WO2022071716A1

WO2022071716A1 - Chamber for nucleic acid reaction, method for nucleic acid reaction using the same, and cartridge for sample processing comprising the same

Info

Publication number: WO2022071716A1
Application number: PCT/KR2021/013209
Authority: WO
Inventors: Jae Young Kim
Original assignee: Seegene, Inc.
Priority date: 2020-09-29
Filing date: 2021-09-28
Publication date: 2022-04-07
Also published as: KR20230058722A

Abstract

The disclosure relates to a nucleic acid reaction chamber. There is provided a nucleic acid reaction chamber including a body portion comprising a flow path and a sample region, wherein the flow path includes a first flow path and a second flow path, wherein the sample region is individually connected to lower portions of the first flow path and the second flow path, wherein the first flow path and the second flow path communicate with the outside, respectively. According to the disclosure, in spite of the small volume of the sample solution and the viscosity of the reaction mixture, the mixing of the sample solution and the reaction mixture may be uniformly performed, and the reliability of the detection result may be improved.

Description

CHAMBER FOR NUCLEIC ACID REACTION, METHOD FOR NUCLEIC ACID REACTION USING THE SAME, AND CARTRIDGE FOR SAMPLE PROCESSING COMPRISING THE SAME

The disclosure relates to a chamber for nucleic acid reaction, a method for the nucleic acid reaction using the same, and a cartridge for sample processing comprising the same.

As modern people's interest in health increases and life expectancy is prolonged, nucleic acid-based in vitro molecular diagnosis, such as accurate analysis of pathogens and gene analysis of patients becomes more significant and its demand is on the rise.

Nucleic acid-based molecular diagnosis is performed by extracting nucleic acids from a sample and then checking the presence or absence of a target nucleic acid among the extracted nucleic acids. The sample processing process of extracting nucleic acid from a sample includes sequentially mixing the sample and various reagents and removing residues other than the nucleic acid. Such a sample processing process requires an elaborate processing of a small amount of solution and, thus, is mostly performed manually by an experimenter or using a piece of liquid handling equipment that may be precisely controlled. Conventional liquid handling equipment is costly and needs professional manpower. Further, since it is a system that simultaneously processes a large number of samples, it takes a long time from sample collection to confirming the test result when the number of samples generated per hour is small, so it is not suitable for use in local hospitals and clinics where a small number of samples are to be processed.

The POC system for nucleic acid detection processes extraction and nucleic acid detection from a sample in one-step in one cartridge. The POC system is designed to proceed with the sample processing process immediately after sample collection, so it has strengths in the local medical field. The POC-based extraction process and the POC-based nucleic acid detection process are performed by sequentially moving the sample to a plurality of sample processing chambers and a plurality of a nucleic acid reaction chamber. The movement of the sample or its processed material between the chambers includes a method of forming an inter-chamber flow path and controlling it with a valve, and a method of moving a solution between the chambers by a liquid transport means. A cartridge used in the POC system for detecting nucleic acid includes an extraction chamber for performing a sample processing process for extracting nucleic acid, as well as a nucleic acid reaction chamber in which amplification of the extracted nucleic acid and optical measurement for detecting a target nucleic acid are performed.

Nucleic acid amplification reaction well known as polymerase chain reaction (PCR) includes repeated cycles of doube-stranded DNA denaturation, annealing of the oligonucleotide primers to DNA templates, and extension/elongation of the primers with the DNA polymerase (Mullis et al., U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354).

A sample solution containing the extracted nucleic acid is mixed with a reaction mixture containing dNTPs and the like to perform an amplification reaction, and the temperature of the mixed solution is changed according to a predetermined sequence and an amplification reaction is performed. The DNA denaturation is performed at about 95°C, and the annealing and primer extension are performed at a temperature lower than 95°C, i.e. a temperature ranging from 55°C to 75°C. In this case, the temperature and reaction time for each step of denaturation, annealing, and extension should be set differently depending on each sample, the nucleic acid to be analyzed, and the sequences of oligonucleotides such as primers and probes used for analysis. The sample solution and the reaction mixture used for this reaction are highly viscous, since they include various substances such as dNTPs, enzymes, and buffers. Furthermore, it is difficult to uniformly mix the sample solution and the reaction mixture due to the nature of the POC system that performs the amplification reaction with a small amount of solution. If the sample solution and the reaction mixture are not uniformly mixed, there is a problem in that it is difficult to detect the target nucleic acid by the amplification reaction. In addition, in the POC system, since the amplification reaction is performed with a small amount of solution, there is a problem of causing a fatal error in the detection result if even the slightest evaporation of the mixed solution occurs.

Therefore, a need arises for developing a nucleic acid reaction chamber and a nucleic acid reaction method capable of uniformly mixing the sample solution and the reaction mixture and preventing evaporation of the mixed solution during the amplification reaction.

In light of the above-described background, the inventors have made intensive researches to develop a nucleic acid reaction chamber and a nucleic acid reaction method capable of uniformly mixing a sample solution containing the extracted nucleic acid and a reaction mixture for performing a nucleic acid amplification reaction. Also efforts have been made to develop a nucleic acid reaction chamber and a nucleic acid reaction method capable of preventing evaporation of the mixed solution during the nucleic acid amplification reaction. As a result, the inventors have developed a nucleic acid reaction chamber including a body portion comprising a flow path and a sample region, wherein the flow path includes a first flow path and a second flow path, wherein the sample region is individually connected to lower portions of the first flow path and the second flow path, wherein the first flow path and the second flow path communicate with the outside, respectively, a cartridge for sample analysis including the same, and a nucleic acid reaction method using the same.

Accordingly, the disclosure aims to provide the nucleic acid reaction chamber including a body portion comprising a flow path and a sample region, wherein the flow path includes a first flow path and a second flow path, whererin individually connected to lower portions of the first flow path and the second flow path, wherein the first flow path and the second flow path communicate with the outside, respectively.

The disclosure also aims to provide the cartridge for the sample analysis including the nucleic acid reaction chamber.

The disclosure also aims to provide the nucleic acid reaction method using the nucleic acid reaction chamber.

The other objectives and advantages of the disclosure will be apparent to the embodiments and claims described below in detail with reference to the accompanying drawings.

According to an embodiment of the disclosure, there is provided a nucleic acid reaction chamber including a body portion comprising a flow path and a sample region, wherein the flow path includes a first flow path and a second flow path, wherein the sample region is individually connected to lower portions of the first flow path and the second flow path, wherein the first flow path and the second flow path communicate with the outside, respectively.

According to an embodiment of the disclosure, there is provided a cartridge for the sample analysis including the nucleic acid reaction chamber.

According to an embodiment of the disclosure, there is provided nucleic acid reaction method using the chamber for nucleic acid reaction, the method comprising: an injection step of injecting a sample solution through the flow path, a mixing step of mixing the reaction mixture in the nucleic acid reaction chamber and the sample solution to form a mixed solution, a covering step of forming a liquid layer of a water-immiscible material on the top side of the mixed solution and a nucleic acid reaction step of performing a nucleic acid reaction by controlling the temperature of the sample region.

According to the disclosure, in spite of the small volume of the sample solution and the viscosity of the reaction mixture, the mixing of the sample solution and the reaction mixture may be uniformly performed, and the reliability of the detection result may be improved.

In addition, according to the disclosure, it is possible to prevent evaporation in the mixed solution of the sample solution and the reaction mixture due to high temperature during the nucleic acid amplification reaction, thereby improving the accuracy of the detection result.

In addition, according to the disclosure, the mixed solution may be easily heated or cooled during the nucleic acid amplification reaction, and target nucleic acid detection using the fluorescence of the optical module may be easily performed.

FIG. 1 is a perspective view of a nucleic acid reaction chamber according to an embodiment of the disclosure.

FIG. 2 is a front view of a nucleic acid reaction chamber according to an embodiment of the disclosure.

FIG. 3 is a perspective view of a nucleic acid reaction chamber according to an embodiment of the disclosure.

FIG. 4 is a front view of a nucleic acid reaction chamber according to a nucleic acid reaction step according to an embodiment of the disclosure.

FIG. 5 illustrates a part of a nucleic acid reaction chamber according to an embodiment of the disclosure.

FIG. 6 is a side view of a cartridge for sample analysis according to an embodiment of the disclosure.

FIG. 7 is a flowchart of a nucleic acid reaction method according to an embodiment of the disclosure.

The configuration and effects of the present disclosure are now described in further detail in connection with embodiments thereof. The embodiments are provided merely to specifically describe the present disclosure, and it is obvious to one of ordinary skill in the art that the scope of the present disclosure is not limited to the embodiments.

The same or substantially the same reference denotations are used to refer to the same or substantially the same elements throughout the specification and the drawings. When determined to make the subject matter of the present disclosure unclear, the detailed description of the known configurations or functions may be skipped.

Such denotations as "first," "second," "A," "B," "(a)," and "(b)," may be used in describing the components of the present disclosure. These denotations are provided merely to distinguish a component from another, and the essence of the components is not limited by the denotations in light of order or sequence. When a component is described as "connected," "coupled," or "linked" to another component, the component may be directly connected or linked to the other component, but it should also be appreciated that other components may be "connected," "coupled," or "linked" between the components.

As used herein, the term "sample" may encompass biological samples (e.g., cells, tissues, or fluids from biological sources) and non-biological samples (e.g., foods, water, and soil). The biological samples include virus, germs, tissues, cells, blood (e.g., whole blood, plasma, and serum), lymph, bone marrow fluid, saliva, sputum, swab, aspiration, milk, urine, stool, ocular humor, semen, brain extracts, spinal fluid, joint fluid, thymus fluid, bronchoalveolar lavage fluid, ascites, and amniotic fluid. The sample may also include a natural nucleic acid molecule isolated from biological sources and a synthetic nucleic acid molecule. According to one embodiment of the present disclosure, the sample may include additional substances such as water, deionized saline, pH buffer, acidic solution, and basic solution.

Sample processing refers to a series of processes to primarily separate an analyte from the sample to thereby obtain a material in the state capable of detection reaction. The sample processing' may further include the process of detecting a target analyte from the substance in the detection reaction-capable state. The analyte may be, for example, a nucleic acid. According to an embodiment, the sample processing may include the process of extracting a nucleic acid.

A nucleic acid reaction refers to a series of physical and chemical reactions that generate a signal depending on the presence or amount of a nucleic acid of a specific sequence in a sample. The nucleic acid reaction may be a reaction including binding of a nucleic acid of a specific sequence in the sample to another nucleic acid or substance, and replication, cleavage or degradation of the nucleic acid of the specific sequence in the sample. The nucleic acid reaction may be a reaction involving a nucleic acid amplification reaction. The nucleic acid amplification reaction may include amplification of a target nucleic acid. The nucleic acid amplification reaction may be a reaction for specifically amplifying the target nucleic acid.

The nucleic acid reaction may be a signal-generating reaction, which is a reaction capable of generating a signal depending on the presence/absence or amount of the target nucleic acid in the sample. This signal-generating reaction may be a genetic analysis process such as PCR, real-time PCR, microarray.

Various methods are known for generating an optical signal indicative of the presence of the target nucleic acid using the nucleic acid reaction. Representative examples include: TaqMan™ probe method (US Pat. No. 5,210,015), molecular beacon method (Tyagi et al., Nature Biotechnology v.14 MARCH 1996), Scorpion method (Whitcombe et al., Nature Biotechnology 17:804- 807 (1999)), Sunrise or Amplifluor method (Nazarenko et al., 2516-2521 Nucleic Acids Research, 25(12):2516-2521 (1997), and U.S. Patent No. 6,117,635), Lux method (U.S. Patent No. 7,537,886), CPT (Duck P, et al. Biotechniques, 9:142-148 (1990)), LNA method (U.S. Patent No. 6,977,295), Plexor method (Sherrill CB, et al., Journal) of the Amerimay Chemical Society, 126:4550-4556 (2004)), Hybeacons™ (DJ French, et al., Molecular and Cellular Probes (2001) 13, 363-374 and U.S. Patent No. 7,348,141), double-labeled self- Quenched probe (U.S. Patent No. 5,876,930), hybridization probe (Bernard PS, et al., Clin Chem 2000, 46, 147-148), PTO cleavage and extension (PTOCE) method (WO 2012/096523), PCE-SH (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide Hybridization) method (WO 2013/115442), PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) method (PCT/KR2013/012312) and the CER method (WO 2011/ 037306).

A target nucleic acid may be amplified in a variety of ways. For example, as a method for amplification of a target nucleic acid molecule, the 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 3(1):1-6 (1993)), transcription-mediated amplification (Phyffer, et al., J. Clin. Microbiol. 34:834-841 (1996); 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. 12(1):75-99 (1999); Hatch et al., Genet. Anal. 15(2): 35-40 (1999)) and Q-Beta Replicase (Lizardi et al., BiolTechnology 6:1197 (1988)), and the like.

In particular, the nucleic acid amplification reaction may be performed while accompanying a change in temperature, and for example, the nucleic acid amplification device may include a denaturing step, an annealing step, an extension (or amplification) step (extension step) in order to amplify DNA (deoxyribonucleic acid) having a specific nucleotide sequence.

Referring to FIG. 1, the nucleic acid reaction chamber 100 according to an embodiment of the present disclosure includes a body portion 110. The body portion 110 includes a flow path and a sample region 130. The flow path includes a first flow path 130 and a second flow path 140. In other words, the body portion 110 includes a first flow path 130, a second flow path 140, and a sample region 150. A nucleic acid reaction of the sample solution is performed in the sample region 150.

First, an embodiment of the cartridge 600 for sample analysis of the present disclosure will be described with reference to FIG. 6. The nucleic acid reaction chamber 100 may configure a part of the cartridge 600 for analyzing a sample. The port 120 of the nucleic acid reaction chamber 100 may be coupled to the cartridge 600 for sample analysis. The cartridge 600 for the sample analysis may include a plurality of chambers, and some of the plurality of chambers may be the nucleic acid reaction chamber 100. The plurality of chambers may be disposed in one direction such as the longitudinal direction of the cartridge 600. The plurality of chambers are spaces for storing materials necessary for a process of extracting a substance to be detected from a sample and performing physical and chemical processes for extraction. The detection target substance may be, for example, a nucleic acid.

The plurality of chambers may include a sample chamber, a holding chamber, and the like. The sample chamber is a chamber in which the collected sample is accommodated, and the holding chamber is a chamber for holding materials necessary for the sample processing reaction, such as magnetic particles, crushing solution, washing solution, and elution solution, respectively.

The number of chambers included in the cartridge 600 of the present disclosure may be configured differently depending on the sample processing method. The number of chambers is not particularly limited, but may be, for example, 5, 6, 7, or 8 or more, and may be 20, 15, 14, 13, or 12 or less. In addition, the number of nucleic acid reaction chambers 100 included in the cartridge 600 of the present disclosure is not particularly limited, but may be, for example, 1 or 2 or more, and may be 5, 10, or 20 or less.

The cartridge 600 of the present disclosure is operated by a sample processing device (not shown) to perform a desired reaction. The cartridge 600 is accommodated in the sample processing device and may interact with various parts, such as a moving module or a heat transfer module included in the sample processing device.

The solution between each chamber may be transferred by a pipette module and the moving module of the sample processing device. After the cartridge 600 is accommodated in the sample processing device, the pipette module may move between the plurality of chambers by the moving module to transfer the solution.

The sample solution containing the extracted nucleic acid is injected into the nucleic acid reaction chamber 100 by the pipette module. That is, the sample solution may be injected into the nucleic acid reaction chamber 100 through a first opening 121 of the first flow path 130 and/or a second opening 122 of the second flow path 140.

Next, the nucleic acid reaction chamber 100 of the present disclosure will be described with reference to FIGS. 1 to 4 and the nucleic acid reaction method 700 of the present disclosure with reference to FIG. 7 will be described.

The nucleic acid reaction method 700 of the present disclosure is a nucleic acid reaction method using the nucleic acid reaction chamber 100, and includes an injection step S710, a mixing step S720, a covering step S730, and a nucleic acid reaction step S740.

The injection step S710 is a step of injecting the sample solution through the first flow path 130 and/or the second flow path 140 (refer to (A) and (B) of FIG. 4), and the injected sample solution is located in the sample region 150. The mixing step S720 is a step of mixing the reaction mixture (R) and the sample solution for the nucleic acid reaction to form a mixed solution (refer to (B) to (E) of FIG. 4), and the reaction mixture may be present in the nucleic acid reaction chamber 100. The covering (masking, coating, anti-vaporizing) step is a step of forming a liquid layer of a water-immiscible (hydrophobia, anti-vaporizing) material on the top side of the mixed solution (refer to (E) and (F) of FIG. 4). The nucleic acid reaction step S740 is a step to prevent evaporation of the mixed solution. The nucleic acid reaction step S740 is a step of performing a nucleic acid reaction by controlling the temperature of the sample region 150. For example, a thermal module of the sample processing device heats or cools the nucleic acid reaction chamber 100.

According to one embodiment of the nucleic acid reaction chamber 100 of the present disclosure, the body portion 110 further includes an accommodating portion 160, a partition portion 112 and the like as well as the first flow path 130, the second flow path 140, and the sample region 150.

According to an embodiment of the present disclosure, the body portion 110 is configured in a thin plate shape, and the first flow path 130, the second flow path 140, and the sample region 150 are configured to be engraved on one surface. The first flow path 130, the second flow path 140, the sample region 150 and the like may be covered by a barrier layer 310 attached to the one surface (refer to FIG. 3).

The first flow path 130 and the second flow path 140 may be paths which connect the first opening 121 and the first sample region 151 and connect the second opening 122 and the second sample region 152, respectively. At least one accommodating portion 160 configured to communicate with the flow path is provided in the body portion 110. That is, the body portion 110 is provided with at least one accommodating portion 160 configured to communicate with the at least one of the first flow path 130 and the second flow path 140. According to an embodiment of the present disclosure, the body portion 110 is provided with at least one accommodating portion 160 communicating with the first flow path 130 and the second flow path 140 and the first accommodating portion 161 communicates with the first flow path 130 and the second accommodating portion 162 communicates with the second flow path 140. The first flow path 130 includes a first auxiliary sample region 131, and the second flow path 140 includes a second auxiliary sample region 141, and the first auxiliary sample region 131 is located on the first sample region 151, and the second auxiliary sample region 141 is located on the second sample region 152.

The sample region 150 is individually connected to the lower portions of the first flow path 130 and the second flow path 140. That is, the lower portion of the first flow path 130 and the lower portion of the second flow path 140 are respectively connected to the sample region 150, and the lower portion of the first flow path 130 is connected to the first sample region 151 and the lower portion of the second flow path 140 is connected to the second sample region 152, respectively. The body portion 110 may include a partition wall 111 provided between the first flow path 130 and the second flow path 140, and the first flow path 130 and the second flow path 140 may be partitioned by the partition wall 111. In addition, the first flow path 130 and the second flow path 140 communicate with the outside, respectively so that the sample solution may be injected, and the sample solution is injected into the first flow path 130 and/or the second flow path 140 in the injection step S710. The sample solution is located in the sample region 150. In order for the injected sample solution to be positioned in the sample region 150, the first flow path 130 and the second flow path 140 may be configured to be inclined upwardly or vertically from one end toward the other end.

According to an embodiment of the present disclosure, the nucleic acid reaction chamber 100 may further include a port portion 120 provided on the top side of the body portion 110, and the first opening 121 through which the first flow path 130 communicates and the second opening 122through which the second flow path 140 communicates is provided on the top surface of the port portion 120. The body portion 110 and the port portion 120 may be integrally configured. The first flow path 130 and the second flow path 140 may communicate with the outside through the first opening 121 and the second opening 122 provided on the top surface of the port portion 120, respectively. Injection of the sample solution may be performed by the pipette module of the sample processing device, and the pipette tip of the pipette module accommodating the sample solution is inserted into the first opening 121 and/or the second opening 122, and the sample solution may be injected. In addition, air is injected or sucked through the first opening 121 or the second opening 122, and the sample solution may be mixed with the reaction mixture (R), and a nozzle for injecting or sucking the air may be inserted into the first opening 121 or the second opening 122.

In addition, the first passage 171 connecting the top portion of the first flow path 130 to the first opening 121 and the second passage 172 connecting the top portion of the second flow path 140 to the second opening 122 may be configured in the inside of the body portion 110. The first flow path 130, the second flow path 140, and the sample region 150 may be engraved on one surface of the body portion 110 as described above, and the first passage 171 and the second passage 172 may connect the passage on one surface of the body portion 110 and the opening on the top surface of the port portion 120 in the inside of the body portion 110 and the port portion 120, respectively.

In addition, the port portion 120 may include at least one recess 123 formed along the periphery of the first opening and/or second opening. The recess 123 may be configured on the top surface of the port portion 120 around the both of the periphery of the first opening 121 and the periphery of the second opening 122, respectively, or one of the periphery of the first opening 121 and the periphery of the second opening 122. The nucleic acid reaction chamber 100 may be configured of a material having a predetermined elasticity (e.g., polycarbonate (PC) or polypropylene (PP)). When the pipette tip or the nozzle is inserted into the opening of the port portion 120, the opening is opened outward by the recess 123, and the pipette tip or the nozzle may be closely coupled to the opening by the elastic force.

The sample solution located in the sample region 150 is mixed with the reaction mixture (R) through the mixing step S720. The reaction mixture (R) refers to a mixture of substances required for the nucleic acid reaction. The reaction mixture (R) may include at least one or more substances from the group consisting of dNTPs, primers, probes, buffers, salts and DNA polymerases. The mixed solution in which the sample solution and the reaction mixture (R) are mixed is heated or cooled in the nucleic acid reaction step S740, and the nucleic acid amplification reaction is performed.

According to one embodiment of the present disclosure, the reaction mixture (R) may be provided in the sample region 150. The reaction mixture (R) may be provided in a solid state in the sample region 150, and is injected in the injection step S710 and mixed with the sample solution located in the sample region 150. The solid reaction mixture may be freeze-dried or lyophilization to form pellets or cakes. The lyophilization of the reagent may be performed by a method known in the art, and includes dehydration by sublimation in a vacuum environment. The solid reaction mixture may contain a lyoprotectant such as sugar or polyalcohol for the lyophilization. The reaction mixture (R) may be injected through the opening of the first flow path 130 and/or the opening of the second flow path 140, but it is more convenient and quick to prepare the nucleic acid in advance in the body portion 110. The reaction may be performed, and the amount of the reaction mixture mixed with the sample solution may be more precisely controlled.

For example, the first flow path 130, the second flow path 140, and the sample region 150 are configured by being engraved on one surface of the body portion 110 and covered by the barrier layer 310 attached to the engraved surface. In this case, by providing the reaction mixture (R) in the sample region 150 through the engraved surface and attaching the barrier layer 310, the reaction mixture (R) may be prepared in advance inside the body portion 110. The reaction mixture (R) may be provided in the first sample region 151.

After the sample solution is injected into the sample region 151 where the reaction mixture is located, it is necessary to mix the sample solution in the sample region 151 so that the reaction mixture is uniformly mixed with the sample solution. According to one embodiment of the present disclosure, the mixing of the sample solution and the reaction mixture (R) may be performed by injecting the air or sucking the air through the first flow path 130 or the second flow path 140. That is, according to one embodiment of the present disclosure, the mixing step S720 includes an injection step of injecting the air and a suction step of sucking the air through the first flow path 130 or the second flow path 140. In the mixing step S720, the injection step and the suction step may be alternately performed at least two times or more, respectively. The injection step and the suction step are performed, and the sample solution located in the sample region 150 moves from the first flow path 130 toward the second flow path 140 or from the second flow path 140 toward the first flow path 130 and accordingly, the sample solution and the reaction mixture (R) are mixed.

According to an embodiment of the present disclosure, the body portion 110 of the nucleic acid reaction chamber 100 further includes a partition portion 112 partitioning the sample region 150 into the first sample region 151 and the second sample region 152 in in the longitudinal direction so that the sample solution and the reaction mixture (R) may be uniformly mixed, wherein the lower end of the partition portion 112 and the lower surface of the sample region 150 are spaced apart from each other and the first sample region 151 and the second sample region 152 are connected to each other.

Since the body portion 110 includes the partition portion 112, even when the sample solution is small, the sample solution and the reaction mixture (R) may be uniformly mixed. Despite the viscosity of the reaction mixture (R) the sample solution and the reaction mixture (R) may be uniformly mix. When the nucleic acid reaction step S740 is performed on a solution in which the sample solution and the reaction mixture (R) are not uniformly mixed, the nucleic acid amplification reaction is normally performed only in the region where the content of some reaction mixture (R) is relatively high, and no amplification reaction is performed or only a very small number of amplification reactions are performed in the region where the content of the remaining reaction mixture (R) is relatively low, and even if the amplification reaction is performed, there may be cases in which the target nucleic acid is not amplified, which may reduce the reliability of the detection result. The partition portion 112 is provided to prevent non-uniform mixing of the sample solution and the reaction mixture (R).

The partition portion 112 partitioning the sample region 150 in the longitudinal direction means that the sample region 150 is partitioned in the longitudinal direction of the first flow path 130 and the second flow path 140. That is, the sample region 150 is divided into the first sample region 151 connected to the first flow path 130 and the second sample region 152 connected to the second flow path 140 by the partition portion 112.

The first sample region 151 and the second sample region 152 are provided to be connected to each other, and the sample solution moves from the first flow path 130 toward the second flow path 140 or from the second flow path 140 toward the first flow path 130, and is mixed with the reaction mixture (R) as it reciprocates between the first sample region 151 and the second sample region 152.

The partition portion 112 may be configured in a vertical direction, and may be configured from the lower end of the partition wall 111 downward. That is, the partition wall 111 divides the first flow path 130 and the second flow path 140, and the partition portion 112 divides the first sample region 151 and the second sample region 152. The top end of the partition portion 112 is connected to the partition wall 111 and the lower end thereof is provided to be spaced apart from the lower surface of the sample region 150, and the first sample region 151 and the second sample region 152 are connected to each other.

In addition, according to an embodiment of the present disclosure, the partition portion 112 may be configured such that the first sample region 151 is larger than the second sample region 152. The first sample region 151 is partitioned larger than the second sample region 152 means that the sample region 150 is not exactly divided in half by the partition portion 112, but that one is larger than the other. The first sample region 151 is larger than the second sample region 152 means that the volume of the first sample region 151 is larger than that of the second sample region 152. Preferably, the first sample region 151 has an average cross-sectional region larger than that of the second sample region 152. In other words, the volume ratio of the first sample region 151 and the second sample region 152 may be greater than a length ratio between a path between a portion connected to the first flow path 130 of the first sample region 151 and a portion connected to the second sample region 152, and the path between the portion connected to the second flow path 140 of the second sample region 152 and the portion connected to the first sample region 151.

As the first sample region 151 is partitioned larger than the second sample region 152, when the sample solution reciprocates between the first sample region 151 and the second sample region 152, a flow rate in the second sample region 152 is configured faster than that in the first sample region 151, and the sample solution and the reaction mixture (R) are quickly and uniformly mixed by the difference in their flow rates. That is, when the sample solution moves from the first sample region 151 to the second sample region 152, a slow flow rate is configured in the first sample region 151, but when the sample solution moves from the second sample region 152 to the first sample region 151, the high flow rate configured in the second sample region 152 generates a strong flow in the first sample region 151, and the sample solution and the reaction mixture (R) are quickly and uniformly mixed.

According to an embodiment of the present disclosure, the partition portion 112 may be located offset from the center of the sample region 150. As the partition portion 112 is located offset from the center of the sample region 150, one of the first sample region 151 and the second sample region 152 may be partition larger than the other. The partition portion 112 is positioned away from the center of the sample region 150 means that it is positioned to be biased toward the flow path 130 or the second flow path 140 based on the center of the portion connected to the first flow path 130 and the portion connected the second flow path 140 of the sample region 150. The partition portion 112 is positioned adjacent to the inner surface of the sample region 150 and the second sample region 152 may be configured with a narrow passage. Accordingly, the first sample region 151 is may be configured with a passage wider than the second sample region 152 as the remaining region.

In addition, as the partition portion 112 is positioned away from the center of the sample region 150, the target nucleic acid detection using the fluorescence of the optical module may be easily performed. For example, the sample processing device may include an optical module including a light source and a photodetector to detect the target nucleic acid, emit excitation light to the solution that has been subjected to the nucleic acid reaction step S740, and detect the fluorescence emitted from the nucleic acid. In order to prevent the optical path of the excitation light and the fluorescence from being obstructed, it is preferable that the partition portion 112 is located away from the center of the sample region 150.

According to an embodiment of the present disclosure, the first flow path 130 includes a first auxiliary sample region 131 provided on the first sample region 151, and the second flow path 140 includes a second auxiliary sample region 141 provided on the second sample region 152. The first auxiliary sample region 131 and the second auxiliary sample region 141 may prevent the sample solution moving between the first sample region 151 and the second sample region 152 from leaking into the opening of the first flow path 130 or the second flow path 140.

That is, by injecting or sucking the air into the opening of the first flow path 130 or the second flow path 140 in the mixing step S720, the sample solution moves between the first sample region 151 and the second sample region 152. When the sample solution in the second sample region 152 moves to the first sample region 151, the amount of the sample solution in the first sample region 151 increases, so that a portion of the sample solution may be introduced into the first flow path 130. Conversely, when the sample solution in the first sample region 151 moves to the second sample region 152, the amount of the sample solution in the second sample region 152 increases, so that a portion of the sample solution may be introduced into the second flow path 140. When the solution introducing into the first flow path 130 from the first sample region 151 leaks into the opening of the first flow path 130 or the solution introducing to the second flow path 140 from the second sample region 152 leaks into the opening of the flow path 140, the nozzle for injecting the air is contaminated or the sample solution is lost, which may cause an error in the detection result. The prepared solution is accommodated in the first auxiliary sample region 131 (refer to (C) of FIG. 4), and the solution introduced into the second flow path 140 from the second sample region 152 is accommodated in the second auxiliary sample region 141(refer to Fig. 4 (D)) and prevents the nozzle from contaminating or the sample solution from being lost.

The first auxiliary sample region 131 and the second auxiliary sample region 141 is provided between the portion connected to the first sample region 151 of the first flow path 130 and the first opening 121, and between a portion connected to the second sample region 152 of the second flow path 140 and the second opening 122, respectively. The first auxiliary sample region 131 may be located adjacent to the first sample region 151, and the second auxiliary sample region 141 may be located adjacent to the second sample region 152. The first auxiliary sample region 131 and the second auxiliary sample region 141 are parts of the first flow path 130 and the second flow path 140, respectively, and are configured to have a larger cross-sectional region than the remaining of the first flow path 130 and the second flow path 140 and may accommodate the sample solution. For example, the first auxiliary sample region 131 and the second auxiliary sample region 141 may be configured by recessing the partition wall 111.

According to an embodiment of the present disclosure, the second auxiliary sample region 141 may have a larger volume than the second sample region 152. That is, since the first sample region 151 is partitioned larger than the second sample region 152, the volume of the solution moved from the first sample region 151 to the second sample region 152 in the mixing step S720 may be larger than that of the second sample region 152. Accordingly, the volume of the solution introducing into the second flow path 140 from the second sample region 152 may be larger than that of the second sample region 152. In order to prevent the solution from leaking int the opening of the second flow path 140, the volume of the second auxiliary sample region 141 is preferably larger than that of the second sample region 152.

Also, according to an embodiment of the present disclosure, the second auxiliary sample region 141 may be equal to or larger than the first auxiliary sample region 131. In other words, the first auxiliary sample region 131 may be equal or smaller than the second auxiliary sample region 141. Since the first sample region 151 is partitioned larger than the second sample region 152, the amount of the sample solution contained in the first sample region 151 is greater than the amount of the sample solution contained in the second sample region 152. Therefore, when the solution moves between the first sample region 151 and the second sample region 152 in the mixing step S720, the volume of the solution flowing from the second sample region 152 into the second flow path 140 is larger than the volume of the solution introducing from the sample region 151 into the first flow path 130, so the volume of the second auxiliary sample region 141 is preferably equal to or larger than the volume of the first auxiliary sample region 131.

After mixing the sample solution and the reaction mixture (R) through the mixing step S720, a liquid layer of the water-immiscible material (C) is formed on the top side of the mixed solution through the covering step S730 (refer to (F) of FIG. 4). In the nucleic acid reaction step S740, which is the next step of the covering step S730, the nucleic acid reaction is performed by controlling the temperature of the sample region. During the denaturation step of the nucleic acid reaction, the mixed solution is heated to a temperature higher than about 95℃. Evaporation of the mixed solution due to the high temperature as described above may cause fatal errors in the detection result. By forming the liquid layer of the water-immiscible material (C) on the top side of the mixed solution in the covering step S730, which is the previous step of the nucleic acid reaction step S740, the evaporation of the mixed solution may be prevented. This water-immiscible material (C) should not substantially evaporate even at 110℃, should have a lower specific gravity than the mixed solution, and should not affect the nucleic acid reaction. The water-immiscible material (C) may be, for example, paraffin, wax or oil, but it is not limited thereto.

The water immiscible material (C) is injected into the body portion 110 through the openings of the first flow path 130 and the second flow path 140 after forming the mixed solution of the sample solution and the reaction mixture (R). However, like the reaction mixture (R), it is more convenient and quicker to perform the nucleic acid reaction by preparing the water-immiscible material (C) in advance in the inside of the body portion 110.

According to one embodiment of the present disclosure, the body portion 110 is provided with at least one accommodating portion 160 configured to communicate with the flow path. The accommodating portion 160 may be provided with the water-immiscible material (C) in a solid state as will be described later in detail. The accommodating portion 160 is configured to communicate with the first flow path 130 and the second flow path 140. At least one accommodating portion 160 is provided, and when one accommodating portion 160 is provided, the single accommodating portion 160 communicates with both the first flow path 130 and the second flow path 140. When two or more accommodating portions 160 are provided, at least one accommodating portion communicating with the first flow path 130 and one or more accommodating portions communicating with the second flow path 140 are respectively provided. The accommodating portion 160 is configured to be recessed inside the body portion 110. For example, the accommodating portion 160 is provided in the partition wall 111 and communicates with the flow path by the opening facing the flow path as shown in the drawing.

As will be described later, the water-immiscible material (C) is provided in the accommodating portion 160 in the solid state, and the solid water-immiscible material (C) is liquefied and moved to the sample region 150. The portion communicating with the flow path of the accommodating portion 160 may be inclined in the direction toward the flow path so that the miscible material (C) may be completely moved from the accommodating portion 160 to the sample region 150 in the accommodating portion 160. For example, when the first flow path 130 and the second flow path 140 are configured in the vertical direction, the portion where the accommodating portion 160 communicates with the first flow path 130 and the second flow path 140 may be configured to be inclined downward without a portion protruding upward. Referring to FIG. 5, the opening communicating with the flow path of the accommodating portion 160 is configured between the top and lower surfaces facing in the vertical direction, and the lower surface be configured to be at least flat in the horizontal direction (refer to reference numeral 512) or configured to be inclined downward (refer to reference numeral 514).

According to one embodiment of the present disclosure, the accommodating portion 160 may include a first accommodating portion 161 communicating with the first flow path 130 and the second accommodating portion 162 communicating with the second flow path 140. The first accommodating portion 161 and the second accommodating portion 162 may communicate with the first flow path 130 and the second flow path 140 at an upstream position of the sample region 150, respectively. Here, the term "upstream" refers to the direction from the first flow path 130 and the second flow path 140 toward the sample region 150. That is, as shown in the drawing, when the first flow path 130 and the second flow path 140 is configured in the vertical direction, the first accommodating portion 161 and the second accommodating portion 162 communicate the first flow path 130 and the second flow path 140 at the top side of the sample region 150, respectively.

The accommodating portion 160 is provided with the water-immiscible material (C). That is, the accommodating portion 160 is configured by being engraved on one surface of the body portion 110 like the first flow path 130, and a water-immiscible material (C) is provided in the accommodating portion 160 through the engraved surface. By attaching the barrier layer 310, the water-immiscible material (C) may be pre-positioned in the accommodating portion 160. The water-immiscible material (C) provided in the accommodating portion 160 may be provided in an amount sufficient to cover both the top sides of the mixed solution in the first sample region 151 and the second sample region 152.

When the water-immiscible material (C) is provided in advance in the accommodating portion 160, the water-immiscible material (C) is preferably provided in the accommodating portion 160 in the solid state. That is, the water-immiscible material (C) in the liquid state may leak from the accommodating portion 160 due to shaking of the nucleic acid reaction chamber 100, etc. In such a case, since it flows out through the opening of the flow path and does not cover all of the top side of the mixed solution, so evaporation occurs or it flows into the sample area 150 and may interfere with the mixing of the sample solution and the reaction mixture (R) in the mixing step S720, it is preferable to provide the water-immiscible material (C) in the solid state in the accommodating portion (160). The shape of the solid water-immiscible material (C) is not particularly limited, and may be, for example, a circle, an oval, a triangle, a square, a polygon, or an irregular shape.

The water-immiscible material (C) provided in the solid state in the accommodating portion 160 is heated in the covering step S730 and phase-changed to the liquid state. A thermal module for heating or cooling the mixed solution may heat and liquefy the water-immiscible material (C) in the solid state. The liquefied water-immiscible material (C) is separated from the accommodating portion 160 and moves to the sample region 150, and forms the liquid layer on the top side of the mixed solution. The water-immiscible material (C) accommodated in the first accommodating portion 161 forms the liquid layer on the top side of the mixed solution located in the first sample region 151, and the water-immiscible material (C) accommodated in the second accommodating portion 162 forms the liquid layer on the top side of the mixed solution located in the second sample region 152. The water-immiscible material (C) is required to have a low specific gravity so that the liquid layer may be located on the top side of the mixed solution (e.g., the specific gravity of paraffin is 0.9 g/cm^3). Since the first accommodating portion 161 and the second accommodating portion 162 communicate with the first flow path 130 and the second flow path 140 upstream from the sample region 150, respectively, the liquefied water-immiscible material (C) naturally moves to the sample region 150 without going through a separate device or step and forms the liquid layer.

The heating of the water-immiscible material (C) may be performed separately from the heating of the mixed solution, or may be automatically performed in the process of heating the sample region 150 to perform the denaturation step. That is, the denaturation step is performed at about 95℃. When using a water-immiscible material (C) with a melting point lower than 95℃ (for example, the melting point of paraffin is 47~64℃), the solid water-immiscible material (C) is heated and liquefied together in the process of thermal module to heat the body portion 110, and the liquefied water-immiscible material (C) is moved to the sample region 150 and the liquid layer may be configured on the top side of the mixed solution before the mixed solution reaches 95℃ and the evaporation may be prevented.

However, a structure that restricts the movement of the material located at the accommodating portion 160 may be configured in the body portion 110 so that the water-immiscible material (C) provided in the accommodating portion 160 in the solid state is not separated from the first flow path 130 or the second flow path 140. That is, as described above, the portion where the accommodating portion 160 and the flow path communicates with the accommodating portion 160 is inclined downward so that all of the liquefied water-immiscible material (C) moves from the accommodating portion 160 to the sample region 150 without remaining. There is a need to provide a movement limiting structure to the body portion 110 to prevent the solid water-immiscible material (C) before being liquefied from being separated from the accommodating portion 160. If the solid water-immiscible material (C) is separated from the accommodating portion 160 before the sample solution is injected, the solid water-immiscible material (C) blocks the flow path and may prevent the sample solution from being injected into the sample region.

Referring to FIG. 5, the opening communicating with the flow path of the accommodating portion 160 is configured between the top and lower surfaces facing each other in the vertical direction. The top surface protrudes in the direction toward the lower surface and a locking protrusion is formed, thereby forming the movement limiting structure in the body portion 110 (refer to reference numeral 511). Alternatively, the locking protrusion may extend in a direction toward the lower surface to form a narrow opening (refer to reference numeral 513). Alternatively, a blocking portion may be provided between the top and lower surfaces to form the movement limiting structure (refer to reference numeral 515).

After forming the liquid layer of the water-immiscible material (C) on the top side of the mixed solution, there is performed the nucleic acid reaction step S740 of controlling the temperature of the sample region 150 to perform the nucleic acid reaction. The temperature control of the sample region 150 may be performed, for example, by the thermal module of the sample processing device. The thermal module may heat the sample region 150 using a thermal conductive film, a peltier device, or the like, or cool the sample region 150 using a heat dissipation fan, or a heat sink.

As described above, the flow path and the sample region 150 are configured by being engraved on one surface of the body portion 110, the barrier layer 310 covering the flow path and the sample region 150 may be attached to the engraved surface of the body portion 110, and the temperature control of the sample region 150 may be performed by heat exchange through the barrier layer 310. That is, the barrier layer 310 is configured of a thermally conductive material, and for example, the thermal module is in contact with the barrier layer 310 to heat or cool the sample region 150. The barrier layer 310 may be, for example, an aluminum thin film. The barrier layer 310 may be attached only to the engraved surface of the body portion 110 as shown in (A) of FIG. 3 or the engraved surface and both side surfaces of the body portion 110 as shown in (B) of FIG.3. When the plurality of nucleic acid reaction chambers 100 are disposed in the longitudinal direction in the sample analysis cartridge 600, both side surfaces of the body portion 110 may be surfaces facing 100 faces each other in the nucleic acid reaction chamber as shown in FIG. 6. By attaching the barrier layer 310 to both side surfaces, the heat exchange is performed not only on the engraved side surface of the body portion 110 but also on both side surfaces when the thermal module adjusts the temperature of the sample region 150 in the nucleic acid reaction step S740, so that the heat exchange may be made more quickly and heat loss in the heating process may be prevented.

In addition, the body portion 110 may be configured of a transparent or translucent material, which is to facilitate the detection of the target nucleic acid after amplifying the nucleic acid through the nucleic acid reaction step S740. That is, as described above, the detection of the target nucleic acid may be performed, for example, by the optical module of the sample processing device. In order to detect the excitation light emitted by the optical module and the emission light emitted from the nucleic acid, the body portion 110 may be configured of the transparent or translucent material, for example, PC (Polycarbonate) or PP (Polyproplene). The optical module may detect the emitted excitation light or the emitted light through the opposite side surface of the engraved surface of the body portion 110. Therefore, it is preferable that the barrier layer is not attached to the opposite side surface thereof.

The examples described herein may be expanded to individual elements and concepts described herein, independently from other concepts, ideas, or systems and may be combined with elements cited anywhere in the present invention. Although some examples have been described in detail with reference to the accompanying drawings, the concept is not limited to such examples. Thus, the scope of the concept is intended to be defined by the appended claims and their equivalents. Further, specific features described individually or as some examples may be combined with other features described individually or other examples although not specifically mentioned for the specific features. Thus, the absence of a description of such combination should not be interpreted as excluding such combination from the scope of the present invention.

[CROSS-REFERENCE TO RELATED APPLICATIONS]

This application claims priority from Korean Patent Application No. 10-2020-0127008, filed on September 29, 2020, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference in its entirety.

Claims

A chamber for nucleic acid reaction, comprising:

a body portion comprising a flow path and a sample region, wherein the flow path includes a first flow path and a second flow path, wherein the sample region is individually connected to lower portions of the first flow path and the second flow path,

wherein the first flow path and the second flow path communicate with the outside, respectively.
The chamber for the nucleic acid reaction according to claim 1, wherein the body portion is provided with at least one accommodating portion configured to communicate with the flow path.
The chamber for the nucleic acid reaction according to claim 2, wherein the accommodating portion comprising a first accommodating portion communicating with the first flow path and a second accommodating portion communicating with the second flow path.
The chamber for the nucleic acid reaction according to claim 3, wherein the first accommodating portion and the second accommodating portion communicate with the first flow path and the second flow path upstream from the sample region, respectively.
The chamber for the nucleic acid reaction according to claim 2, wherein the accommodating portion is provided with a water-immiscible material.
The chamber for the nucleic acid reaction according to claim 5, wherein the water immiscible material is provided in a solid state in the accommodating portion.
The chamber for the nucleic acid reaction according to claim 2, wherein a structure that restricts the movement of the material located in the accommodating portion is configured in the body portion.
The chamber for the nucleic acid reaction according to claim 1, wherein the sample region is provided with a reaction mixture for the nucleic acid reaction.
The chamber for the nucleic acid reaction according to claim 1, further comprising: a partition portion dividing the sample region into a first sample region and a second sample region in the longitudinal direction,

wherein the lower end of the partition portion and the lower surface of the sample region are spaced apart each other, and the first sample region and the second sample region is connected to each other.
The chamber for the nucleic acid reaction according to claim 9, wherein the partition portion is configured that the first sample region is larger than the second sample region.
The chamber for the nucleic acid reaction according to claim 9, wherein the partition portion is located offset from the center of the sample region.
The chamber for the nucleic acid reaction according to claim 9, wherein the first flow path comprises a first auxiliary sample region provided on the first sample region, and the second flow path comprises a second auxiliary sample region provided on the second sample region.
The chamber for the nucleic acid reaction according to claim 12, wherein a volume of the second auxiliary sample region is larger than that of the second sample region.
The chamber for the nucleic acid reaction according to claim 12, wherein a volume of the second auxiliary sample region is the same as or larger than that of the first auxiliary sample region.
The chamber for the nucleic acid reaction according to claim 1, further comprising:

a port portion provided on the top side of the body and comprising the first opening through which the first flow path communicates and the second opening through which the second passage communicates.
The chamber for the nucleic acid reaction according to claim 15, wherein a first passage connecting a top portion of the first flow path to the first opening and a second passage connecting a top portion of the second passage to the second opening are configured in the body portion.
The chamber for the nucleic acid reaction according to claim 15, further comprising:

at least one recess formed along the periphery of the first opening and/or second opening.
The chamber for the nucleic acid reaction according to claim 1, wherein the flow path and the sample region are configured by being engraved on one surface of the body portion, and a barrier layer covering the flow path and the sample region is attached to the engraved surface of the body portion.
The chamber for the nucleic acid reaction according to claim 18, wherein the barrier layer is configured with a thermally conductive material.
The chamber for the nucleic acid reaction according to claim 1, wherein the body portion is configured with a transparent or translucent material.
A cartridge for sample analysis comprising the nucleic acid reaction chamber according to claim 1.
A nucleic acid reaction method using a chamber for nucleic acid reaction comprising a body portion comprising a flow path and a sample region, wherein the flow path includes a first flow path and a second flow path, wherein the sample region is individually connected to lower portions of the first flow path and the second flow path, wherein the first flow path and the second flow path communicate with the outside, respectively,

the method comprising:

an injection step of injecting a sample solution through the flow path;

a mixing step of mixing the reaction mixture in the nucleic acid reaction chamber and the sample solution to form a mixed solution;

a covering step of forming a liquid layer of a water-immiscible material on the top side of the mixed solution; and

a nucleic acid reaction step of performing a nucleic acid reaction by controlling the temperature of the sample region.
The method according to claim 22, wherein the covering step forms the liquid layer by heating the water-immiscible material provided in a solid state in at least one accommodating portion configured to communicate with the flow path inside the body portion.
The method according to claim 22, wherein the mixing step comprises an injection step of injecting air through the first flow path or the second flow path and a suction step of sucking the air.
The method according to claim 24, wherein the mixing step is that the injection step and the suction step are alternately performed at least two times or more.