Nucleic acid amplification chip

Abstract

The application provides a nucleic acid amplification chip, comprising a substrate and a heat-conducting metal plate, an amplification cavity for accommodating a nucleic acid sample and a flow channel for guiding the nucleic acid sample to flow into and out of the amplification cavity are arranged on a first side of the substrate, a cavity opening of the amplification cavity is on the first side, and the heat-conducting metal plate is sealingly connected to the cavity opening of the amplification cavity. The application can effectively realize synchronous conduction of heating or cooling of a temperature control device by arranging the amplification cavity on the substrate and sealing the amplification cavity with the heat-conducting metal plate, can effectively shorten the time consumption of temperature control cycles, greatly improves the amplification efficiency, and can effectively prevent sample leakage and cross contamination in the operation process of EP tubes in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nucleic acid amplification chip. Background Technology

[0002] PCR is a common molecular biology technique for amplifying DNA fragments. The conventional PCR method involves transferring the sample to be amplified into EP tubes (Eppendorf Micro Test Tubes) and placing them in a standard PCR instrument for cyclic heating and cooling. Due to their extremely high chemical and biological stability, commercially available EP tubes are mostly made of PP (polypropylene). Because thin-walled EP tubes are difficult to mold and prone to breakage and quality control issues, existing commercially available EP tubes have relatively thick walls, typically above 0.5 mm. This thick wall means that when using a temperature control device (such as a commonly used Peltier temperature controller), it takes about 3 minutes to complete one temperature-controlled cycle of heating-holding (for example, 30 seconds)-cooling of the nucleic acid sample inside the tube. Therefore, to complete 40 cycles, it would take up to two hours. Based on the aforementioned problems, existing technologies often reduce the tube wall thickness to accelerate the amplification process. Currently, the minimum wall thickness can be reduced to 0.3 mm, which corresponds to a temperature control cycle time of approximately 70-90 seconds. As mentioned earlier, this thickness of tube wall places extremely high demands on the injection molding process, making manufacturing quality control difficult. Furthermore, the excessively thin tube wall is prone to breakage during sample transfer or EP tube use. In another related technology, EP tubes are made of metal with good thermal conductivity. EP tubes made of this material can effectively reduce the overall time of temperature control cycles, but they are prone to sample leakage and cross-contamination during sample collection, transfer, and amplification operations. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to provide a nucleic acid amplification chip to overcome the shortcomings of the prior art when using EP tubes to amplify nucleic acid samples, such as excessively long temperature control cycle time, easy sample leakage and cross-contamination during the use of EP tubes.

[0004] To address the aforementioned problems, the present invention provides a nucleic acid amplification chip, comprising a substrate and a thermally conductive metal plate. The first side of the substrate is provided with an amplification cavity for accommodating a nucleic acid sample and a flow channel for guiding the nucleic acid sample into and out of the amplification cavity. The opening of the amplification cavity is located on the first side, and the thermally conductive metal plate is sealed to the opening of the amplification cavity.

[0005] In some embodiments, the bottom wall of the amplification cavity has a plurality of spaced support pillars, which extend along the thickness direction of the substrate to form a support array, and the thermally conductive metal plate is connected to the free end face of the support pillars.

[0006] In some embodiments, the substrate is made of PP plastic, the thermally conductive metal plate is an aluminum foil with an outer PP film, and the thermally conductive metal plate is heat-sealed to the cavity and / or the free end face of the support column.

[0007] In some embodiments, the aluminum foil has a thickness of 50 μm to 100 μm.

[0008] In some embodiments, the flow channel includes an inlet flow channel and an outlet flow channel, both of which have an opening on the first side. The nucleic acid amplification chip also includes a cover plate with a central through hole corresponding to the position of the amplification chamber. The cover plate is connected to the first side to form a seal for the opening. The thermally conductive metal plate is located within the central through hole, and the periphery of the thermally conductive metal plate is at least sealed to the hole wall of the central through hole.

[0009] In some embodiments, an annular heat-sealing plane capable of heat-sealing the thermally conductive metal plate is formed between the wall of the central through hole and the opening of the amplification cavity; and / or, the cover is made of black PP material and the substrate is made of transparent PP material.

[0010] In some embodiments, both the inlet and outlet channels have on / off valve structures that can control their opening and closing.

[0011] In some embodiments, the on / off valve structure includes a control groove constructed on the substrate, a receiving through hole constructed on the cover, an elastic sealing diaphragm disposed within the receiving through hole, and a force-applying member matched with the receiving through hole. The force-applying member can be controlled to move up and down to apply force to the elastic sealing diaphragm. After the elastic sealing diaphragm is forceped, the elastic sealing diaphragm contacts the bottom wall and side wall of the control groove. And / or, the sample inlet channel has a sample inlet located upstream of the corresponding on / off valve structure, and the sample outlet channel has a sample outlet located downstream of the corresponding on / off valve structure.

[0012] In some embodiments, the shape of the control groove and the shape of the receiving through hole are both matching ellipses, and the major axis of the ellipse is parallel to the sample flow direction of the inlet or outlet channel.

[0013] In some embodiments, the accommodating through hole is a stepped hole, and a pressure ring is built into the larger section of the stepped hole. The pressure ring and the stepped surface of the stepped hole clamp the elastic sealing diaphragm and are welded together.

[0014] This invention provides a nucleic acid amplification chip. By constructing an amplification cavity on a substrate and sealing the cavity with a thermally conductive metal plate, when amplifying the nucleic acid sample in the cavity, the heat dissipation surface of the temperature control device is attached to the outer side of the thermally conductive metal plate. Since the thermally conductive metal plate has better thermal conductivity than PP material, it can effectively achieve synchronous conduction of heating or cooling of the temperature control device, effectively shortening the time of temperature control cycle and greatly improving amplification efficiency. At the same time, since the technical solution of this application adopts on-chip amplification, it can also effectively eliminate the potential risks of sample leakage and cross-contamination in the operation process of EP tubes in the prior art. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the nucleic acid amplification chip (after assembly) according to an embodiment of the present invention;

[0016] Figure 2 for Figure 1 Exploded structural diagram;

[0017] Figure 3 for Figure 1 Partial exploded view of the on / off valve structure in the image;

[0018] Figure 4 for Figure 3 A schematic diagram of the pressure ring structure in the middle;

[0019] Figure 5 for Figure 3 A schematic diagram of the force-applying component.

[0020] The attached figures are labeled as follows:

[0021] 1. Substrate; 11. Amplification chamber; 111. Support column; 12. Sample inlet channel; 13. Sample outlet channel; 14. Control groove; 15. Sample inlet; 16. Sample outlet; 2. Thermally conductive metal plate; 3. Cover plate; 31. Central through hole; 32. Accommodation through hole; 4. Elastic sealing diaphragm; 5. Force application component; 51. Push rod; 52. Telescopic valve body; 6. Pressure ring; 61. Diaphragm accommodating cavity; 62. Welding wire; 7. On / off valve structure. Detailed Implementation

[0022] See also Figures 1 to 5As shown, according to an embodiment of the present invention, a nucleic acid amplification chip is provided, including a substrate 1 and a thermally conductive metal plate 2. An amplification cavity 11 for accommodating a nucleic acid sample and a flow channel for guiding the nucleic acid sample into and out of the amplification cavity 11 are constructed on a first side of the substrate 1. The opening of the amplification cavity 11 is located on the first side, and the thermally conductive metal plate 2 is sealed to the opening of the amplification cavity 11. In this technical solution, by constructing the amplification cavity 11 on the substrate 1 and sealing it with the thermally conductive metal plate 2, when amplifying the nucleic acid sample in the amplification cavity 11, the heat dissipation surface of the temperature control device is attached to the outer surface of the thermally conductive metal plate 2. Since the thermally conductive metal plate 2 has better thermal conductivity than PP material, it can effectively achieve synchronous conduction of heating or cooling of the temperature control device, effectively shortening the time consumed by the temperature control cycle and greatly improving the amplification efficiency. Furthermore, since the technical solution of this application adopts on-chip amplification, it can effectively eliminate the potential risks of sample leakage and cross-contamination during the operation process using EP tubes in the prior art. It should be noted that the amplification cavity 11 in this application has a planar structure, and its size can be reasonably designed according to the actual needs of sample detection to accommodate different sample volumes. The size of the heat-conducting metal plate 2 matches and adapts to the amplification cavity 11. The depth of the amplified sample (i.e., the depth of the amplification cavity 11) can be designed to be very small (e.g., 1 mm). The size of the heat dissipation surface of the temperature control device can be consistent with or slightly larger than the size of the heat-conducting metal plate 2. Of course, multiple control devices can also be laid out on a plane to achieve comprehensive and uniform temperature adjustment of the heat-conducting metal plate 2, thus achieving efficient temperature control of nucleic acid samples. It is understood that the aforementioned temperature control device includes a heating device and a cooling device to meet the alternating hot and cold cycle requirements of nucleic acid sample amplification.

[0023] It should be noted that the nucleic acid amplification chip of the present invention can be simultaneously constructed with other microfluidic structures required for nucleic acid extraction. In another feasible embodiment, the nucleic acid amplification chip is connected to relevant microfluidic chips or components upstream or downstream of the process through corresponding interfaces (such as the sample inlet 15 and sample outlet 16 mentioned below) to form a pipeline, without the need for manual sample transfer and other operations, effectively eliminating the potential risks of sample leakage and cross-contamination in the operation process using EP tubes in the prior art.

[0024] In one specific embodiment, the volume of the aforementioned amplification chamber 11 is designed to be 300 μL. Taking Peltier as an example, the temperature rise and fall of 65-95°C can be controlled within 5-10 seconds. In extreme cases, one cycle can be completed in 40 seconds, and 30 cycles can be completed in 20 minutes. Compared with the prior art, the amplification speed is greatly improved.

[0025] See Figure 2As shown, the bottom wall of the amplification cavity 11 has multiple spaced support pillars 111. These support pillars 111 extend along the thickness direction of the substrate 1 to form a support array. A thermally conductive metal plate 2 is connected to the free end face of the support pillars 111. Especially when the area (volume) of the amplification cavity 11 is relatively large, the multiple support pillars 111 forming the support array can reliably support the thermally conductive metal plate 2, improving the structural reliability of the chip and preventing damage to the thermally conductive metal plate 2 due to external forces. The axial cross-section of the aforementioned support pillars 111 is preferably circular to reduce obstruction to sample flow.

[0026] In one specific embodiment, the substrate 1 is made of PP plastic, the support column 111 is integrally injection molded onto the substrate 1, and the heat-conducting metal plate 2 is an aluminum foil with an outer PP film laminate. The heat-conducting metal plate 2 is heat-sealed to the cavity opening and / or the free end face of the support column 111. Using heat sealing to connect the heat-conducting metal plate 2 facilitates the connection between the heat-conducting metal plate 2 and the substrate 1, and also provides a better sealing effect after connection. The aluminum foil thickness is 50μm-100μm to give it a certain degree of toughness, making it less susceptible to damage from accidental operation, while ensuring synchronous heat conduction.

[0027] See again Figure 1 and Figure 2 As shown, the aforementioned flow channels include an inlet flow channel 12 and an outlet flow channel 13, both of which have openings on the first side. The nucleic acid amplification chip also includes a cover plate 3, which has a central through hole 31 corresponding to the position of the amplification chamber 11. The cover plate 3 is connected to the first side to form a seal for the openings. A thermally conductive metal plate 2 is located within the central through hole 31, and the periphery of the thermally conductive metal plate 2 is at least sealed to the wall of the central through hole 31. That is, the thermally conductive metal plate 2 and the cover plate 3 together form a seal for the first side of the substrate 1. In this technical solution, both the inlet flow channel 12 and the outlet flow channel 13 have openings on the first side. The cover plate 3 can reliably seal the openings of the flow channels, reducing the difficulty of constructing the flow channels (e.g., injection molding, draft molding, or direct machining).

[0028] In some embodiments, an annular heat-sealing plane capable of heat-sealing the thermally conductive metal plate 2 is formed between the wall of the central through hole 31 and the opening of the amplification cavity 11. Figure 1 The diagram shows a hexagonal annular heat-sealing plane. The annular width of the heat-sealing plane should not be too small (in one specific embodiment, the width is 3 mm) to ensure a reliable sealed connection between the heat-conducting metal plate 2 and the first side of the substrate 1.

[0029] The cover plate 3 is made of black PP material and the substrate 1 is made of transparent PP material to facilitate laser bonding between the cover plate 3 and the substrate 1. Specifically, the combination of transparent PP material and black PP material can facilitate the heating of the laser at the interface between the two, thereby improving the quality of laser bonding between the two.

[0030] See also Figure 1 and Figure 3 As shown, both the sample inlet channel 12 and the sample outlet channel 13 are equipped with on / off valve structures 7 that can control their opening and closing. These on / off valve structures 7 are controlled to be in a cut-off state during the nucleic acid sample amplification temperature control process to prevent liquid cross-contamination during amplification heating. They are controlled to be in a connected state during sample injection or waste liquid discharge. At this time, the aforementioned sample inlet 15 is located upstream of its corresponding on / off valve structure 7, and the sample outlet 16 is located downstream of its corresponding on / off valve structure 7.

[0031] See Figure 3 As shown, the on / off valve structure 7 includes a control groove 14 constructed on the substrate 1, a receiving through hole 32 constructed on the cover plate 3, an elastic sealing diaphragm 4 placed in the receiving through hole 32, and a force-applying member 5 matched with the receiving through hole 32. The force-applying member 5 can be controlled to lift and apply force to the elastic sealing diaphragm 4. After the elastic sealing diaphragm 4 is forceped, it contacts the bottom wall and side wall of the control groove 14. At this time, the elastic sealing diaphragm 4 is clamped between the force-applying member 5 and the groove wall of the control groove 14, thereby achieving reliable cutoff of the flow channel. It is understood that when the force-applying member 5 is controlled to detach from the elastic sealing diaphragm 4, the elastic sealing diaphragm 4 recovers to its initial state by its own elasticity, thereby opening the flow channel. The on / off valve structure 7 in this technical solution is simple in structure, easy to control, and low in cost. The aforementioned elastic sealing diaphragm 4 can specifically be a silicone diaphragm. The aforementioned force-applying member 5 can specifically be a controlled lifting structure. In one embodiment, see Figure 5 The force-applying component 5 includes a push rod 51 and a telescopic valve body 52 located at the free end of the push rod 51. The push rod 51 can be connected to a lifting device (not shown in the figure). The telescopic valve body 52 can be a valve plate. The width of the valve plate matches the width of the control groove 14, so that it can be used together with the elastic sealing diaphragm 4 to cut off the flow channel.

[0032] The groove shape of the control groove 14 and the shape of the receiving through hole 32 are both elliptical and matched with each other. The major axis of the ellipse is parallel to the sample flow direction of the sample inlet channel 12 or the sample outlet channel 13. The elliptical design with the major axis parallel to the sample flow direction eliminates the generation of dead volume and also has good flow smoothness.

[0033] In some embodiments, the accommodating through hole 32 is a stepped hole, and a pressure ring 6 is built into the larger section of the stepped hole. An elastic sealing diaphragm 4 is sandwiched between the pressure ring 6 and the stepped surface of the stepped hole and welded (ultrasonic welding) to form a single unit. See [reference needed]. Figure 4 As shown, the pressure ring 6 has a corresponding diaphragm receiving cavity 61 on the side of the stepped surface facing the stepped hole. The aforementioned elastic sealing diaphragm 4 is at least partially housed in the diaphragm receiving cavity 61. A circumferentially extending welding line 62 is formed on the end face of the pressure ring 6 facing the stepped surface, which can form a reliable sealed welding connection with the stepped surface.

[0034] It will be readily understood by those skilled in the art that the aforementioned advantageous methods can be freely combined and superimposed without conflict.

[0035] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention. The above are merely preferred embodiments of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention.

Claims

1. A nucleic acid amplification chip, characterized in that, The device includes a substrate (1) and a thermally conductive metal plate (2). The first side of the substrate (1) has an amplification cavity (11) for containing a nucleic acid sample and a flow channel for guiding the nucleic acid sample into and out of the amplification cavity (11). The opening of the amplification cavity (11) is located on the first side, and the thermally conductive metal plate (2) is sealed to the opening of the amplification cavity (11). The bottom wall of the amplification cavity (11) has multiple spaced support pillars (111). These support pillars (111) extend along the thickness direction of the substrate (1) to form a support array. The thermally conductive metal plate (2) is connected to the support array. On the free end face of the column (111); the flow channel includes an inlet flow channel (12) and an outlet flow channel (13), both of which have an opening on the first side. The nucleic acid amplification chip also includes a cover plate (3), which has a central through hole (31) corresponding to the position of the amplification chamber (11). The cover plate (3) is connected to the first side to form a seal on the opening. The heat-conducting metal plate (2) is located in the central through hole (31) and the periphery of the heat-conducting metal plate (2) is at least sealed to the hole wall of the central through hole (31).

2. The nucleic acid amplification chip according to claim 1, characterized in that, The substrate (1) is made of PP plastic, and the heat-conducting metal plate (2) is an aluminum foil with an outer layer of PP film. The heat-conducting metal plate (2) is heat-sealed to the cavity and / or the free end face of the support column (111).

3. The nucleic acid amplification chip according to claim 2, characterized in that, The aluminum foil has a thickness of 50μm-100μm.

4. The nucleic acid amplification chip according to claim 1, characterized in that, The hole wall of the central through hole (31) and the cavity opening of the amplification cavity (11) form an annular heat-sealing plane that can heat-seal the heat-conducting metal plate (2); and / or, the cover plate (3) is made of black PP material and the substrate (1) is made of transparent PP material.

5. The nucleic acid amplification chip according to claim 1, characterized in that, Both the sample inlet channel (12) and the sample outlet channel (13) have on / off valve structures (7) that can control their on / off states.

6. The nucleic acid amplification chip according to claim 5, characterized in that, The on / off valve structure (7) includes a control groove (14) constructed on the substrate (1), a receiving through hole (32) constructed on the cover plate (3), an elastic sealing diaphragm (4) placed in the receiving through hole (32), and a force-applying member (5) matched with the receiving through hole (32). The force-applying member (5) can be controlled to lift and apply force to the elastic sealing diaphragm (4). After the elastic sealing diaphragm (4) is applied force, the elastic sealing diaphragm (4) contacts the bottom wall and side wall of the control groove (14); and / or, the sample inlet channel (12) has a sample inlet (15) located upstream of the on / off valve structure (7) corresponding to it, and the sample outlet channel (13) has a sample outlet (16) located downstream of the on / off valve structure (7) corresponding to it.

7. The nucleic acid amplification chip according to claim 6, characterized in that, The groove shape of the control groove (14) and the shape of the accommodating through hole (32) are both elliptical shapes that match each other, and the major axis of the ellipse is parallel to the sample flow direction of the sample inlet channel (12) or the sample outlet channel (13).

8. The nucleic acid amplification chip according to claim 6, characterized in that, The accommodating through hole (32) is a stepped hole, and a pressure ring (6) is built into the large hole section of the stepped hole. The pressure ring (6) and the stepped surface of the stepped hole clamp the elastic sealing diaphragm (4) and are welded together.

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