded perspective view illustrating a part of the thermal cycler according to an embodiment of the present disclosure.

FIG. 4 is a plan view illustrating a part of the thermal cycler according to an embodiment of the present disclosure.

FIGS. 5 and 6 are cross-sectional views illustrating operation states of the thermal cycler according to an embodiment of the present disclosure.

Hereinafter, the present disclosure will be described in more detail with reference to embodiments. It will be apparent to those having ordinary knowledge in the art that the following embodiments are for illustrative purposes only and the scope of the present disclosure is not limited by the embodiments.

In designating elements of the drawings by reference numerals, the same elements will be designated by the same reference numerals if possible although they are shown in different drawings. Further, in the following description of the present disclosure, a detailed description of known functions and configurations incorporated herein will be omitted in the situation in which the subject matter of the present disclosure may be rendered unclear thereby.

In addition, terms, such as "first", "second", "A", "B", "(A)", or "(B)" may be used herein to describe elements of the present invention. Each of these terms is not used to define essence, order, sequence, or number of elements, etc., but is used merely to distinguish the corresponding element from other elements. When it is mentioned that a first element is "connected", "coupled", or "linked" to a second element, it should be interpreted that, not only can the first element be directly connected, coupled, or linked to the second element, but a third element can also be "connected", "coupled", or "linked" between the first and second elements.

The term "sample" as used herein refers to a substance estimated including or supposed to include an analyte.

The "sample" may include biological samples (e.g., cells, tissue, and body fluid from biological supply sources) and non-biological samples (e.g., food, water, and soil). The biological samples may include, but are not limited to, virus, bacteria, tissue, cells, blood (e.g., whole blood, plasma, and serum), lymph, bone marrow fluid, saliva, sputum, swab, aspiration, milk, urine, feces, ocular fluid, semen, brain extract, spinal cord fluid (SCF), joint fluid, extracts from appendix, spleen and tonsil tissue, thymic fluid, bronchial lavage fluid, ascitic fluid, and amniotic fluid. Further, the samples may include natural nucleic acid molecules and synthetic nucleic acid molecules isolated from biological sources. According to an embodiment of the present disclosure, the "sample" may include substances used for preservation, processing, detection, etc. of the sample. The "sample" may include, but is not limited to, additional substances such as reagents for amplification, reagents for detection, preservatives, water, deionized water, saline water, pH buffer solutions, acid solutions, and basic solutions.

The term "sample processing" as used herein refers to a series of processes of primarily separating an analysis target substance from the sample and obtaining a substance from which a detection reaction is possible. The sample processing may be used as having a meaning that additionally includes a process of detecting a target analysis substance from the substance from which the detection reaction is possible. The analysis target substance may be, for example, nucleic acid.

The nucleic acid detection may be performed by a signal-generating reaction.

The term "signal-generating reaction" as used herein means any reaction capable of generating signals in a dependent manner on properties of a target analyte in a sample. The properties may be, for instance, activity, amount of presence (or absence) of the target analyte, in particular, the presence (or absence) of the target analyte in a sample. According to an embodiment of the present disclosure, the signal-generating reaction includes a biological reaction and a chemical reaction. The biological reaction includes a genetic analysis process such as PCR (Polymerase Chain Reaction), real-time PCR, microarray analysis and invader analysis, an immunological analysis process, and a bacterial growth analysis process. According to an embodiment of the present disclosure, the signal-generating reaction is a genetic analysis process. The chemical reaction includes the process of analyzing the creation, change or destruction of a chemical substance. According to an embodiment of the present disclosure, the signal-generating reaction is a signal amplification reaction.

The term "signal amplification reaction" as used herein means a reaction that increases or decreases a signal generated by the signal-generating means. According to an embodiment of the present disclosure, the amplification reaction means an increase (or amplification) reaction of a signal generated by the signal-generating means depending on the presence of a target analyte. The amplification reaction may or may not be accompanied by amplification of a target analyte (eg, a nucleic acid molecule). Particularly, in the present disclosure, the amplification reaction means a signal amplification reaction accompanied by amplification of a target analyte.

There have been known various methods of generating an optical signal indicating presence of a target nucleic acid using a signal0generating reaction. Representative examples of the various methods are as follows: a PTO cleavage and extension (PTOCE) method (WO 2012/096523), a TaqMan^TM probe method (U.S. Pat. No. 5,210,015), a molecule beacon method (Tyagi et al., Nature Biotechnology v. 14 MARCH 1996), a scorpion method (Whitcombe et al., Nature Biotechnology 17: 804-807 1999)), a sunrise or amplifluor method (Nazarenko et al., Nucleic Acids Research, 2512: 2516-2521 1997) and U.S. Pat. No. 6,117,635), a lux method (U.S. Pat. No. 7,537,886), a CPT (Duck P, et al., Biotechniques, 9: 142-148 1990)), an LNA method (U.S. Pat. No. 6,977,295), a Plexor method (Sherrill C B, et al., Journal of the American Chemical Society, 126: 4550-4556 (2004)), Hybeacons^TM (D. J. French, et al., Molecular and Cellular Probes (2001) 13, 363-374 and U.S. Pat. No. 7,348,141), a dual-labeled self-quenched probe (U.S. Pat. No. 5,876,930), a hybridization probe (Bernard P.S., et al., Clin Chem 2000; 46, 147-148), Detection of target nucleic acid sequences by PTO cleavage and extension (PTOCE) assay (WO2012/096523), Detection of target nucleic acid sequence by PTO cleavage and extension-dependent signaling oligonucleotide hybridization (PCE-SH) assay (WO2013/115442), Detection of target nucleic acid sequence by PTO cleavage and extension-dependent non-hybridization (PCE-NH) assay (PCT/KR2013/012312), Detection of target nucleic acid sequences by cyclic exonucleolytic reactions (CER) (WO2011/037306), and Assimilating probe method (PCT/US2011/041540).

Various types of nucleic acid amplification reactions may be performed using the thermal cylinder according to the present disclosure. For example, the nucleic acid amplification reactions include polymerase chain reaction (PCR), ligase chain reaction (LCR) (U.S. Patent Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), Strand displacement amplification (SDA) (Walker, et al. Nucleic Acids Res. 20(7): 1691-6, 1992); Walker PCR Methods Appl. 31): 1-6, 1993)), transcription-mediated amplification (Phyffer, et al., J. Clin. Microbiol. 34: 834-841, 1996); and Vuorinen, et al., J. Clin. Microbiol. 33: 1856-1859, 1995)), Nucleic acid sequence-based amplification (NASBA) (Compton, Nature 350 (6313): 91-2 ,1991)), Rolling circle amplification (RCA) (Lisby, Mol. Biotechnol. 121): 75-99, 1999), Hatch et al., Genet. Anal. 15(2): 35-40, 1999)), Q-beta Replicase (Lizardi et al., BiolTechnology 6: 1197, 1988)), loop-mediated isothermal amplication (LAMP, Y. Mori, H. Kanda and T. Notomi, J. Infect. Chemother., 2013, 19, 404-411), and recombinase polymerase amplication(RPA, J. Li, J. Macdonald and F. von Stetten, Analyst, 2018, 144, 31-67)etc.

Especially, the thermal cycler according to the present disclosure is put to good use in a nucleic acid amplification reaction based on a polymerase chain reaction. Various nucleic acid amplifying methods using a polymerase chain reaction have been known. For example, the various nucleic acid amplifying methods include quantitative PCR, digital PCR, asymmetric PCR, reverse transcription polymerase chain reaction (RT-PCR), differential display PCR (DD-PCR), nested PCR, arbitrarily primed polymerase chain reaction (AP-PCR), multiplex PCR, SNP genome typing PCR, etc.

The term "cycle" as used herein means a unit of changes of conditions in a plurality of measurements accompanied with changes of conditions. The change of the conditions means, for example, an increase or decrease in temperature, reaction time, reaction number, concentration, pH, and replication number of a measurement target (e.g., nucleic acid). Thus, a cycle may be a time or process cycle, a unit operation cycle or a reproductive cycle.

As one example, when analyzing a substrate decomposition capacity of an enzyme according to a substrate concentration, the substrate decomposition capacity of the enzyme is analyzed therefrom after measuring the substrate decomposition degree of the enzyme several times by varying the substrate concentration. At this time, the change of the condition is an increase in the substrate concentration, and the used substrate concentration increase unit is set as one cycle.

As another example, when performing isothermal amplification of nucleic acid, several measurement may be performed with different reaction times for a sample. In this case, the reaction time is the change of the condition, and the reaction time unit is set as one cycle. For example, the measurement may be performed several times with changing the reaction time, such as 1 minute, 2 minutes, 3 minutes, etc. In this case, a cycle has a unit of time, and one cycle is set as a reaction time unit of 1 minute.

More specifically, the term "cycle" may refer to one unit of repetition when a reaction of a constant process is repeated or when a reaction is repeated on the basis of a fixed time interval.

For example, in the case of the polymerase chain reaction (PCR), one cycle refers to a reaction including an operation of denaturing nucleic acid, an operation of binding (i.e., hybridizing or annealing) the nucleic acid and a primer, and an operation of extending the primer. In this case, a constant change in conditions includes an increase in the number of repetitions of reactions, and a unit of repetitions of reactions including the series of operations is set as one cycle.

FIGS. 1 and 2 are perspective views illustrating a thermal cycler according to an embodiment of the present disclosure. FIG. 3 is an exploded perspective view illustrating a part of the thermal cycler according to an embodiment of the present disclosure. FIG. 4 is a plan view illustrating a part of the thermal cycler according to an embodiment of the present disclosure. FIGS. 5 and 6 are cross-sectional views illustrating operation states of the thermal cycler according to an embodiment of the present disclosure.

A sample is held in a sample holder 10. The sample holder 10 may be, for example, a tube, a vessel, or a cuvette. A target nucleic acid is included in the sample held in the sample holder 10 in a state in which the target nucleic acid can react and be detected in a sample processing process. The sample holder 10 may include a container part in which the sample is held, and further include a heat conduction part. According to an embodiment, the heat conduction part may be thermally connected to a thermal module 110, and the thermal module 110 may heat or cool the sample held in the container part. According to another embodiment, the sample holder may only include the container part. According to another embodiment, the sample holder may include a container part, a heat conduction part thermally connected to the thermal module 110, and a heat conduction part thermally connected to a heating part 121. In addition, a sample space in which the sample is held may be provided by carving one area of the container part, and the heat conduction part may be coupled to one area of the container part while covering the sample space. As will be described below, the sample holder 10 may be provided in the shape of a square pillar. The container part may be provided in the shape of square pillars, the sample space may be provided in one area of the square pillar by carving, and the heat conduction part may be coupled to the one area while covering the sample space. According to this structure, the sample held in the sample space may be directly thermally connected to the heat conduction part, and thus, the sample can be more rapidly heated and cooled. The heat conduction part of the sample holder 10 may be implemented as a metal layer, for example, aluminum (Al), copper (Cu) silver (Ag) or combinations thereof, an Al-Mg alloy, an Al-Si alloy, gold (Au), or tungsten (W).

First, a description will be made with reference to FIGS. 1 and 2.

A thermal cycler 100 according to the present disclosure includes a thermal module 110 and a thermal pusher 120. One or more sample holders 10 are positioned in a slot space 200 defined between the thermal module 110 and the thermal pusher 120. The slot space 200 is a hollow space defined between the thermal module 110 and the thermal pusher 120. The sample holders 10 may be positioned in or removed from the slot space 200. The slot space 200 may position one or more sample holders 10 therein. When a plurality of sample holders 10 are provided in the slot space 200, respective sample holders of the plurality of sample holders 10 may be separated from or coupled integrally with each other. The respective sample holders 10 may be positioned in or removed from the slot space 200, simultaneously or in a predetermined order. In the figures, an embodiment in which three sample holders 10 are positioned in the slot space 200 is illustrated. The sample holders 10 are positioned into the slot space 200 from outside the slot space 200. According to an embodiment, the sample holders 10 may be inserted into and removed from the slot space 200 through a space above the slot space 200.

The form and size of the slot space 200 may be configured different depending on the form and number of the sample holders 10. According to an embodiment, the slot space 200 may be configured in a form capable of positioning the sample holders 10 in a longitudinal positioning. The form capable of positioning the sample holders 10 in a longitudinal positioning refers to a form by which the sample holders 10 having a form elongated in a longitudinal positioning can be positioned in a form standing in a longitudinal positioning. A plurality of sample holders 10 may be positioned in the slot space 200 so as to stand in the longitudinal positioning. The plurality of sample holders 10 may be arranged parallel to each other in the lateral direction. According to an embodiment, the slot space 200 may be configured in a form capable of positioning the sample holders 10 having a square pillar shape therein. The slot space 200 may be configured in a form capable of positioning the sample holders 10 having a square pillar shape in a longitudinal positioning. The sample holders 10 having a square pillar shape may be positioned in the slot space 200 in a longitudinal positioning. Each of the sample holders 10 having a square pillar shape may be configured such that two opposite areas of four lateral areas have a greater area than the remaining two opposite areas of the four lateral areas, and may be positioned such that the two opposite areas face the thermal module 110 and the thermal pusher 120, respectively. A plurality of sample holders 10 having a square pillar shape may be positioned in the slot space 200, and may be disposed such that the remaining two areas of each of the plurality of sample holders 10 oppose each other.

Specifically, the slot space 200 is defined between a thermal conductor 111 of the thermal module 110 and a thermal conductor 123 of the thermal pusher 120, such that the sample holders 10 positioned in the slot space 200 are contacted to the

thermal conductors

111 and 123 on both sides to be thermally connected thereto. Here, elements "thermally connected" to each other mean that the elements are provided in forms capable of exchanging heat with each other, and a thermal conductor mediating heat exchange between the elements may further be provided. For example, the sample holders 10 may be thermally contacted to the thermal conductor 111 of the thermal module 110 and the thermal conductor 123 of the thermal pusher 120. In a case in which the plurality of sample holders 10 are positioned in the slot space 200, each of the sample holders 10 may be contacted to the

thermal conductors

111 and 123 on both sides.

As a support unit 125 of the thermal pusher 120 is moved by a movement unit 126, the

thermal conductors

111 and 123 on both sides move away from or get closer to each other, thereby increasing or reducing the size of the slot space 200 (see FIGS. 5 and 6). In a situation in which the

thermal conductors

111 and 123 on both sides have moved away from each other, the sample holder 10 is positioned in the slot space 200. As the

thermal conductors

111 and 123 on both sides move closer to each other, the sample holder 10 may be press-contacted to the

thermal conductors

111 and 123 on both sides. In the figures, the distance between the

thermal conductors

111 and 123 on both sides is illustrated in an exaggerated manner for the sake of understanding. A change by which the distance between the

thermal conductors

111 and 123 on both sides is increased or reduced may be smaller than the thickness of the sample holder 10.

In addition, the slot space 200 defined between the

thermal conductors

111 and 123 on both sides may be divided from an external space by a cover part 130. That is, the slot space 200 may be defined by the

thermal conductors

111 and 123 on both sides and the cover part 130. The cover part 130 may be positioned on the thermal module 110 or the thermal pusher 120, and may be comprised of two or more components, such that some of the two or more components may be positioned on the thermal module 110, and the remaining ones of the two or more components may be positioned on the thermal pusher 120. Alternatively, the cover part 130 may be positioned on any component other than the thermal module 110 or the thermal pusher 120. According to an embodiment, the cover part 130 may include a first cover 130a coupled to a metal block 113 of the thermal module 110 and a second cover 130b coupled to the support unit 125 of the thermal pusher 120. As illustrated in the figures, the first cover 130a may define the bottom# and one side of the slot space 200, and the second cover 130b may define another side of the slot space 200 opposite the first cover 130a.

The sample holder 10 positioned in the slot space 200 may be contacted to the

thermal conductors

111 and 123 on both sides and supported by the cover part 130. The cover part 130 supports the sample holder 10 positioned in the slot space 200. In addition, the cover part 130 may guide the sample holder 10 being inserted into the slot space 200 or prevent the sample holder 10 from being removed from the slot space 200.

In addition, the cover part 130 thermally or optically block the sample holder 10 positioned in the slot space 200 from the outside (see FIG. 6). That is, the cover part 130 is positioned in the slot space 200 to cover at least a portion of areas of the sample holder 10 press-contacted to the thermal module 110, not covered with the thermal module 110 or a pressure part 124. As the pressure part 124 press-contacts the sample holder 10 to the thermal module 110, one area 11 and the opposite area 12 of the sample holder 10 are covered with the

thermal conductors

111 and 123 on both sides, respectively. At least a portion of the remaining areas of the sample holder 10, i.e., the bottom area, the top area, and side areas, may be covered with the cover part 130. In a case in which the sample holder 10 has a square pillar shape, the cover part 130 may cover the bottom area and both side areas of the square pillar. In a case in which a plurality of sample holders 10 are arranged in parallel to each other, the cover part 130 may cover the outermost side areas of the sample holders 10. The cover part 130 can prevent heat from being lost to the outside when the sample holder 10 is being heated, thereby improving energy efficiency. In addition, the cover part 130 can prevent excitation light generated by an optics mechanism and emission light generated by the samples from leaking to the outside, thereby improving the accuracy of detection. In the figures, an embodiment in which the slot space 200 is open upward such that the first cover 130a covers the bottom area and one side area of the sample holder 10 and the second cover 130b covers the opposite side area of the sample holder 10 opposite one side area covered with the first cover 130a is illustrated.

The thermal module 110 performs the amplification reaction of the target nucleic acid included in the sample held in the sample holder 10 by heating or cooling the sample holder 10. More specifically, the thermal module 110 heats or cools one area 11 of the sample holder 10. The thermal module 110 includes the thermal conductor 111 contacted to one area 11 of the sample holder 10. The heating or cooling of the sample holder 10 may be indirectly performed on the sample holder 10 through the thermal conductor 111. According to an embodiment, the thermal module 110 may include a heating element 112 thermally connected to the first thermal conductor 111 to perform a heating operation to heat the sample holder 10 and a thermoelectric element 310 thermally connected to the first thermal conductor 111 to perform a cooling operation to cool the sample holder 10 (see FIG. 3). The thermal cycler 100 according to the present disclosure may include a control module to control the heating element 112 of the thermal module 110 and the thermoelectric element 310. The control module may additionally control a heating element 122 of the thermal pusher 120 and the movement unit 126. According to an embodiment, the control module may be implemented as printed circuit boards (PCBs, see

reference numerals

160a and 160b).

In addition, the heating element 112 of the thermal module 110 may be a resistance heating module, for example, a resistance heating module of 12 V and 30 W. The heating element 112 of the thermal module 110 may be appropriately selected from 12V/24V and 10W/20W/30W/40W/50W/60W. The thermoelectric element 310 may be a Peltier element or a thermoelectric cooler (TEC). The thermoelectric element 310 may be thermally connected to a heat sink 114, and may be thermally connected to the heat sink 114 through the metal block 113. In FIGS. 5 and 6, an embodiment in which the thermoelectric element 310 is connected to the heat sink 114 through the metal block 113 is illustrated. A blower fan may be coupled to the heat sink 114. In addition, the thermal module 110 may be provided with a temperature sensor to measure the temperature of at least one of the sample holder 10 and the thermal conductor 111. Thus, the thermal module 110 may sense whether or not the sample holder 10 is heated or cooled to a proper temperature. The temperature sensor may be provided in the thermal pusher 120.

In addition, the thermal conductor 111 of the thermal module 110 may be implemented as a metal layer. Thus, the heating or cooling of the sample holder 10 by the thermal module 110 can be rapidly performed. The thermal conductor 111 is implemented as a metal layer having low heat capacity and a wide surface area, thereby allowing the heating of the sample holder 10 by the heating element 112 of the thermal module 110 and the cooling of the sample holder 10 by the thermoelectric element 310 to be rapidly performed. This metal layer is made of a metal having high thermal conductivity, for example, aluminum (Al), copper (Cu), silver (Ag), or combinations thereof, an Al-Mg alloy, an Al-Si alloy, gold (Au), or tungsten (W).

Next, the thermal pusher 120 includes the pressure part 124 press-contacting the sample holder 10 to the thermal module 110 and the heating part 121 heating the opposite area of one area 11 of the sample holder 10.

The sample holder 10 is press-contacted to the thermal module 110 by the pressure part 124, and thus, the heating part 121 coupled to the area of the support unit 125 facing the thermal module 110 is also press-contacted to the sample holder 10. Specifically, the sample holder 10 is press-contacted to the thermal conductor 111 of the thermal module 110 and the thermal conductor 123 of the heating part 121 by the pressure part 124. The pressure part 124 includes the support unit 125 supporting the sample holder 10 and press-contacting the sample holder 10 to the thermal module 110 and the movement unit 126 moving the support unit 125 in the direction of the thermal module 110. In response to the movement unit 126 moving the support unit 125 in the direction of the thermal module 110, the width of the slot space 200 is narrowed, the

thermal conductors

111 and 123 on both sides move closer to each other, and the sample holder 10 is press-contacted to the

thermal conductors

111 and 123 on both sides. As the sample holder 10 is press-contacted to the

thermal conductors

111 and 123 on both sides, the heating or cooling of the sample holder 10 is rapidly performed and the loss of heat to the outside is prevented.

The support unit 125 may be configured such that the lower portion is coupled to the movement unit 126 and the upper portion opposes the sample holder 10. The heating part 121 may be coupled to the top area of the support unit 125 facing the thermal module 110 and be press-contacted to the opposite area 12 of the sample holder 10 by the pressure part 124. The lower portion of the support unit 125 may be screw-engaged with a bolt screw 412 of the movement unit 126, and a nut screw screw-engaged with the bolt screw 412 may be provided on the lower portion of the support unit 125. Although a area of the support unit 125 facing the thermal module 110 is illustrated as being a flat surface in the figures, the form of the support unit 125 is not limited thereto. Rather, the support unit 125 may be configured to correspond to the form or number of the sample holders 10. That is, the support unit 125 may have a form capable of being moved by the movement unit 126 and press-contacting the sample holder 10 to the

thermal conductors

111 and 123 on both sides. The form of the support unit 125 may be appropriately changed depending on the form or number of the sample holders 10.

Referring to FIG. 4, the movement unit 126 is coupled to the lower portion of the support unit 125, and moves the support unit 125 in the direction of the sample holder 10. According to an embodiment, the movement unit 126 includes: a rail 413 providing a movement path to the support unit 125; a motor 415 and a gear part 414 rotating the bolt screw 412; and a base 411 supporting the rail 413, the bolt screw 412, the motor 415, and the gear part 414. The rail 413 penetrates the lower portion of the support unit 125, and both ends of the rail 413 are coupled to the base 411. A pair of rails 413 may be provided on both sides of the bolt screw 412 and coupled to both sides of the lower portion of the support unit 125. The bolt screw 412 is screw-engaged with while penetrating the lower portion of the support unit 125, and both ends of the bolt screw 412 are rotatably coupled to the base 411. For example, both ends of the bolt screw 412 may be coupled to the base 411 through bearings. As the motor 415 rotates the bolt screw 412 through the gear part 414, the support unit 125 is moved by screw-engagement of the support unit 125 and the bolt screw 412. The movement unit 126 may be implemented as a means capable of moving the support unit 125, and may be configured to include a cam, a cylinder, a linear actuator, etc.

The heating part 121 may heat the sample holder 10 together with the thermal module 110. That is, the thermal module 110 heats one area 11 of the sample holder 10, and the heating part 121 heats the opposite area 12 of the sample holder 10. The heating operation of the thermal module 110 and the heating operation of the heating part 121 may be performed simultaneously. The heating element 122 of the heating part 121 may be controlled to heat the sample holder 10 at one of at least two temperature points. For example, to perform a denaturation operation, the heating operation of the thermal module 110 and the heating operation of the heating part 121 may be performed simultaneously. The heating of the sample holder 10 caused by the heating operation of the heating part 121 may be additionally performed with respect to the heating of the sample holder 10 caused by the heating operation of the thermal module 110. That is, the thermal module 110 may operate as a main heating part of the sample holder 10, while the heating part 121 may operate as an auxiliary heating part of the sample holder 10.

In addition, during the cooling operation of the thermal module 110, the heating part 121 may heat the sample holder 10 at a temperature lower than that at which the sample holder 10 is heated during the heating operation. That is, the heating part 121 may heat the sample holder 10 by performing the heating operation simultaneously with the heating operation of the thermal module 110, and may also heat the sample holder 10 during the cooling operation of the thermal module 110. For example, in a case in which the thermal module 110 performs the cooling operation to cool the sample holder 10 in order to perform the annealing and extension operations of the primer after the denaturation operation is performed, the heating part 121 may heat the sample holder 10 at a temperature lower than that at which the sample holder 10 is heated in the heating operation performed simultaneously with the heating operation of the thermal module 110 in the denaturation operation. The denaturation operation is performed at a temperature of about 95°C, and the annealing and extension operations are performed at a temperature of about from 55°C to 75°C. Thus, when the sample holder 10 is cooled by the thermal module 110, the heating part 121 may heat the sample holder 10 so that the sample holder 10 maintains the temperature at which the annealing and extension operations are performed.

Referring to FIG. 3, the heating part 121 is coupled to a area of the support unit 125 facing the thermal module 110 so as to be press-contacted to the sample holder 10 by the pressure part 124. Specifically, the heating part 121 may be coupled to the upper portion of the support unit 125.

The heating part 121 includes the thermal conductor 123 contacted to the sample holder 10 and the heating element 122 to heat the thermal conductor 123. Like the heating element 112 of the first heating element 120, the heating element 122 of the heating part 121 is a component performing the heating operation to heat the sample holder 10. The heating element 122 of the heating part 121 may be a resistance heating module, for example, a resistance heating module of 12V and 10W. The heating element 122 may be appropriately selected from among 12V/24V and 10W/20W/30W/40W/50W/60W. Each of the sample holder 10 and the heating element 122 is thermally contacted to the thermal conductor 123 of the heating part 121. For example, the heating element 122 may be thermally contacted to the thermal conductor 123. The heating of the sample holder 10 caused by the heating element 122 is indirectly performed through the thermal conductor 123. That is, the heating element 122 is not directly thermally connected to the sample holder 10, but is indirectly thermally connected thereto through the thermal conductor 123. The thermal conductor 123 of the heating part 121 may be implemented as a metal layer, and thus, heat exchange between the sample holder 10 and the heating element 122 may be rapidly performed. The thermal conductor 123 is implemented as a metal layer having low heat capacity and a wide surface area, and thus, the sample holder 10 may be rapidly heated during the heating operation caused by the heating element 122. This metal layer is made of a metal having high thermal conductivity, for example, aluminum (Al), copper (Cu), silver (Ag), or combinations thereof, or an Al-Mg alloy, an Al-Si alloy, gold (Au), or tungsten (W).

Referring to FIGS. 5 and 6, the thermal pusher 120 includes an optic path 140 penetrating the pressure part 124 and the heating part 121. One end of the optic path 140 is open in the direction of the thermal module 110. The optic path 140 provides an optic path through which excitation light radiating on the sample in the sample holder 10 and emission light emitted from the sample are transmitted and received. The optic path 140 penetrates the upper portion of the support unit 125, the heating element 122, and the thermal conductor 123 and is open in the direction of the thermal module 110. Thus, as the sample holder 10 is press-contacted to the

thermal conductors

111 and 123 on both sides by the pressure part 124, one end of the optic path 140 is press-contacted to the opposite area 12 of the sample holder 10. More specifically, the optic path 140 is configured such that one end thereof is press-contacted to an internal position of the sample holder 10 in which the sample is positioned, such that excitation light can be accurately provided to the sample held in the sample holder, and emission light emitted from the sample can be detected in real time during the amplification reaction.

The excitation light generated by the optics mechanism (not shown) passes through the opposite area 12 of the sample holder 10 so as to be incident on the sample through one end of the optic path 140 open in the direction of the thermal module 110, while emission light emitted from the sample passes through the opposite area 12 of the sample holder 10 so as to be detected by the optics mechanism. That is, one or more optic fibers 150 are inserted into the optic path 140, and the optics mechanism may radiate excitation light on the sample through the optic fibers 150 or detect emission light emitted by the sample through the optic fibers 150. Here, it is preferable that one end of each of the optic fibers 150 does not protrude from one end of the optic path 140. The optic fibers 150 inserted into the optic path 140 may be a bundle of optic fibers. The excitation light radiated by the optics mechanism may include one or more wavelengths, and the emission light may also include one or more wavelengths corresponding to those of the excitation light. Thus, the optic fibers 150 may be a single optic fiber or a plurality of optic fibers such that the excitation light and the emission light each including one or more wavelengths can be transmitted and received through the optic fibers 150. A lens may be provided at open one end of optic path 140 in the direction of the thermal module 110. The excitation light and the emission light may be radiated on the sample or detected by the optics mechanism through the lens.

The optic fibers 150 inserted into the optic path 140 move along with the support unit 125 being moved by the movement unit 126. Since the optics mechanism radiates or detects light through the optic fibers 150 inserted into the optic path 140, it is not required to move the optics mechanism, thereby improving structural stability. In addition, a movement part for moving the optics mechanism may be simplified into the movement unit 126 for moving the support unit 125, thereby reducing fabrication costs.

In addition, the optic path 140 may be provided in a position corresponding to the sample holder 10. That is, in a case in which the slot space 200 is configured such that a plurality of sample holders 10 are positioned therein, the optic paths 140 are provided in positions corresponding to the plurality of sample holders 10, respectively. For example, as illustrated in the figures, in a case in which three sample holders 10 are positioned in the slot space 200, three optic paths 140 are provided in positions corresponding to the three sample holders 10, respectively.

The other end of the optic path 140 may be open in the direction of one of the rear area, the top area, the bottom area, the left side area, or the right side area of the pressure part 124. That is, the other end of the optic path 140 may be open to one of the rear area, the top area, the bottom area, the left side area, or the right side area of the support unit 125. The optic fibers 150 are inserted into the other end of the optic path 140, and the distal ends of the optic fibers 150 may be positioned on one end of the optic path 140. In the figures, an embodiment in which the other end of the optic path 140 is open in the direction of the rear area of the support unit 125 is illustrated.

Hereinafter, a method of operating the thermal cycler according to the present disclosure will be described. The method of operating the thermal cycler according to the present disclosure is an operation method for performing an amplification reaction of target nucleic acid included in a sample by heating or cooling the sample holder 10.

The method of operating the thermal cycler according to the present disclosure is a method of operating the thermal cycler 100 including the thermal module 110 to heat or to cool one area 11 of the sample holder 10 containing the sample, the thermal pusher 120 including the pressure part 124 press-contacting for the sample holder 10 to the thermal module 110 and the heating part 121 heating the opposite area 12 of the sample holder 10, and the slot space 200 defined between the thermal module 110 and the thermal pusher 120 and capable of positioning one or more sample holders 10 therein. The method includes the operations of: positioning the sample holder 10 in the slot space 200; press-contacting the sample holder 10 to the thermal module 110; heating the sample holder 10; and cooling the sample holder 10.

In the positioning operation, the sample holder 10 is positioned in the slot space 200. One or more sample holders 10 may be positioned in the slot space 200.

In the press-contacting operation, the sample holder 10 is press-contacted to the thermal module 110. That is, the sample holder 10 positioned in the slot space 200 by the thermal pusher 120 is press-contacted to the thermal module 110. As the movement unit 126 of the pressure part 124 moves the support unit 125 in the direction of the thermal module 110, the heating part 121 is press-contacted to the opposite area 12 of the sample holder 10, and then, as the support unit 125 is continuously moved, one area 11 of the sample holder 10 is press-contacted to the thermal module 110. Specifically, the thermal conductor 123 of the heating part 121 is press-contacted and thermally connected to the opposite area 12 of the sample holder 10, and one area 11 of the sample holder 10 is press-contacted and thermally connected to the thermal conductor 111 of the thermal module 110. Thus, one area 11 and the opposite area 12 of the sample holder 10 are thermally connected to the

thermal conductors

111 and 123 on both sides.

In the heating operation, the thermal module 110 heats one area 11 of the sample holder 10. The heating element 112 thermally connected to the thermal conductor 111 of the thermal module 110 may indirectly heat one area 11 of the sample holder 10 by heating the thermal conductor 111. In addition, in the heating operation, the heating part 121 of the thermal pusher 120 may heat the opposite area 12 of the sample holder 10. The heating element 122 thermally connected to the thermal conductor 123 of the heating part 121 may indirectly heat the opposite area 12 of the sample holder 10 by heating the thermal conductor 123.

In the cooling operation, the thermal module 110 cools one area 11 of the sample holder 10. The thermoelectric element 310 thermally connected to the thermal conductor 111 of the thermal module 110 may indirectly cool one area 11 of the sample holder 10 by cooling the thermal conductor 111. In addition, in the cooling operation, the heating part 121 may heat the opposite area 12 of the sample holder 10 at a temperature lower than that at which the sample holder 10 is heated in the heating operation.

According to an embodiment of the present disclosure, the operation of the heating part may be repeatedly performed a plurality of times by the number of cycles of the heating operation and the cooling operation. That is, one cycle of performing each step of the nucleic acid amplification process, i.e., the denaturation operation, the primer binding operation, and the extension operation, includes the heating operation and the cooling operation. As a result, since the nucleic acid amplification is performed by performing a plurality of cycles, the thermal module may repeat the heating operation and the cooling operation a plurality of times.

[CROSS-REFERENCE TO RELATED APPLICATIONS]

This application claims priority from Korean Patent Application No. 10-2020-0161328, filed on November 26, 2020, and No. 10-2020-0187060, filed on December 30, 2020, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference in its entirety.

Claims

A thermal cycler comprising:

a thermal module to heat or cool one area of a sample holder containing a sample;

a thermal pusher comprising a pressure part for press-contacting the sample holder to the thermal module and a heating part heating an opposite area of the sample holder opposite to the one area; and

a slot space defined between the thermal module and the thermal pusher and capable of positioning one or more sample holders therein.
The thermal cycler according to claim 1, wherein the pressure part comprises:

a support unit supporting the sample holder and press-contacting the sample holder to the thermal module; and

a movement unit moving the support unit in a direction of the thermal module,

wherein the heating part is coupled to a area of the support unit facing the thermal module and is press-contacted to the sample holder by the pressure part.
The thermal cycler according to claim 1, wherein the heating part comprises:

a thermal conductor to which the sample holder is to be thermally connected; and

a heating element to heat the thermal conductor.
The thermal cycler according to claim 3, wherein the thermal conductor comprises a metal layer.
The thermal cycler according to claim 3, wherein the heating element is controlled to heat the sample holder at one of at least two temperature points.
The thermal cycler according to claim 3, wherein the heating element comprises a resistance heating module.
The thermal cycler according to claim 1, wherein the slot space is configured in a form capable of positioning the sample holder in a longitudinal positioning.
The thermal cycler according to claim 1, wherein the slot space is configured in a form capable of positioning the sample holder having a square pillar shape therein.
The thermal cycler according to claim 1, wherein the thermal pusher comprises an optic path penetrating the pressure part and the heating part, and having one end being open in a direction of the thermal module.
The thermal cycler according to claim 9, wherein one or more optic fibers are inserted into the optic path.
The thermal cycler according to claim 9, wherein the optic path is provided in a position corresponding to each of the sample holders.
The thermal cycler according to claim 9, wherein a lens is provided in the open end of the optic path facing the thermal module.
The thermal cycler according to claim 9, wherein the other end of the optic path is open to one of a rear area, a top area, a bottom area, a left side area, or a right side area of the pressure part.
The thermal cycler according to claim 1, further comprising a cover part positioned in the slot space to cover at least a portion of areas of the sample holder not covered with the thermal module or the pressure part.
A method of operating a thermal cycler comprising a thermal module to heat or cool one area of a sample holder containing a sample, a thermal pusher comprising a pressure part for press-contacting the sample holder to the thermal module and a heating part heating an opposite area of the sample holder opposite the one area, and a slot space defined between the thermal module and the thermal pusher and capable of positioning one or more sample holders therein, the method comprising the operations of:

positioning the sample holder in the slot space;

press-contacting the sample holder to the thermal module;

heating the sample holder; and

cooling the sample holder.
The method according to claim 15, wherein, in the operation of the press-contacting, the heating part is press-contacted to the opposite area of the sample holder.
The method according to claim 16, wherein the one area of the sample holder is press-contacted to a thermal conductor of the thermal module.
The method according to claim 16, wherein a thermal conductor of the heating part is press-contacted to the opposite area of the sample holder.
The method according to claim 15, wherein, in the operation of the heating, the thermal module heats the one area of the sample holder.
The method according to claim 15, wherein, in the operation of the cooling, the thermal module cools the one area of the sample holder.
The method according to claim 15, wherein, in the operation of the heating, the heating part heats the opposite area of the sample holder.
The method according to claim 21, wherein, in the operation of the cooling, the heating part heats the opposite area of the sample holder at a temperature lower than a temperature to heat the sample holder in the operation of the heating.
The method according to claim 15, wherein the operation of the heating and the operation of the cooling are each repeatedly performed a plurality of time.