PCR module

Abstract

Disclosed is a PCR module in which a sample can be injected using a capillary effect. The PCR module includes a microfluidic chamber including an inlet portion for inputting a sample and manufactured by injection molding, a well array provided on a lower surface of the microfluidic chamber and including a plurality of micro wells, upper and lower portions of the micro wells being through, and a capillary member providing a path such that the sample input through the inlet portion reaches the micro wells by a capillary effect. When a PCR solution is moved to a reaction space using the capillary effect and the reaction space is filled with the solution, it is possible to prevent an air trap from being formed at a corner or an edge region of the well array, which is the reaction space. Accordingly, it is possible to prevent an error caused by the air trap from occurring in a PCR test result. Furthermore, since the well array is located on a CMOS photosensor array, a PCR reaction can be measured in real time.

Description

Technical Field

[0001] This application claims the priority interest based on Korean Patent Application No. 10-2021-0090030, filed on July 8, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] This disclosure relates to a PCR module; and more specifically, to a PCR module that prevents gas traps from appearing in the corners or edge regions of the reaction space of a microfluidic chamber, while enabling real-time PCR measurements. Background Technology

[0003] Gene amplification technology is an important process in molecular diagnostics, and it is a technique for repeatedly replicating and amplifying specific base sequences of trace amounts of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in a sample. Polymerase chain reaction (PCR) is a representative gene amplification technique, and it includes three steps: DNA denaturation, primer annealing, and DNA replication. Because each step depends on the sample temperature, DNA can be amplified by repeatedly changing the sample temperature.

[0004] Previously, PCR was typically performed in 96-well or 384-well microplates. When higher yields are required, conventional microplate PCR methods are neither cost-effective nor efficient. Meanwhile, reducing the PCR reaction volume reduces reagent consumption and amplification time while decreasing the thermal mass of the reaction volume. This strategy can also be implemented in an array format (m×n), allowing for many smaller reaction volumes. Furthermore, the use of any array allows for scalable high-yield analysis with increased quantitative sensitivity, dynamic range, and specificity.

[0005] To date, digital polymerase chain reaction (dPCR) has been performed using an array format. dPCR results can be used to detect and quantify the concentration of rare alleles, provide absolute quantification of nucleic acid samples, and also measure fold changes at low nucleic acid concentrations. Generally, increasing the replication count increases the accuracy and reproducibility of dPCR results.

[0006] Most quantitative polymerase chain reaction (qPCR) platforms are designed with array formats for sample-by-sample analysis, where PCR results must be available for subsequent analysis. However, for dPCR, the specific location or well for each PCR result is non-critical, and only the number of positive and negative copies per sample can be analyzed.

[0007] In dPCR, solutions containing small amounts of the target polynucleotide or nucleic acid sequence are partitioned into small test samples, each typically containing one molecule of the target nucleotide sequence or none at all. When the samples are thermally cycled in the PCR protocol, process, or experiment, samples containing the target nucleotide sequence are amplified and produce a positive detection signal, while samples not containing any target nucleotide sequence are not amplified and do not produce a detection signal.

[0008] In the case of this type of digital PCR, the reaction space is extremely small in size and extremely numerous, making it difficult to introduce samples into each reaction space.

[0009] Furthermore, because the sample is not injected into the corners or edges of the reaction space, air traps can form in those areas. These air traps expand or contract due to temperature changes during the PCR process, causing errors in the test results.

[0010] Existing main dPCR methods utilize endpoint PCR, which separates the process of introducing a sample into the reaction space from the process of amplifying the base sequence, making it impossible to measure the reaction in which the base sequence is amplified in real time in each reaction space. Summary of the Invention

[0011] Technical goals

[0012] Therefore, one technical challenge of this disclosure is to solve these conventional problems, and one objective of this disclosure is to provide a PCR module that allows real-time PCR measurement by injecting samples into the reaction space using capillary principles, while preventing air traps from appearing in the corners or edge regions of the reaction space in the microfluidic chamber.

[0013] Technical solutions

[0014] To achieve the objectives of the present disclosure as described above, a PCR module according to one embodiment of the present disclosure includes a microfluidic chamber, a pore array, and a capillary member; the microfluidic chamber includes an inlet unit and can be manufactured by injection molding, the inlet unit being formed for introducing a sample; the pore array includes a plurality of micropores having a through upper portion and a lower portion, and is disposed on the lower surface of the microfluidic chamber; the capillary member is configured to provide a path such that a sample introduced through the inlet unit reaches the micropores through capillary action.

[0015] Beneficial effects

[0016] According to this PCR module, when the PCR solution moves into the reaction space of the microfluidic chamber using capillary principles and fills the reaction space, it is possible to prevent air traps from forming in the corners or edges of the well array, which is located within the reaction space. Therefore, it is possible to prevent errors in PCR test results caused by air traps. Furthermore, because the well array is located above the CMOS photoelectric sensor array, it is possible to measure the PCR reaction in real time. Attached Figure Description

[0017] Figure 1 According to one embodiment of this disclosure, a perspective view is provided for interpreting a PCR module.

[0018] Figure 2 For the purpose of explanation Figure 1 The PCR module shown is a perspective view after decomposition.

[0019] Figure 3 For the purpose of explanation Figure 1 An exploded perspective view of the capillary component shown.

[0020] Figure 4 For the purpose of explanation Figure 1 The cross-sectional view of the PCR module shown. Detailed Implementation

[0021] To achieve the objectives of the present disclosure as described above, a PCR module according to one embodiment of the present disclosure includes a microfluidic chamber, a pore array, and a capillary member; the microfluidic chamber includes an inlet unit and can be manufactured by injection molding, the inlet unit being formed for introducing a sample; the pore array includes a plurality of micropores having a through upper portion and a lower portion, and is disposed on the lower surface of the microfluidic chamber; the capillary member is configured to provide a path such that a sample introduced through the inlet unit reaches the micropores through capillary action.

[0022] In one embodiment, the capillary component may include a first strip disposed between the microfluidic chamber and the pore array, including an inlet orifice, an input orifice, and a connecting orifice configured to connect the inlet orifice and the input orifice; the inlet orifice is formed corresponding to an inlet unit, the input orifice is formed corresponding to the pore array, and the connecting orifice has a width narrower than the diameter of the inlet orifice and is configured to connect the inlet orifice and the input orifice.

[0023] In one embodiment, the first strip may have a rectangular shape and a circular shape, with the circular shape superimposed on the corner region of the rectangular shape; wherein the rectangular shape may correspond to an array of holes, and the circular shape may correspond to an inlet unit of a microfluidic chamber.

[0024] In one embodiment, the input hole may be formed in a rectangular shape, the inlet hole may be formed in a circular shape, and the connection hole may be formed in the area where the rectangular and circular shapes overlap.

[0025] In one embodiment, the first strip may include a double-sided strip with an adhesive layer formed on the surface of the contact microfluidic chamber and the surface of the contact hole array, respectively.

[0026] In one embodiment, the input aperture may have a rectangular shape that is larger than the rectangular shape of the aperture array in order to expose the micropores of the aperture array.

[0027] In one embodiment, the aperture array may have four edge regions attached to the first strip.

[0028] In one embodiment, the side surface of the connecting hole forming the first strip may be hydrophilic.

[0029] In one embodiment, the capillary member may further include a second strip disposed on the underside of the aperture array and covering the connection aperture and input aperture formed in the first strip, wherein the second strip may correspond to the circular shape of the first strip.

[0030] In one embodiment, the surface of the second strip corresponding to the connecting hole may be hydrophilic.

[0031] In one embodiment, the thickness of the second strip may be the same as the thickness of the hole array.

[0032] In one embodiment, the second strip may be a single-sided strip with an adhesive layer formed on the surface in contact with the first strip.

[0033] In one embodiment, the microfluidic chamber may comprise a polydimethylsiloxane (PDMS) material.

[0034] In one embodiment, the microfluidic chamber may include a rectangular base member and a rectangular tower member; the base member includes a rectangular first flat unit, a rectangular support hole, and a circular hole, the support hole being formed in the central region of the first flat unit and the hole being formed in the corner region of the first flat unit; the tower member includes a rectangular second flat unit, a disc-shaped membrane switch, and a closed-loop inlet unit, and is disposed on the base member.

[0035] In one embodiment, the PCR module may further include a CMOS photoelectric sensor array disposed below the well array and capturing real-time reaction images of samples filling the microwells of the well array.

[0036] Methods of implementing the present invention

[0037] In the following description, the invention will be illustrated in more detail with reference to the accompanying drawings. Because this disclosure is subject to various modifications and forms, specific embodiments will be shown in the drawings and described in detail in the specific embodiments. However, this is not intended to limit the disclosure to any particular form; and it should be understood that all modifications, equivalents, and alternatives are included within the spirit and scope of this disclosure.

[0038] In describing each figure, similar reference numerals are used for similar parts. In the figures, for clarity of this disclosure, the dimensions of the structures are enlarged from the actual dimensions.

[0039] Terms such as "first" and "second" may be used to describe various components, but these components should not be limited by such terms. Such terms are used only for the purpose of distinguishing components from one another. For example, a first component may be referred to as a second component without departing from the scope of this disclosure; and similarly, a second component may also be referred to as a first component. Singular expressions include plural expressions unless the context clearly indicates otherwise.

[0040] In this specification, it should be understood that terms such as “comprising” or “having” are intended to specify the presence of features, quantities, steps, operations, components, parts or combinations thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, quantities, steps, operations, components, parts or combinations thereof.

[0041] Furthermore, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those generally understood by one of ordinary skill in the art to which this disclosure pertains. Terms as defined in commonly used dictionaries should be interpreted as having a meaning matching the contextual meaning in the relevant field and should not be interpreted as having an ideal or overly formal meaning, unless otherwise defined herein.

[0042] Figure 1 According to one embodiment of this disclosure, a perspective view is provided for interpreting the PCR module 100. Figure 2 For the purpose of explanation Figure 1 The PCR module 100 shown is a perspective view after decomposition. Figure 3 For the purpose of explanation Figure 1 An exploded perspective view of the capillary component 130 shown. Figure 4 For the purpose of explanation Figure 1 A cross-sectional view of the PCR module 100 shown.

[0043] refer to Figures 1 to 4 According to one embodiment of the present disclosure, a PCR module 100 includes a microfluidic chamber 110, a pore array 120, and a capillary component 130.

[0044] The microfluidic chamber 110 includes a base member 112 and a tower member 114. The base member 112 and the tower member 114 may be integrally formed. In the bottom region of the microfluidic chamber 110, a recessed space is formed to accommodate the pore array 120 and the capillary member 130. The microfluidic chamber 110 may include transparent and elastic materials. For example, the microfluidic chamber 110 may include a polydimethylsiloxane (PDMS) material. Therefore, the microfluidic chamber 110 can be manufactured by injection molding.

[0045] The base member 112 includes a rectangular first flat unit 112a, a rectangular support hole 112b, a circular first flat hole 112c, and a circular second flat hole 112d. The support hole 112b is formed in the central region of the lower portion of the first flat unit 112a, the first flat hole 112c is formed in the first corner region of the lower portion of the first flat unit 112a, and the second flat hole 112d is formed in the second corner region of the lower portion of the first flat unit 112a. The first and second corner regions face each other. The support hole 112b may correspond to the hole array 120; the first flat hole 112c may correspond to an inlet unit through which a sample is injected; and the second flat hole 112d may correspond to an outlet unit through which a sample is discharged.

[0046] The tower component 114 includes a disc-shaped membrane switch 114a and a closed-loop inlet unit 114b, and is disposed on the base component 112.

[0047] A membrane switch 114a is disposed in the central region of the tower member 114 and protrudes upward. The lower region of the membrane switch 114a corresponds to the support hole of the base member 112. Therefore, a recessed space is formed in the bottom portion of the microfluidic chamber 110. The CMOS image sensor and the aperture array 120 can be accommodated in this recessed space.

[0048] The inlet unit 114b has a fence shape and is formed for sample input. The inlet unit 114b protrudes upward on one side of the membrane switch 114a and prevents the sample from flowing out. An inlet is formed in the central region of the inlet unit 114b in the vertical direction of the base member 112. The sample is injected into the pore array 120 through this inlet via the capillary member 130.

[0049] The tower component 114 may also include a gripping unit 114c; from an observer's perspective, the gripping unit 114c protrudes upward on the opposite side of the membrane switch 114a. The gripping unit 114c may be configured to face the inlet unit 114b relative to the membrane switch 114a. The height of the gripping unit 114c may be greater than the height of the inlet unit 114b. The height of the inlet unit 114b and the height of the membrane switch 114a may be the same.

[0050] The pore array 120 includes a plurality of micropores having a through upper and lower portion and can be disposed on the lower surface of the microfluidic chamber 110. The pore array 120 can be disposed above the CMOS photoelectric sensor array 140 and can be inserted into a substrate hole formed in the PCB 150. The pore array 120 may include a plurality of micropores. The shape, size, and number of micropores can be varied. Each micropore can contain an analytical sample, such as a powder sample or a liquid sample. The analytical sample is a specific component used to analyze biological material. In other words, the analytical sample is a component used for quantitative or qualitative analysis of specific biological materials (such as proteins, DNA, RNA, etc.), referring to primers, probes, antibodies, aptamers, DNA or RNA polymerases, etc., and specifically referring to components necessary for real-time execution of polymerase chain reactions, isothermal enzyme reactions, ligase chain reactions (LCR), etc.

[0051] The capillary component 130 includes a first strip 132 and a second strip 134. The first strip 132 is disposed between the microfluidic chamber 110 and the pore array 120, and the second strip 134 is disposed on the underside of the pore array 120. It provides a path for a sample introduced through the inlet unit 114b to reach the micropores via capillary action. Here, capillary action refers to the property of movement along a narrow space (such as a tube or porous material), regardless of external forces such as gravity.

[0052] The first strip 132 may have a rectangular shape and a circular shape, with the circular shape superimposed on the corner region of the rectangular shape; wherein the rectangular shape may correspond to the aperture array 120, and the circular shape may correspond to the inlet unit 114b of the microfluidic chamber 110.

[0053] The first strip 132 may include an inlet hole 132a, an input hole 132b, and a connecting hole 132c; the inlet hole 132a is formed corresponding to the inlet unit 114b; the input hole 132b is formed corresponding to the hole array 120; the connecting hole 132c has a width narrower than the diameter of the inlet hole 132a and is configured to connect the inlet hole 132a and the input hole 132b. The input hole 132b may be formed in a rectangular shape, the inlet hole 132a may be formed in a circular shape, and the connecting hole 132c may be formed in the region where the rectangular and circular shapes overlap. The input hole 132b may have a larger rectangular shape than the rectangular shape of the hole array 120 and expose the micropores of the hole array 120.

[0054] The first strip 132 may include a double-sided strip with an adhesive layer formed on the surface of the contact microfluidic chamber 110 and the surface of the contact aperture array 120, respectively. Therefore, the aperture array 120 may have four edge regions attached to the first strip 132. The side surfaces of the connecting holes 132c forming the first strip 132 may be hydrophilically treated. Therefore, samples injected through the inlet can more easily reach the aperture array 120.

[0055] The second strip 134 may be disposed on the underside of the aperture array 120 and may cover the connecting aperture 132c and the input aperture 132b formed in the first strip 132. The second strip 134 may correspond to the circular shape of the first strip 132. The surface of the second strip 134 corresponding to the connecting aperture 132c may be hydrophilically treated. Therefore, the sample injected through the inlet can reach the aperture array 120 more smoothly. The second strip 134 may be a single-sided strip with an adhesive layer formed on the surface in contact with the first strip 132.

[0056] According to this disclosure, by forming a first strip 132 (disposed between the microfluidic chamber 110 and the pore array 120) and a second strip 134 (disposed on the lower side of the pore array 120) on the capillary member 130, it is possible to prevent air traps from forming in the corner or edge regions of the pore array 120.

[0057] Typically, air traps expand or contract due to temperature changes during the PCR process, causing errors in PCR test results. However, according to this disclosure, when the sample is moved via capillary action using the capillary member 130 and fills the reaction space of the microfluidic chamber 110, the formation of air traps is prevented at the corners or edges of the well array 140, which serves as the reaction space. Therefore, it is possible to prevent errors in the test results due to air traps in the PCR test results. Furthermore, because the well array 120 is located above the CMOS photoelectric sensor array 140, the PCR reaction can be measured in real time.

[0058] The PCR module 100 according to this disclosure may further include a CMOS photoelectric sensor array 140. The CMOS photoelectric sensor array 140 may be disposed below the well array 120 and may capture images of the PCR reaction products in real time, the PCR reaction being performed by filling the microwells of the well array 120.

[0059] The CMOS photodetector array 140 can be configured via an insertion substrate aperture formed in the PCB 150. The CMOS photodetector array 140 can receive emitted light and capture images of the products of the PCR reaction performed in the PCR apparatus in real time. In other words, the CMOS photodetector array 140 detects fluorescence (emission light) generated from multiple probes by excitation light. The fluorescence detection described above can be performed using time-separation or wavelength-separation methods.

[0060] In the case of the time separation method described above, as the fluorescent material emits emission light in response to excitation light, a fluorescent sensor array or a single sensor forming the array detects the emitted light passing through the emission filter and determines the time constant of the detected emitted light to sense the fluorescence.

[0061] For this purpose, the PCR module 100 may also include an emission filter (not shown) that selects light with a predetermined wavelength. The emission filter may be disposed on the CMOS photoelectric sensor array 140.

[0062] Meanwhile, in the case of the wavelength separation method described above, as the fluorescent material responds to the excitation light and emits emission light, the fluorescence sensor array or a single sensor forming the array detects the emitted light passing through the emission filter described above and senses the fluorescence by spectral analysis of the detected emitted light.

[0063] The PCR module 100 according to this disclosure may further include a PCB 150. The PCB 150 is disposed below the microfluidic chamber 110 and can accommodate the mounted CMOS photoelectric sensor array 140. The PCB 150 may be configured to contact the bottom edge region of the microfluidic chamber 110.

[0064] When a sample is introduced into the inlet, it is supplied through the capillary member 130 to a local region, an upper region, and a lower region of the pore array 120.

[0065] In this embodiment, the PCR module 100 may further include a light-providing unit (not shown) configured to direct excitation light toward a probe housed in each of the micropores of the well array 120. In one embodiment, the light-providing unit may include a light source (emitting light), such as a light-emitting diode (LED) light source, a laser light source, etc. Light emitted from the light source passes through or is reflected away from the micropores of the well array 120; and in this case, the CMOS photodetector array 140 can detect the light signal generated by nucleic acid amplification.

[0066] As described above, according to this disclosure, because the PCR module releases reagents built into the well array, there is no need for a separate process for setting the reagents, thereby significantly reducing the possibility of contamination and eliminating the need for a separate process for preparing the test.

[0067] Furthermore, in the case of digital real-time PCR, even though the micropores of the well array (which is the reaction space) are extremely small in size and extremely numerous, it is possible to inject the sample into each of the reaction spaces through capillary action.

[0068] Furthermore, because the sample is injected into the corners or edges of the pore array, the formation of gas traps in the corners or edges of the pore array (which is the reaction space) is prevented, and the reliability of the test results is improved.

[0069] Although the foregoing description is based on examples, those skilled in the art will understand that various modifications and alterations may be made to this disclosure without departing from the spirit and scope of the disclosure as set forth in the appended claims.

[0070] <Explanation of Symbols in the Attached Drawings>

[0071] 100: PCR module

[0072] 110: Microfluidic Chamber

[0073] 112a: First flat element

[0074] 112b: Support hole

[0075] 112c: First flat hole

[0076] 112: Base components

[0077] 112d: Second flat hole

[0078] 114: Tower components

[0079] 114a: Membrane switch

[0080] 114b: Entry Unit

[0081] 120: Hole Array

[0082] 130: Capillary components

[0083] 132: First band

[0084] 132a: Inlet hole

[0085] 132b: Input port

[0086] 132c: Connecting hole

[0087] 134: Second band

[0088] 140: CMOS photoelectric sensor array

[0089] 150: PCB

[0090] Industrial applicability

[0091] According to the PCR module of this disclosure, when the PCR solution moves into the reaction space of the microfluidic chamber using capillary principles and fills the reaction space, it is possible to prevent air traps from forming in the corners or edges of the well array disposed within the reaction space. Therefore, it is possible to prevent errors in PCR test results caused by air traps. Furthermore, because the well array is located above a CMOS photoelectric sensor array, it is possible to measure the PCR reaction in real time.

Claims

1. A PCR module, comprising: A microfluidic chamber, the microfluidic chamber including an inlet unit formed for introducing a sample and capable of being manufactured by injection molding; A pore array comprising a plurality of micropores having a through upper portion and a lower portion and disposed on the lower surface of the microfluidic chamber; and A capillary component configured to provide a path such that the sample introduced through the inlet unit reaches the micropore via capillary action; The capillary component includes a first strip disposed between the microfluidic chamber and the pore array, including an inlet hole, an input hole, and a connecting hole. The inlet hole is formed corresponding to the inlet unit, the input hole is formed corresponding to the pore array, and the connecting hole has a width narrower than the diameter of the inlet hole and is configured to connect the inlet hole and the input hole. The first strip has both rectangular and circular shapes, with the circular shape superimposed on the corner region of the rectangular shape. The rectangular shape corresponds to the aperture array, and the circular shape corresponds to the inlet unit of the microfluidic chamber; The input hole is formed in the rectangular shape, the inlet hole is formed in the circular shape, and the connection hole is formed in the area where the rectangular shape and the circular shape overlap.

2. The PCR module of claim 1, wherein the first band comprises a double-sided band having an adhesive layer formed on the surface contacting the microfluidic chamber and the surface contacting the pore array, respectively.

3. The PCR module according to claim 1, wherein the input well has a rectangular shape that is larger than the rectangular shape of the well array to expose the micropores of the well array.

4. The PCR module of claim 1, wherein the well array has four edge regions attached to the first band.

5. The PCR module of claim 1, wherein the side surface of the connection hole forming the first band is hydrophilically treated.

6. The PCR module of claim 1, wherein the capillary component further comprises a second strip, the second strip being disposed on the underside of the well array and covering the connection well and the input well formed in the first strip, and The second strip corresponds to the circular shape of the first strip.

7. The PCR module according to claim 6, wherein the surface of the second band corresponding to the connecting hole is hydrophilically treated.

8. The PCR module according to claim 6, wherein the thickness of the second band is the same as the thickness of the well array.

9. The PCR module of claim 6, wherein the second band is a one-sided band having an adhesive layer formed on the surface in contact with the first band.

10. The PCR module according to claim 1, wherein the microfluidic chamber comprises polydimethylsiloxane (PDMS) material.

11. The PCR module of claim 1, wherein the microfluidic chamber comprises: A rectangular base component, the base component comprising a rectangular first flat unit, a rectangular support hole, and a circular hole, the support hole being formed in the central region of the first flat unit, and the hole being formed in the corner region of the first flat unit; and A rectangular tower component, comprising a rectangular second flat unit, a disc-shaped membrane switch, and a closed-loop inlet unit, is disposed on the base component.

12. The PCR module according to claim 1, further comprising: A CMOS photoelectric sensor array is disposed below the aperture array and captures reaction images of the sample filling the micropores of the aperture array in real time.

Images