Microfluidic device for detecting nucleic acids

Abstract

This invention relates to a microfluidic device for detecting nucleic acids, comprising: a chip body; a sample chamber formed inside the chip body and into which a sample is injected; a waste chamber spaced apart from the sample chamber and formed inside the chip body; a connecting channel formed inside the chip body connecting the sample chamber and the waste chamber to form a flow path for the sample in the sample chamber, and having an inlet connected to the sample chamber and an outlet connected to the waste chamber; a nucleic acid detection layer provided at the inlet of the connecting channel and having at least one micropore passing through the nucleic acid detection layer in the sample flow direction; and a probe connector formed on the surface of the nucleic acid detection layer, wherein the probe connector amplifies to detect the target nucleic acid by complementary binding with a target nucleic acid in the sample. Therefore, by installing the nucleic acid detection layer at the inlet of the connecting channel so that it flows through the micropores of the nucleic acid detection layer when flowing from the sample chamber to the connecting channel, bubble generation can be eliminated, thus enhancing the reproducibility of the detection.

Description

Technical Field

[0001] This disclosure relates to microfluidic devices for detecting nucleic acids, and more specifically, to microfluidic devices for detecting nucleic acids (capable of detecting nucleic acids such as RNA). Background Technology

[0002] Efficient amplification of target nucleic acids, such as those of viruses, is crucial not only for nucleic acid detection but also for DNA sequencing and cloning. Several methods have been proposed for nucleic acid amplification. Examples of nucleic acid amplification include polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (SSR), uncleic acid sequence based amplification (NASBA), and strand displacement amplification (SDA).

[0003] Many of these methods are less accurate in quantitative measurements and require expensive equipment. The accuracy is even lower when analyzing one or more target nucleic acids simultaneously.

[0004] Isothermal nucleic acid amplification reactions have been developed to overcome these shortcomings. Among isothermal reactions, rolling circle amplification (RCA) has received widespread attention. That is, several techniques such as PCR have traditionally been used to amplify circular DNA. However, these methods are time-consuming, inefficient, and costly in terms of manpower and resources.

[0005] The PCR method consists of the following processes: a denaturation process, in which DNA is separated into single strands in a reaction solution including primer pairs, template, polymerase, and dNTPs while the temperature is raised to a high temperature; an annealing process, in which primers complementary to each DNA single strand bind to the template while the temperature is lowered; and a polymerization process in which the new strands are polymerized using polymerase under a polymerization reaction while the temperature is raised again. Through this amplification, the DNA strands grow exponentially. However, because the PCR process involves the above steps, temperature changes are inevitable, and therefore, temperature controllers and heating devices must be provided in the PCR apparatus. However, when PCR is used for the amplification of target nucleic acids in lab-on-a-chip (LOC) systems, in addition to devices such as detection devices for LOCs, a separate temperature controller and heater are required for the PCR reaction. Therefore, there are disadvantages such as complex equipment and high equipment cost.

[0006] Several isothermal amplification methods have been proposed to address these drawbacks. Loop-mediated isothermal amplification (LAMP) is one such method, using six amplification primers to generate a branched, multi-loop product. This LAMP method has some limitations when used for early diagnosis or as a biosensor because it uses early reverse transcriptase (RT) to detect the target RNA.

[0007] As another isothermal amplification method, the RCA method has been proposed. The advantage of the RCA method is that it does not require the temperature changes needed in PCR amplification as described above, and therefore allows for the amplification of the target nucleic acid under isothermal conditions. Thus, amplification can be performed without a separate temperature control device, reducing the complexity and cost of the equipment.

[0008] In linear rolling circle amplification (LRCA), the target DNA sequence and an open circular probe hybridize to form a complex. This complex then ligates to form a target amplification loop. Primer sequences and DNA polymerase are then placed into the target amplification loop. The target amplification loop then forms a template, on which new DNA is formed. The template extends from the primers and into consecutive repeating sequences complementary to the target amplification loop, thereby generating thousands of nucleic acid copies per hour.

[0009] Exponential RCA (ERCA) is an improved version of LRCA (linear rolling circle amplification). ERCA uses additional primer sequences that bind to a clonal sequence complementary to the target loop to provide a new amplification center, thus providing exponentially increasing amplification. In ERCA, strand substitution is sequential. However, ERCA is limited to using the initial single-stranded RCA product as a template for another DNA synthesis, which uses a separate single-stranded linear primer attached to the product, without additional RCA.

[0010] Another approach is to use molecular padlock probes (MPPs) and rolling circle amplification (RCA) (C. Larsson et al., Nature Methods, 2004, 1, 227). This approach has several advantages. Specifically, it is highly specific and performs the amplification of complementary nucleic acids in the circular MPP by distinguishing the target nucleic acid sequence. In particular, it provides improved sensing sensitivity without a separate purification process due to the direct coupling of the RCA products. This method involves immobilizing the target nucleic acid probe on a surface such as gold or quartz through a simple chemical surface treatment, on which the RCA reaction can be initiated.

[0011] The following paper published in 2015 by Ho Yeon Lee et al., “DhITACT: DNA Hydrogel Formation by Isothermal Amplification of Complementary Target in Fluidic Channels (June 17, 2015, Advanced Materials, Vol. 27, Issue 23, Pages 3513-3517),” discloses a scheme for preparing an RCA reaction surface on the bottom surface of a microchannel and waiting approximately two hours for it to react with a sample solution to generate long single-stranded DNA. This scheme utilizes multiple dumbbell-shaped self-assemblies, allowing the DNA to form like a hydrogel, and thus preventing flow into the corresponding channels.

[0012] However, in the technique disclosed in Li Haoyan's paper, the RCA reaction surface is formed on the bottom surface of the microchannel, and this method requires waiting for the reaction period until the entire microchannel is blocked due to amplification from the RCA reaction surface. Therefore, the test time is 2 hours or longer.

[0013] Therefore, the applicant of this application has proposed a "microfluidic device for detecting a target gene" as disclosed in Korean Patent No. 10-1799192. In the microfluidic device disclosed in the Korean Patent, a microbead filler containing microbeads and a probe linker complementary to the target gene of the microbeads are formed, such that the target gene binds to the probe linker. The target gene is detected by utilizing the phenomenon that the gaps between the microbeads are blocked due to the amplification of the target gene bound to the probe linker.

[0014] Compared to existing RCA methods, microfluidic devices, such as those disclosed in a Korean patent registration, offer the advantage of reducing examination time by up to 15 minutes. However, the demand for faster examinations is increasing. This is because, in the event of a pandemic caused by a viral infectious disease, faster examination speeds are required.

[0015] Furthermore, in practical applications of microbeads disclosed in Korean patents, the microbeads are based on hydrogels and therefore cannot be stored in a dry state. Long-term storage will lead to performance degradation. Summary of the Invention

[0016] Technical Purpose

[0017] Therefore, this disclosure is intended to solve the above-mentioned problems and to provide a microfluidic device for detecting nucleic acids, wherein the testing time is reduced when detecting the target nucleic acid, and the nucleic acid detection layer can be stored in a dry state.

[0018] Technical solution

[0019] One aspect of the present invention provides a microfluidic device for detecting nucleic acids, the microfluidic device comprising: a chip body; a sample chamber defined in the chip body for receiving a sample therein; a waste chamber spaced apart from the sample chamber and defined in the chip body; a connecting channel defined in the chip body for connecting the sample chamber and the waste chamber to each other, the connecting channel serving as a flow path for the sample in the sample chamber and having an inlet connected to the sample chamber and an outlet connected to the waste chamber; a nucleic acid detection layer disposed in the inlet of the connecting channel, the nucleic acid detection layer having at least one or more micropores extending through the nucleic acid detection layer in the sample flow direction; and a probe connector formed on the surface of the nucleic acid detection layer, the probe connector being amplified and detecting the target nucleic acid via complementary binding to a target nucleic acid in the sample.

[0020] In one implementation, the micropores of the nucleic acid detection layer are blocked due to amplification via complementary binding between the probe linker and the target nucleic acid, or the size of the micropores is reduced due to amplification via complementary binding between the probe linker and the target nucleic acid, such that at least one of the final arrival distance of the sample to the linker channel, the arrival time of the sample to the final arrival distance, or the flow rate of the sample is altered, and the target nucleic acid is detected based on at least one of the final arrival distance, arrival time, or flow rate.

[0021] In one implementation, the microfluidic device further includes a stirring device installed in the sample chamber to stir the sample injected into the sample chamber. When the stirring device stirs the sample in the sample chamber, the probe linkers of the nucleic acid detection layer and the target nucleic acid in the sample complementarily bind to each other.

[0022] In one implementation, the sample in the sample chamber is stirred by a stirring device for a predetermined stirring duration and then flows along the connecting channel.

[0023] In one implementation, the microfluidic device further includes a sample heater for heating the sample in the sample chamber to a preset temperature range.

[0024] In one implementation, the preset temperature range is set to a value within the range of 30°C to 37°C, and the stirring duration is set to a value within the range of 5 minutes to 30 minutes.

[0025] In one implementation, the hydrodynamic force for causing the sample in the sample chamber to flow along the connecting channel includes at least one of the following: negative pressure from the waste chamber; gravity based on the tilt of the chip body; or head difference between the sample chamber in which the sample is received and the empty waste chamber.

[0026] In one implementation, after the sample is injected into the sample chamber, an oil immiscible with the sample is injected into the sample chamber. The oil is placed on the top surface of the sample to block the sample from the outside and increase the gravity or head difference that allows the sample to flow into the connecting channel.

[0027] In one implementation, the probe ligand includes: a coating portion coated on the surface of a nucleic acid detection layer; a primer that binds to the coating portion; and a template that binds to the primer in a complementary manner. The template includes: a first binding site that binds to the target nucleic acid; a second binding site that binds to the primer in a complementary manner; and a complementary third binding site in the template to form a dumbbell shape. The first binding sites are formed at two opposite ends of the template to be separated from each other, and the second binding site is formed between the separated first binding sites. A ligase that promotes complementary binding to the target nucleic acid is present at the first binding site.

[0028] In one implementation, the microfluidic device further includes a sample sensor for detecting a sample flowing along the connecting channel, wherein at least one of the final arrival distance, arrival time, or flow rate is measured based on the detection results of the sample sensor.

[0029] In one implementation, the connection channels include multiple connection channels defined in the chip body to individually connect the sample chamber and the waste chamber to each other, with each of the nucleic acid detection layers installed in the inlet of each of the connection channels; probe connectors formed on the surfaces of the nucleic acid detection layers are made of different materials that bind to different target nucleic acids respectively.

[0030] In one implementation, the probe connector is attached to the entrance of a nucleic acid detection layer installed in one of a plurality of connector channels, wherein one of the plurality of connector channels serves as a negative reference channel.

[0031] In one implementation, a probe linker formed on a nucleic acid detection layer mounted in one of a plurality of linking channels is configured such that nucleic acid amplification occurs regardless of the presence of the target nucleic acid, with one of the plurality of linking channels serving as a positive reference channel.

[0032] In one implementation, the nucleic acid detection layer includes a membrane or mesh in which micropores are formed.

[0033] Technical effect

[0034] According to the above configuration, and according to this disclosure, the nucleic acid detection layer is installed in the inlet of the connecting channel, so that when the sample flows from the sample chamber to the connecting channel, the sample flows directly through the micropores of the nucleic acid detection layer, thereby eliminating the bubble generation phenomenon and thus improving the reproducibility of the detection.

[0035] In addition, with the elimination of bubble formation, not only can the limitations of flow pressure control be eliminated, but the use of a single nucleic acid detection layer also allows for flow control at lower pressures than when using microbead packing.

[0036] Furthermore, existing microbead fillers are in the form of hydrogels, making them difficult to dry and store. However, the nucleic acid detection layer according to this disclosure is made of nylon or the like, allowing for dry storage. Attached Figure Description

[0037] Figure 1 This is a diagram illustrating a microfluidic device for detecting nucleic acids according to an embodiment of the present disclosure.

[0038] Figure 2 This is a schematic diagram illustrating a cross-section of a microfluidic device for detecting nucleic acids according to an embodiment of the present disclosure.

[0039] Figure 3This is a diagram illustrating an example of a nucleic acid detection layer in a microfluidic device for detecting nucleic acids according to an embodiment of the present disclosure.

[0040] Figure 4 This is a diagram illustrating an example of applying fluid dynamics to a sample in a microfluidic device for detecting nucleic acids according to an embodiment of the present disclosure.

[0041] Figure 5 This is a diagram illustrating an example of a sample sensor for a microfluidic device for detecting nucleic acids according to an embodiment of the present disclosure.

[0042] Figure 6 This is a diagram illustrating an example of a probe connector of a microfluidic device for detecting nucleic acids according to an embodiment of the present disclosure.

[0043] Figure 7 This is a diagram illustrating a microfluidic device for detecting nucleic acids according to another embodiment of the present disclosure. Detailed Implementation

[0044] In the following description, embodiments according to this disclosure will be described in detail with reference to the accompanying drawings.

[0045] Figure 1 This is a diagram illustrating a microfluidic device 100 for detecting nucleic acids according to an embodiment of the present disclosure. Figure 2 This is a schematic cross-sectional view of a microfluidic device 100 for detecting nucleic acids according to an embodiment of the present disclosure.

[0046] refer to Figure 1 and Figure 2 According to embodiments of the present disclosure, a microfluidic device 100 for detecting nucleic acids includes a chip body 110, a sample chamber 120, a waste chamber 150, a connection channel 140, a nucleic acid detection layer 130, and a probe connector 200 (see [link to relevant documentation]). Figure 6 ).

[0047] The chip body 110 is provided in the form of a microfluidic chip, which defines a sample chamber 120, a waste chamber 150, and a connection channel 140. The upper and lower bodies of the chip body can be connected to each other, so that the sample chamber 120, the waste chamber 150, the connection channel 140, etc. are defined therein.

[0048] A sample chamber 120 is defined within a chip body 110, and a sample S is injected into the sample chamber 120. An example is illustrated where the sample chamber 120 is provided in an edge region on one side of the chip body 110, according to this disclosure. Figure 2 As shown, an example is illustrated in which the sample chamber 120 is formed at a height relatively higher than the height of the connecting channel 140.

[0049] The waste chamber 150 is spaced apart from the sample chamber 120 and is formed in another edge region of the chip body 110 and within the chip body 110.

[0050] A connection channel 140 is defined in the chip body 110 to connect the sample chamber 120 and the waste chamber 150 to each other. In this respect, the connection channel 140 serves as a flow path through which the sample S injected into and received in the sample chamber 120 flows toward the waste chamber 150.

[0051] That is, the connecting channel 140 is formed in the form of a microchannel that communicates the sample chamber 120 and the waste chamber 150 with each other. According to this disclosure, an example in which the connecting channel has an inlet connected to the sample chamber 120 and an outlet connected to the waste chamber 150 is illustrated.

[0052] The nucleic acid detection layer 130 is installed in the inlet of the connecting channel 140. In this respect, at least one or more micropores 131 extending in the flow direction of the sample S are defined in the nucleic acid detection layer 130. Therefore, even when the nucleic acid detection layer 130 is installed to block the inlet of the connecting channel 140, the sample S may still flow toward the connecting channel 140 through the micropores 131.

[0053] Figure 3 This is a diagram illustrating an example of a nucleic acid detection layer 130 of a microfluidic device 100 for detecting nucleic acids according to an embodiment of the present disclosure. Figure 3 The embodiment shown is an example in which the nucleic acid detection layer 130 is provided in the form of a grid. A plurality of micropores 131 are defined in a square-shaped membrane. The nucleic acid detection layer 130 can be manufactured by stamping through the plurality of micropores 131 in a square nylon membrane. In another example, a membrane defining a plurality of micropores 131 can be used as the nucleic acid detection layer 130 according to the present disclosure.

[0054] In this respect, the micropores 131 of the nucleic acid detection layer 130 can be formed to have various sizes depending on the size of the target nucleic acid. Elements other than the target nucleic acid can pass through the micropores 131, thereby preventing the micropores 131 from being blocked by other elements. According to this disclosure, the diameter of the micropores 131 is, for example, determined in the range of 50 nm to 50 μm.

[0055] Probe connector 200 (see) Figure 6The probe linker 200 is formed on the surface of the nucleic acid detection layer 130. Preferably, the probe linker 200 may also be formed on the inner surface of the micropores 131 formed on the nucleic acid detection layer 130. In this regard, the hydrogel formed by amplification resulting from the complementary binding between the probe linker 200 formed on the surface of the nucleic acid detection layer 130 and the target nucleic acid can block the micropores formed in the nucleic acid detection layer 130, or the size of the micropores can be reduced. Therefore, when the sample S contained in the sample chamber 120 flows through the connection channel 140, the final arrival distance of the sample S, the arrival time to the final arrival distance, and the flow rate can be changed. Furthermore, at least one of the final arrival distance, arrival time, and flow rate can be used to detect the target nucleic acid.

[0056] Figure 4 This is a diagram illustrating an example of applying fluid dynamics to a sample S in a microfluidic device 100 for detecting nucleic acids according to an embodiment of the present disclosure.

[0057] In such Figure 4 In the embodiment shown in (a), with the waste chamber 150 blocked by a plug, the stirring device 121 stirs the sample S in the sample chamber 120 during the stirring time described above. Then, an object such as an injection needle 171 is used to puncture the waste chamber cover 151, forming an vent hole in the cover 151. Therefore, a head difference is created between the empty waste chamber 150 and the sample chamber 120, causing the sample S in the sample chamber 120 to flow through the micropores 131 of the nucleic acid detection layer 130 into the connecting channel 140.

[0058] In such Figure 4 In the embodiment shown in (b), an example is illustrated where a negative pressure device 172 (e.g., an injection pump) is used to provide negative pressure for the flow of sample S into waste chamber 150. Figure 4 In the embodiment shown in (c), the chip body 110 itself is tilted downward toward the waste chamber 150 to provide fluid dynamics to the sample S in the sample chamber 120, so that the sample flows toward the connection channel 140 under gravity.

[0059] In this regard, after the sample S is injected into the sample chamber 120, unmixed oil O (such as mineral oil O) can be injected into the sample chamber 120. Therefore, the oil O disposed on the top surface of the sample S blocks the sample S from the outside. That is, the oil not only prevents foreign substances from flowing into the sample S from the outside, but also prevents the sample S from contacting the outside air, so that the sample S does not evaporate into the air during the stirring process. Furthermore, the oil O provides an increased flow force due to the aforementioned gravity or head difference used for the flow of the sample S to the connecting channel 140.

[0060] Refer again Figure 1 ,exist Figure 1 In the embodiment shown, the sample sensor 160 has an LED module 161 for illuminating light and a photodiode 162 for detecting light emitted from the LED module 161. The LED module 161 is positioned on one side of the connection channel 140 to illuminate the interior of the connection channel 140. The photodiode 162 is disposed on the opposite side of the connection channel 140 opposite to the LED module 161 to receive light illuminating from the LED module 161. In this respect, the LED module 161 and the photodiode 162 can be arranged such that the connection channel 140 is disposed between them. Therefore, when the sample S flows along the connection channel 140, the sample S blocks the light illuminating from the LED module 161. Therefore, the maximum travel distance of the sample S along the connection channel 140 or the arrival time corresponding to the maximum travel distance can be detected.

[0061] As described above, the nucleic acid detection layer 130 according to the embodiments of this disclosure is installed in the inlet of the connection channel 140. Therefore, the sample S contained in the sample chamber 120 flows into the connection channel 140 through the micropores 131 of the nucleic acid detection layer 130 installed in the inlet of the connection channel 140. This prevents air bubbles from forming as the sample S flows along the connection channel 140 and passes through the micropores 131.

[0062] More specifically, in the “microfluidic device 100 for detecting target genes” disclosed in the aforementioned Korean Patent No. 10-1799192, microbead packing is installed in the middle region of the microchannel by way of example.

[0063] However, when sample S passes through the voids formed in the microbead packing, bubbles are generated at high flow rates. These bubbles interfere with the flow of sample S. Therefore, reducing the flow pressure to prevent bubble formation not only reduces the reproducibility of nucleic acid detection but also requires frequent flow pressure control, which limits the detection process.

[0064] Conversely, in the microfluidic device 100 for detecting nucleic acids according to an embodiment of the present disclosure, the nucleic acid detection layer 130 is installed in the inlet of the connecting channel 140. When the sample flows from the sample chamber 120 to the connecting channel 140, the sample S flows directly through the micropores 131 of the nucleic acid detection layer 130, thereby eliminating bubble generation and thus improving the reproducibility of the detection.

[0065] With the elimination of bubble formation, not only are the limitations of flow pressure control eliminated, but the use of a single nucleic acid detection layer 130 also allows for flow control at lower pressures than when using microbead packing.

[0066] In this regard, the microfluidic device 100 for detecting nucleic acids according to embodiments of the present disclosure may further include a stirring device 121 installed in the sample chamber 120, such as... Figure 1 and Figure 2 As shown in the image.

[0067] The stirring device 121 rotates within the sample chamber 120 and stirs the sample S injected into the sample chamber 120. Therefore, the sample S in the sample chamber 120 is uniformly contacted with the nucleic acid detection layer 130 installed at the inlet of the connection channel 140 while being stirred by the stirring device 121. Thus, the likelihood that the target nucleic acid in the sample S binds complementaryly to the probe connective 200 formed on the nucleic acid detection layer 130 during stirring increases.

[0068] Therefore, in the microfluidic device 100 for detecting nucleic acids according to an embodiment of the present disclosure, the sample S in the sample chamber 120 is stirred by the stirring device 121 for a predetermined stirring duration, and then the sample S flows along the connection channel 140. In this regard, the stirring duration is set to a value in the range of, for example, 5 minutes to 30 minutes. The stirring duration is preferably set taking into account the time required for complementary binding between the probe connector 200 and the target nucleic acid, and the testing time.

[0069] Furthermore, the sample S in the sample chamber 120 can be heated to a preset temperature range. In this regard, the temperature range is set to the optimal reaction temperature for the amplification process via complementary binding between the probe linker 200 and the target nucleic acid (i.e., PCA reaction). According to this disclosure, the temperature is set to a value in the range of 30°C to 37°C.

[0070] According to this configuration, during stirring, the target nucleic acid in sample S fully binds to the probe connectors 200 formed on the surface of the nucleic acid detection layer 130. Furthermore, when sample S flows along the connection channel 140 through the micropores 131 of the nucleic acid detection layer 130, the micropores 131 can either be blocked earlier or the time taken to reach the preset distance can be shortened. Therefore, the detection time can be reduced.

[0071] Furthermore, the target nucleic acid and probe linker 200 in sample S are connected to each other before sample S passes through microwell 131. Therefore, the possibility of bubble formation due to the flow of sample S through microwell 131 can be reduced.

[0072] Figure 5 This is a diagram illustrating an example of a sample sensor 160 of a microfluidic device 100 for detecting nucleic acids according to an embodiment of the present disclosure.

[0073] In such Figure 5In the embodiment shown in (a), the sample sensor 160 includes a sample sensing element 181, such as a rod made of a porous material or a litmus strip mounted within the connection channel 140. The sample sensing element 181 changes color when it reacts with the sample S. In this respect, the sample sensor 160 may include a camera and may be configured to detect the flow distance of the sample S based on images captured by the camera.

[0074] exist Figure 5 In the embodiment shown in (b), as an example of the sample sensor 160, the time when the sample S arrives at the waste chamber 150 can be determined based on the color change of the variable color substance 182 when the variable color substance 182, which changes color when reacting with the sample S, is contained in the waste chamber 150.

[0075] Figure 6 This is a diagram illustrating an example of a probe connector 200 of a microfluidic device 100 for detecting nucleic acids according to an embodiment of the present disclosure. (See reference) Figure 6 The illustration shows an example in which the probe connector 200 according to the present disclosure includes a coating portion 210, a primer 220 and a template 230.

[0076] The coating portion 210 is coated on the surface of the nucleic acid detection layer 130. The coating portion 210 is made of a material to which the primer 220 can attach and be fixed. For example, the coating portion 210 may contain carboxyl or amino groups. In a specific example, the coating portion 210 may contain one or more of the following: 5-hydroxydopamine hydrochloride, norepinephrine, epinephrine, pyrogallol, DOPA (3,4-dihydroxyphenylalanine), catechin, tannic acid, pyrogallol, catechol, heparin catechol, chitosan catechol, polyethylene glycol catechol, polyethyleneimine catechol, polymethyl methacrylate catechol, hyaluronic acid catechol, polylysine-catechol, and polylysine.

[0077] Primer 220 is immobilized to coating portion 210, and template 230 binds to primer 220 in a complementary manner. In this respect, template 230 includes a first binding site that binds to the target nucleic acid in a complementary manner, a second binding site that binds to primer 220 in a complementary manner, and a complementary third binding site in template 230, forming a dumbbell shape. Furthermore, the first binding sites are formed at two opposite ends of template 230 to separate them, and the second binding site is formed between the separated first binding sites.

[0078] In this respect, primer 220 may contain at least one group selected from the group consisting of: thiol, amine, hydroxyl, carboxyl, isothiocyanate, NHS ester, aldehyde, epoxide, carbonate, HOBt ester, glutaraldehyde, carbamate, imidazole carbamate, maleimide, aziridine, sulfone, vinylsulfone, hydrazine, phenyl azide, benzophenone, anthraquinone, and diene. Its terminal may be modified.

[0079] In this respect, under the above configuration, the target nucleic acid binds to the probe linker 200 according to the present disclosure and is thus amplified. The amplified target nucleic acids become entangled with each other to form a large-sized hydrogel. As a result, the micropores 131 formed in the nucleic acid detection layer 130 are blocked.

[0080] The probe connector 200 in the above example is configured, for example, based on the configuration disclosed in the paper "DhITACT: DNA hydrogel formation by isothermal amplification of complementary targets in fluid channels" published by Li Haoyan et al. in 2015 (June 17, 2015, Advanced Materials, Vol. 27, No. 23, pp. 3513-3517). Its detailed description is as described in the paper above.

[0081] Figure 7 This is a diagram illustrating a microfluidic device 300 for detecting nucleic acids according to another embodiment of the present disclosure.

[0082] according to Figure 7 The connection channels 340 in the microfluidic device 300 for detecting nucleic acids in the embodiment shown include a plurality of connection channels 340 defined in the chip body 310 for individually connecting the sample chambers 320a and 320b and the waste chamber 350 to each other.

[0083] In this respect, sample chambers 320a and 320b may include a sample receiving chamber 320a and a plurality of stirring chambers 320b. The stirring chambers 320b are connected to the waste chamber 350 via connection channels 340. That is, the inlet of each connection channel 340 can be connected to the corresponding stirring chamber 320b. Each nucleic acid detection layer 330 can be installed in the inlet of each connection channel 340.

[0084] In this regard, the probe connectors 200 formed on the surfaces of the nucleic acid detection layers 330, which are respectively installed in the entrances of the connection channels 340, can be made of different materials that bind to different target nucleic acids. Therefore, multiple target nucleic acids can be detected simultaneously using a single microfluidic device 300 for nucleic acid detection.

[0085] In this respect, according to this disclosure, the waste chamber 350 includes a plurality of water chambers respectively connected to the connection channels 340. However, in another example, a waste chamber 350 may be configured to be connected to a plurality of connection channels 340.

[0086] In another example, the nucleic acid detection layer 330 installed in one of the multiple connection channels 340 may not contain probe connectors. Therefore, the corresponding connection channel can serve as a negative reference channel. That is, in the nucleic acid detection layer 330 without attached probe connectors, no nucleic acid amplification reaction occurs, allowing the sample S to flow smoothly, which can be compared to the flow caused by blockage of other nucleic acid detection layers 330.

[0087] In another example, a probe linker formed on a nucleic acid detection layer 330 mounted on one of a plurality of linking channels 340 can be configured such that nucleic acid amplification occurs regardless of the presence of the target nucleic acid. Therefore, the corresponding linking channel can serve as a positive reference channel. That is, unlike the negative reference channel, blockage occurs in the positive reference channel at the start of sample S flow. This can be compared to the flow of sample S through the other linking channels 340.

[0088] While several embodiments of this disclosure have been shown and described, those skilled in the art to which this disclosure pertain will understand that modifications may be made to these embodiments without departing from the spirit or principles of this disclosure. The scope of this disclosure will be defined based on the appended claims and their equivalents.

[0089] Figure Labels

[0090] 100, 300: Microfluidic device; 110, 310: Chip body

[0091] 120: Sample chamber; 320a: Sample receiving chamber

[0092] 320b: Stirring chamber; 121, 321: Stirring device

[0093] 140, 330: Nucleic acid detection layer; 131: Microwell

[0094] 140, 340: Connecting channels; 150, 350: Waste chamber

[0095] 160: Sample sensor; 161: LED module

[0096] 162: Photodiode; 171: Injection needle

[0097] 172: Negative pressure device; 181: Sample detection component

[0098] 182: Variable color substance; 200: Probe connector

[0099] 210: Coating portion; 220: Primer

[0100] 230: Template

Claims

1. A microfluidic device for detecting nucleic acids, the microfluidic device comprising: Chip body; A sample chamber, defined within the chip body, for receiving a sample within the sample chamber; A waste chamber, which is spaced apart from the sample chamber and defined within the chip body; A connection channel is defined in the chip body to connect the sample chamber and the waste chamber to each other, wherein the connection channel serves as a flow path for the sample in the sample chamber and has an inlet connected to the sample chamber and an outlet connected to the waste chamber; A nucleic acid detection layer, installed in the inlet of the connecting channel at the junction of the sample chamber and the connecting channel, wherein the nucleic acid detection layer comprises a monolayer membrane or a monolayer mesh, the monolayer membrane or the monolayer mesh having at least one or more micropores extending through the monolayer membrane or the monolayer mesh in the flow direction of the sample, such that elements other than the target nucleic acid can pass through the at least one or more micropores; and A probe connector is formed on the surface of the nucleic acid detection layer; The probe linker is amplified and the target nucleic acid is detected via complementary binding with the target nucleic acid in the sample.

2. The microfluidic device according to claim 1, wherein, The micropores of the nucleic acid detection layer are blocked due to amplification via complementary binding between the probe linker and the target nucleic acid, or the size of the micropores is reduced due to amplification via complementary binding between the probe linker and the target nucleic acid, thereby altering at least one of the following: the final distance of the sample to the connection channel, the time of arrival of the sample to the final distance, or the flow rate of the sample. The target nucleic acid is detected based on at least one of the final arrival distance, the arrival time, or the flow rate.

3. The microfluidic device according to claim 1 further includes a stirring device, the stirring device being installed in the sample chamber to stir the sample injected into the sample chamber. in, When the stirring device stirs the sample in the sample chamber, the probe linker of the nucleic acid detection layer and the target nucleic acid in the sample complement each other and bind together.

4. The microfluidic device according to claim 3, wherein, The sample in the sample chamber is stirred by the stirring device for a predetermined stirring duration and then flows along the connecting channel.

5. The microfluidic device according to claim 4, further comprising a sample heater for heating the sample in the sample chamber to a preset temperature range.

6. The microfluidic device according to claim 5, wherein, The preset temperature range is set to a value within the range of 30°C to 37°C, and the predetermined stirring duration is set to a value within the range of 5 minutes to 30 minutes.

7. The microfluidic device according to claim 4, wherein, The hydrodynamic force used to cause the sample in the sample chamber to flow along the connecting channel includes at least one of the following: Negative pressure from the waste chamber; Based on the gravity caused by the tilt of the chip body; or The head difference between the sample chamber containing the sample and the empty waste chamber.

8. The microfluidic device according to claim 7, wherein, After the sample is injected into the sample chamber, an oil that is immiscible with the sample is injected into the sample chamber. The oil is disposed on the top surface of the sample to block the sample from the outside and increase the gravity or head difference that causes the sample to flow into the connecting channel.

9. The microfluidic device according to claim 1, wherein, The probe connector includes: The coating portion applied to the surface of the nucleic acid detection layer; Primers that bind to the coating portion; and A template that binds to the primer in a complementary manner; The template includes: The first binding site that binds to the target nucleic acid; A second binding site that binds to the primer in a complementary manner; and Complementary third binding sites in the template to form a dumbbell shape. The first binding sites are respectively formed at two opposite ends of the template so as to be separated from each other. The second binding site is formed between the separated first binding sites. The ligase that promotes complementary binding with the target nucleic acid is present at the first binding site.

10. The microfluidic device of claim 2, further comprising a sample sensor for detecting the sample flowing along the connecting channel. in, At least one of the final distance reached, the time of arrival, or the flow rate is measured based on the detection results of the sample sensor.

11. The microfluidic device according to claim 1, wherein, The connection channels include a plurality of connection channels defined within the chip body for individually connecting the sample chamber and the waste chamber to each other. Each of the nucleic acid detection layers is installed in the inlet of each of the plurality of connection channels; The probe connectors formed on the surface of the nucleic acid detection layer are made of different materials that bind to different target nucleic acids.

12. The microfluidic device according to claim 11, wherein, The probe connector is attached to the nucleic acid detection layer installed in the entrance of one of the plurality of connection channels, wherein one of the plurality of connection channels serves as a negative reference channel.

13. The microfluidic device according to claim 11, wherein, The probe connector formed on the nucleic acid detection layer mounted in one of the plurality of connection channels is configured such that nucleic acid amplification occurs regardless of the presence of the target nucleic acid, wherein one of the plurality of connection channels serves as a positive reference channel.

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