PCR module

Abstract

A PCR module capable of injecting a sample using the capillary principle is disclosed. The PCR module includes a microfluidic chamber including an inlet portion formed for sample injection and capable of being manufactured by injection molding; a well array including a plurality of microwells penetrating the top and bottom and disposed on the bottom surface of the microfluidic chamber; and a capillary member providing a path for the sample injected through the inlet portion to reach the microwells by capillary action. When a PCR solution is moved into a reaction space using the capillary principle and the solution fills the reaction space, it is possible to prevent the formation of air pockets in the corners and edge regions of the well array, which is the reaction space. This makes it possible to prevent errors caused by air pockets in PCR test results. In addition, since the well array is located on a CMOS photosensor array, real-time PCR reactions can be measured.

Description

[Technical Field]

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0090030, dated July 8, 2021, and all contents disclosed in the documents of this Korean Patent Application are incorporated herein by reference.

[0002] The present invention relates to a PCR module, and more particularly to a PCR module that prevents the occurrence of air pockets in the corners and edge regions of the reaction space of a microfluidic chamber and enables real-time PCR measurement. [Background technology]

[0003] Gene amplification technology is an essential process in molecular diagnosis, and it involves repeatedly replicating and amplifying specific base sequences in trace amounts of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in a sample. Among these, polymerase chain reaction (PCR) is a representative gene amplification technology and consists of three steps: DNA denaturation, primer binding (annealing), and DNA replication (extension). Each step is dependent on the temperature of the sample, so DNA can be amplified by repeatedly changing the temperature of the sample.

[0004] Traditionally, PCR has typically been performed in 96- or 384-well microplates. When higher throughput is required, traditional microplate PCR methods are not cost-effective or efficient. On the other hand, reducing the PCR reaction volume can reduce reagent consumption and, with the reduced thermal mass of the reaction volume, shorten amplification time. This strategy can also be implemented in an array format (mxn), thereby enabling multiple, smaller reaction volumes. Furthermore, the use of an array allows for scalable high-throughput analysis with increased quantitative sensitivity, dynamic range, and specificity.

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

[0006] The array format of most quantitative polymerase chain reaction (qPCR) platforms is designed for sample-by-sample analysis, where PCR results must be addressable for post-hoc analysis. However, for dPCR, the specific location or well of each PCR result is not important; simply the number of positive and negative replicates per sample may be analyzed.

[0007] In dPCR, a solution containing a relatively small number of target polynucleotides or nucleic acid sequences can be divided into small test samples, whereby each sample typically contains one or zero molecules of the target nucleotide sequence. When the samples are subsequently thermally cycled in a PCR protocol, procedure, or experiment, samples containing the target nucleotide sequence will be amplified and produce a positive detection signal, while samples not containing any target nucleotide sequence will not be amplified and will not produce a detection signal.

[0008] In the case of such digital PCR, the size of the reaction spaces is very small and there are a large number of them, so it is difficult to introduce a sample into each of the reaction spaces.

[0009] Furthermore, if the sample is not injected into the corners or edges of the reaction space, air pockets may form in the corners or edges of the reaction space, which may expand or contract due to temperature changes during the PCR process, resulting in errors in the test results.

[0010] The main existing dPCR method uses end-point PCR, which separates the process of adding a sample to 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 [Problem to be solved by the invention]

[0011] The technical problem of the present invention is to solve these conventional problems, and the object of the present invention is to provide a PCR module that uses the capillary principle to inject a sample into a reaction space of a microfluidic chamber, preventing the occurrence of air pockets in the corners and edge regions of the reaction space, and enabling real-time PCR measurement. [Means for solving the problem]

[0012] To achieve the object of the present invention, a PCR module according to one embodiment includes: a microfluidic chamber that includes an inlet portion formed for sample introduction and that can be manufactured by injection molding; a well array that includes a plurality of microwells that are perforated at the top and bottom and that is disposed on the bottom surface of the microfluidic chamber; and a capillary member that provides a path for the sample introduced through the inlet portion to reach the microwells by capillary action. [Effects of the Invention]

[0013] This PCR module uses the capillary principle to move a PCR solution into the reaction space of a microfluidic chamber. When the solution fills the reaction space, air pockets can be prevented from forming at the corners and edges of the well array disposed within the reaction space. This prevents errors in PCR test results due to air pockets. Furthermore, the well array is located on a CMOS photosensor array, allowing real-time PCR reactions to be measured. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a perspective view illustrating a PCR module according to one embodiment of the present invention. [Figure 2] FIG. 2 is an exploded rear perspective view illustrating the PCR module shown in FIG. [Figure 3] FIG. 2 is an exploded perspective view illustrating the capillary member shown in FIG. 1. [Figure 4] FIG. 2 is a cross-sectional view illustrating the PCR module shown in FIG. DETAILED DESCRIPTION OF THE INVENTION

[0015] To achieve the object of the present invention, a PCR module according to one embodiment includes: a microfluidic chamber that includes an inlet portion formed for sample introduction and that can be manufactured by injection molding; a well array that includes a plurality of microwells that are perforated at the top and bottom and that is disposed on the bottom surface of the microfluidic chamber; and a capillary member that provides a path for the sample introduced through the inlet portion to reach the microwells by capillary action.

[0016] In one embodiment, the capillary member may include a first tape disposed between the microfluidic chamber and the well array, the first tape including an inlet hole formed corresponding to the inlet portion, an input hole formed corresponding to the well array, and a connecting hole having a width narrower than the diameter of the inlet hole and connecting the inlet hole and the input hole.

[0017] In one embodiment, the first tape has a rectangular shape with a circular shape superimposed on the edge region of the rectangular shape, the rectangular shape corresponding to the well array, and the circular shape corresponding to the inlet portion of the microfluidic chamber.

[0018] In one embodiment, the input hole may be formed in the square shape, the inflow hole may be formed in the circle shape, and the connection hole may be formed in a region where the square shape and the circle shape overlap.

[0019] In one embodiment, the first tape may include a double-sided tape having an adhesive layer formed on each of a surface that contacts the microfluidic chamber and a surface that contacts the well array.

[0020] In one embodiment, the input hole may have a rectangular shape larger than the rectangular shape of the well array, exposing the microwells of the well array.

[0021] In one embodiment, the well array has four edge regions attached to the first tape.

[0022] In one embodiment, the side of the first tape where the connecting holes are formed is subjected to a hydrophilic treatment.

[0023] In one embodiment, the capillary member further includes a second tape disposed below the well array and covering the connection hole and the input hole formed in the first tape, and the second tape may correspond to the circular shape of the first tape.

[0024] In one embodiment, the surface of the second tape corresponding to the connecting holes is hydrophilically treated.

[0025] In one embodiment, the thickness of the second tape is the same as the thickness of the well array.

[0026] In one embodiment, the second tape is a cross-section tape having an adhesive layer formed on the surface that contacts the first tape.

[0027] In one embodiment, the microfluidic chamber may include a PDMS (Polydimethylsiloxane) material.

[0028] In one embodiment, the microfluidic chamber may include a square base member including a square first flat portion, a square support hole formed in a central region of the first flat portion, and a circular hole formed in an edge region of the first flat portion; and a square top member including a square second flat portion, a dish-shaped membrane switch, and a closed-loop inlet portion, and disposed on the base member.

[0029] In one embodiment, the PCR module may further include a CMOS photosensor array disposed below the well array for capturing reaction images of samples filled in the microwells of the well array in real time.

[0030] The present invention will now be described in more detail with reference to the accompanying drawings. Because the present invention can be modified in various ways and take on various forms, specific embodiments are shown by way of example in the drawings and will be described in detail herein. However, it should be understood that this is not intended to limit the present invention to the specific disclosed form, and that the present invention encompasses all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention.

[0031] In the drawings, like reference numerals have been used to refer to like elements throughout the description, and the dimensions of structures in the drawings have been exaggerated to make the invention clearer.

[0032] Terms such as "first," "second," etc. are used to describe various components, but the components are not limited by these terms. These terms are used only to distinguish one component from another. For example, a first component may be referred to as a "second component," and similarly, a second component may be referred to as a "first component," without departing from the scope of the present invention. A singular term includes a plural term unless the context clearly dictates otherwise.

[0033] In this specification, the terms "comprise" or "have" and the like are intended to specify the presence of a stated feature, number, step, operation, component, part, or combination thereof, but should be understood as not precluding the presence or possible addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0034] Furthermore, unless otherwise defined, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by those skilled in the art. Terms defined in commonly used dictionaries should be interpreted to have a meaning consistent with the context of the relevant art, and should not be interpreted as having an ideal or overly formal meaning unless expressly defined herein.

[0035] FIG. 1 is a perspective view illustrating a PCR module 100 according to one embodiment of the present invention.

[0036] Fig. 2 is an exploded rear perspective view illustrating the PCR module 100 shown in Fig. 1. Fig. 3 is an exploded perspective view illustrating the capillary member 130 shown in Fig. 1. Fig. 4 is a cross-sectional view illustrating the PCR module 100 shown in Fig. 1.

[0037] 1 to 4, a PCR module 100 according to one embodiment of the present invention includes a microfluidic chamber 110, a well array 120, and a capillary member .

[0038] The microfluidic chamber 110 includes a base member 112 and a top member 114. The base member 112 and the top member 114 may be integrally formed. A recessed space is formed in the bottom region of the microfluidic chamber 110 to accommodate the well array 120 and the capillary member 130. The microfluidic chamber 110 may include a transparent and flexible material. For example, the microfluidic chamber 110 may include a PDMS material. This allows the microfluidic chamber 110 to be manufactured by injection molding.

[0039] The base member 112 includes a rectangular first flat portion 112a, a rectangular support hole 112b formed in a central region of the lower portion of the first flat portion 112a, a circular first flat hole 112c formed in a first edge region of the lower portion of the first flat portion 112a, and a circular second flat hole 112d formed in a second edge region of the lower portion of the first flat portion 112a. The first edge region and the second edge region face each other. The support hole 112b may correspond to the well array 120, the first flat hole 112c may correspond to an inlet portion through which a sample is injected, and the second flat hole 112d may correspond to an outlet portion through which a sample is discharged.

[0040] The top member 114 includes a dish-shaped membrane switch 114 a and a closed-loop inlet portion 114 b , and is disposed on the base member 112 .

[0041] The membrane switch 114a is disposed in the central region of the top member 114 and protrudes upward. The lower region of the membrane switch 114a corresponds to the support hole of the base member 112. This forms a recessed space at the bottom of the microfluidic chamber 110. The recessed space accommodates the CMOS image sensor and the well array 120.

[0042] The inlet portion 114b has a fence shape and is formed for introducing a sample. The inlet portion 114b protrudes upward from one side of the membrane switch 114a to block the outflow of the sample. An inlet is formed in the central region of the inlet portion 114b in the vertical direction of the base member 112. The sample is injected into the well array 120 through the inlet via the capillary member 130.

[0043] The top member 114 may further include a grip portion 114c that protrudes upward from the viewer's point of view on the other side of the membrane switch 114a. The grip portion 114c is disposed so as to face the inlet portion 114b with respect to the membrane switch 114a. The height of the grip portion 114c is greater than the height of the inlet portion 114b. The height of the inlet portion 114b and the height of the membrane switch 114a are the same.

[0044] The well array 120 includes a plurality of microwells that are perforated from top to bottom and is disposed on the underside of the microfluidic chamber 110. The well array 120 is disposed on the CMOS photosensor array 140 and inserted into substrate holes formed in the PCB 150. The well array 120 may include a plurality of microwells. The shapes, dimensions, and number of the microwells vary. Each microwell contains an analytical sample, such as a powder or liquid sample. The analytical sample is a specific component for analyzing a biological substance. That is, the analytical sample refers to components for quantitative or qualitative analysis of a specific biological substance, such as protein, DNA, or RNA, such as primers, probes, antibodies, aptamers, DNA or RNA polymerase, and particularly refers to components necessary for performing a real-time polymerase chain reaction, a constant temperature enzyme reaction, or a ligase chain reaction (LCR).

[0045] The capillary member 130 includes a first tape 132 disposed between the microfluidic chamber 110 and the well array 120 and a second tape 134 disposed below the well array 120, and provides a path for a sample introduced through the inlet portion 114b to reach the microwells by capillarity action. Here, capillarity refers to the property of moving along a narrow space such as a thin tube or porous material, regardless of external forces such as gravity.

[0046] The first tape 132 has a square shape with a circle superimposed on the edge region of the square, and the square shape corresponds to the well array 120, and the circle shape can correspond to the inlet portion 114b of the microfluidic chamber 110.

[0047] The first tape 132 may include an inlet hole 132a formed corresponding to the inlet of the inlet portion 114b, an input hole 132b formed corresponding to the well array 120, and a connecting hole 132c having a width narrower than the diameter of the inlet hole 132a and connecting the inlet hole 132a and the input hole 132b. The input hole 132b may be formed in a square shape, the inlet hole 132a may be formed in a circular shape, and the connecting hole 132c may be formed in a region where the square shape and the circle shape overlap. The input hole 132b may have a square shape larger than the square shape of the well array 120, thereby exposing the microwells of the well array 120.

[0048] The first tape 132 is a double-sided tape with adhesive layers formed on both the surface that contacts the microfluidic chamber 110 and the surface that contacts the well array 120. As a result, the four edge regions of the well array 120 are attached to the first tape 132. The side of the first tape 132 that forms the connecting hole 132c is hydrophilically treated. This allows the sample injected through the inlet to reach the well array 120 more smoothly.

[0049] The second tape 134 is disposed below the well array 120 and can cover the connecting hole 132c and the input hole 132b formed in the first tape 132. The second tape 134 can correspond to the circular shape of the first tape 132. The surface of the second tape 134 corresponding to the connecting hole 132c is hydrophilically treated. This allows the sample injected through the inlet to reach the well array 120 more smoothly. The second tape 134 is a cross-sectional tape with an adhesive layer formed on the surface that contacts the first tape 132.

[0050] According to the present invention, the capillary member 130 is formed by a first tape 132 arranged between the microfluidic chamber 110 and the well array 120 and a second tape 134 arranged below the well array 120, thereby preventing the formation of air pockets in the corners and edge regions of the well array 120.

[0051] Typically, air pockets expand or contract due to temperature changes during the PCR process, causing errors in PCR test results. However, according to the present invention, when a sample is moved by capillary action using the capillary member 130 and fills the reaction space of the microfluidic chamber 110, air pockets are prevented from forming in the corners and edges of the well array 140, which is the reaction space. This prevents errors in PCR test results due to air pockets. In addition, since the well array 120 is located on the CMOS photosensor array 140, real-time PCR reactions can be measured.

[0052] The PCR module 100 according to the present invention may further include a CMOS photosensor array 140. The CMOS photosensor array 140 is disposed below the well array 120 and can capture images of PCR reaction products being filled into the microwells of the well array 120 in real time.

[0053] The CMOS photosensor array 140 is inserted into a substrate hole formed in the PCB 150. The CMOS photosensor array 140 receives emitted light and can capture images of PCR reaction products occurring in a PCR device in real time. That is, the CMOS photosensor array 140 detects fluorescence emitted from a number of probes in response to excitation light. The fluorescence detection may be performed using a time-separation method or a wavelength-separation method.

[0054] In the time-resolved mode, as the fluorescent material emits light in response to the excitation light, the fluorescent sensor array, or the single sensors that make up the array, detect the emitted light that passes through an emission filter and sense the fluorescence by determining the time series of the detected emitted light.

[0055] For this purpose, the PCR module 100 may further include an emission filter (not shown) that selects light having a predetermined wavelength. The emission filter is disposed on the CMOS photosensor array 140.

[0056] On the other hand, in the wavelength separation method, as the fluorescent material emits light in response to excitation light, the fluorescent sensor array or individual sensors constituting the array detect the emitted light that passes through the emission filter and sense the fluorescence through spectral analysis of the detected emitted light.

[0057] The PCR module 100 according to the present invention may further include a PCB 150. The PCB 150 is disposed below the microfluidic chamber 110 and can accommodate the mounted CMOS photosensor array 140. The PCB 150 is positioned so as to contact the bottom edge region of the microfluidic chamber 110.

[0058] When a sample is introduced into the inlet, the capillary member 130 provides the sample to a portion of the well array 120, an upper region of the portion, and a lower region of the portion.

[0059] In this embodiment, the PCR module 100 may further include a light providing unit (not shown) arranged to irradiate excitation light toward the probes contained in each microwell of the well array 120. In one embodiment, the light providing unit may include a light source that emits light, such as an LED (Light Emitting Diode) light source or a laser light source. The light emitted from the light source passes through or is reflected by the microwells of the well array 120, and in this case, the CMOS photosensor array 140 can detect optical signals generated by nucleic acid amplification.

[0060] As described above, according to the present invention, the PCR module is sold with the reagents already installed in the well array, eliminating the need for a separate procedure for setting up the reagents, dramatically reducing the possibility of contamination and eliminating the need for a separate procedure for test preparation.

[0061] Furthermore, in the case of digital real-time PCR, even if the size of the microwells in the well array, which are the reaction spaces, is very small and the number of them is very large, it is possible to introduce a sample into each of the reaction spaces by capillary action.

[0062] Furthermore, the sample is injected up to the corners and edge regions of the well array, preventing the formation of air pockets in the corners and edge regions of the well array, which are the reaction spaces, thereby improving the reliability of the test results.

[0063] Although the present invention has been described with reference to the embodiments, those skilled in the art will understand that the present invention can be modified and changed in various ways without departing from the spirit and scope of the present invention as defined in the claims below. [Industrial Applicability]

[0064] The PCR module of the present invention utilizes the capillary principle to move a PCR solution into the reaction space of a microfluidic chamber. When the solution fills the reaction space, air pockets can be prevented from forming at the corners and edges of the well array disposed within the reaction space. This prevents errors in PCR test results due to air pockets. Furthermore, the well array is positioned on a CMOS photosensor array, allowing real-time PCR reaction monitoring. [Explanation of symbols]

[0065] 100: PCR module 110: Microfluidic chamber 112a: First flat section 112b: Support hole 112c: First Flat Hole 112: Base material 112d: 2nd Flat Hole 114: Top member 114a:Membrane switch 114b: Inlet section 120: Well array 130: Capillary member 132: Tape 1 132a: Inflow hole 132b: Throwing hole 132c: Connecting hall 134: Second Tape 140: CMOS photosensor array 150: PCB

Claims

1. a microfluidic chamber that can be manufactured by injection molding and includes an inlet portion formed for sample introduction; a well array disposed on the bottom surface of the microfluidic chamber, the well array including a plurality of microwells that are perforated at the top and bottom; a capillary member that provides a path for the sample introduced through the inlet to reach the microwell by capillary action; The capillary member is a first tape disposed between the microfluidic chamber and the well array, the first tape including an inlet hole formed corresponding to the inlet portion, an input hole formed corresponding to the well array, and a connecting hole having a width narrower than a diameter of the inlet hole and connecting the inlet hole and the input hole; the first tape has a rectangular shape and a circular shape superimposed on an edge region of the rectangular shape, the square shape corresponds to the well array, and the circle shape corresponds to the inlet portion of the microfluidic chamber; The input hole is formed in the square shape, the inflow hole is formed in the circle shape, and the connection hole is formed in a region where the square shape and the circle shape overlap, a second flat hole formed in a second edge region below the first flat portion; and a second flat hole formed in a second edge region below the first flat portion.

2. 2. The PCR module according to claim 1, wherein the first tape comprises a double-sided tape having an adhesive layer formed on each of a surface that contacts the microfluidic chamber and a surface that contacts the well array.

3. 2. The PCR module according to claim 1, wherein the input hole has a rectangular shape larger than the rectangular shape of the well array, and exposes a microwell of the well array.

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

5. The PCR module according to claim 1 , wherein a side surface of the first tape on which the connecting holes are formed is subjected to a hydrophilic treatment.

6. The capillary member is a second tape disposed beside the well array and covering the connection holes and the inlet holes formed in the first tape; The PCR module according to claim 1 , wherein the second tape corresponds to the circular shape of the first tape.

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

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

9. 7. The PCR module according to claim 6, wherein the second tape is a cross-section tape having an adhesive layer formed on the surface that contacts the first tape.

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

11. The microfluidic chamber comprises: a rectangular base member including a rectangular first flat portion, a rectangular support hole formed in a central region of the first flat portion, and a circular hole formed in an edge region of the first flat portion; 2. The PCR module according to claim 1, further comprising: a rectangular top member disposed on the base member, the top member including a rectangular second flat portion, a dish-shaped membrane switch, and a closed-loop inlet portion.

12. 10. The PCR module of claim 1, further comprising a CMOS photosensor array disposed below the well array for capturing a reaction image of a sample filled in the microwell of the well array in real time.

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